Table of Content

15 April 2014, Volume 47 Issue 8

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CROP GENETICS & BREEDING·GERMPLASM RESOURCES·MOLECULAR GENETICS

Development of Drought-Tolerant Rice Germplasm by Screening and Transforming TAC Clones of Oryza officinalis Wall.

LIU Rui, ZHANG Huan-Huan, CHEN Zhi-Xiong, SHAHID Muhammad Qasim, FU Xue-Lin, LIU Yao-Guang, LIU Xiang-Dong, LU Yong-Gen

Scientia Agricultura Sinica. 2014, 47(8):  1445-1457.  doi:10.3864/j.issn.0578-1752.2014.08.001

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【Objective】Oryza officinalis Wall. has many abiotic tolerance-related genes,which are very important germplasms in rice breeding, however, it is difficult to utilize these genes in cultivated rice by interspecific hybridization due to the reproductive isolation. This study was planned to establish an efficient and quick method for transferring useful genes of O. officinalis into elite cultivated rice varieties. 【Method】Positive clones were screened by southern hybridization from the TAC library of O. officinalis with the conservative sequence of AP2/EREBP and bZIP family genes as probes. The probes were labeled with α-32P during the first screening and with Digoxigenin during the second screening. PCR was used to verify the positive clones finally, which were introduced into cultivated rice by Agrobacterium tumefaciens-mediated transformation. The transformed plants were detected by PCR amplification with the primes designed according to the conservative sequence of Hpt and SacB that located on both sides of the fragment in TAC. Southern hybridization was also used to identify the transformation plants. PEG-6000 was used to identify the drought tolerance of T2 at germination and seedling stages.【Result】A total of 1 073 clones were developed from the TAC library with AP2/EREBP and bZIP probes that were labeled with α-P32. A total of 147 clones were obtained from the 1 073 clones with Digoxigenin labeled probes, and 95 clones detected by AP2/EREBP probe and 52 clones by bZIP probe. After PCR detection, a total of 103 clones produced PCR amplified products, among them 63 clones were detected by AP2/EREBP probe and 40 clones by bZIP probe and the percentages of positive clones were 66.32% and 76.92%, respectively. Five positive clones (49R-O14, 55R-A17, 8R- A24, 22D-P2q and 52D-M16, named by the clone’s number) were successfully introduced into Nipponbare by the Agrobacterium tumefaciens-mediated transformation. The result showed that the longer inserted fragment was more difficult to be transformed into O. sativa. The results of PCR and Southern hybridization showed that foreign fragments were transferred successfully into the genome of regenerated plantlets. Further, tests of hygromycin resistance also showed the same results. The transgenic lines showed strong tolerance to drought stress, among which R12-23, R15-41, and R1-8 were more tolerance to drought stress than Nipponbare did through detection by tolerance to drought stress at budstage; M63-9 and R12-23 were better in tolerance to drought stress than Nipponbare did through detection by tolerance to drought stress at seedingstage. It was concluded that R12-23 is high tolerance to drought stress.【Conclusion】 The work demonstrated that introduction of stress-related TAC clones coupled with a transgenic validation approach is an efficient strategy to transfer agronomically important genes from O. officinalis to cultivated rice.

Overexpression of a Gossypium hirsutum Stress-Associated Protein Gene (GhSAP1) Improves Salt Stress Tolerance in Transgenic Tobacco

DING Lin-Yun, ZHANG Wei, WANG Jin-Cheng, TIAN Liang-Liang, LI Ni-Na, GUO Qi, YANG Shu-Ming, HE Man-Lin, GUO Wang-Zhen

Scientia Agricultura Sinica. 2014, 47(8):  1458-1470.  doi:10.3864/j.issn.0578-1752.2014.08.002

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【Objective】Stress associated proteins (SAPs) is a type of novel stress response-inducing zinc finger proteins with A20/AN1 zinc-?nger domain. The objectives of this study are to isolate a member of stress associated protein (SAP) gene family, analyze its responses to abiotic stress, and provide a potential candidate gene for improving abiotic stress tolerance in cotton by biotechnological approaches. 【Method】A cotton zinc protein gene GhSAP1 was isolated based on in silico cloning and confirmed by RT-PCR analysis, further, recombinant plasmid vector with GhSAP1 was transformed into purified cotton mesophyll protoplast mediated with PEG for elucidating the characteristics of encoded proteins and subcellular location by a transient expression system. The expression patterns of GhSAP1 in different tissues and under salt stress were analyzed through real-time RT-PCR. By leaf disc method, Agrobacterium tumefaciens-mediated transformation, and tissue culture, different transgenic tobacco lines with high ectopic overexpression of GhSAP1 were obtained, further, their determination of physiologic index related to abiotic stress was finished and the potential of improving abiotic stress tolerance in plant was elucidated. 【Result】GhSAP1 contained ORF length of 543 bp and encoded a polypeptide containing 180 amino acids with typical A20/AN1 zinc finger structural domain. The theoretical iso-electric point was 8.97 with a calculated molecular weight of 19.3 kD. Subcellular location showed that GhSAP1 was localized in cell nucleus. The expression patterns in different tissues and organs showed that GhSAP1 expressed preferentially in root, stem, and leaf tissues, but the expression level was the lowest in young ovules at five days post anthesis. GhSAP1 was accumulated significantly when induced after 2 h under salt stress treatment. Ectopic overexpression of GhSAP1 in tobacco plants was performed via leaf disc transformation mediated by Agrobacterium tumefaciens, and was confirmed in both genomic and transcriptional level by PCR and RT-PCR in transgenic tobacco seedlings. Ten tobacco transgenic lines were preliminarily obtained through resistance selection with 100 μg•mL-1 Kan culture medium for transgenic progeny, and confirmed by PCR and RT-PCR of GhSAP1. The result showed that GhSAP1 was integrated into tobacco genome validity with high ectopic overexpression. Further, three transgenic tobacco positive lines were selected by random and analyzed under salt stress. Treating the seedlings of germination for seven days using 200 mmol•L-1 NaCl, 12 days later, the average survival rate of transgenic tobacco lines was 81.7%, which was 3 times higher than those of wild-type plants, and the relative root length in transgenic plants was 3.05 cm in average, significantly higher than wild type plants. Further, L2 pure lines with higher GhSAP1 expression was selected to elucidate physiologic index related to abiotic stress. In detail, when treating the plants with 9 to 11 leaves using 200 mmol•L-1 NaCl, 30 days later, the transgenic lines overexpressing GhSAP1 showed better SOD activity and increased total soluble sugar content, with net increase value of 23.89 U•g-1 FW and 8.37 %, respectively, which was significantly higher than non-transgenic plants. Compared with non-stress treatment, the reduction of the chlorophyll content of transgenic plants was only 0.17 mg•g-1 FW, in contrast to the 0.39 mg•g-1 FW of the non-transgenic plants. After treatment under salt stress condition, the MDA content of transgenic plants only increased by 7.42 nmol•g-1 FW, however, the MAD content of the non-transgenic plants increased by 20.85 nmol•g-1 FW. Meanwhile, the transgenic plants had the better transport ability of K+ from plant root to green tissue, the K+/ Na+ ratio of above-ground part was 1.23, with higher transport ability compared with wild type plants.【Conclusion】It was concluded that GhSAP1 plays an important role in response to salinity stress in plants, overexpression of GhSAP1 can improve significantly tolerance to salt stress in transgenic tobacco plants.

QTL Mapping Based on Embryo and Maternal Genetic Systems for Oil and Protein Contents in Rapeseed (Brassica napus L.)

XU Jian-Feng-1, LONG Yan-2, WU Jian-Guo-3, ZHAO Zhi-Gang-4, XU Hai-Ming-1, WEN Juan-1, MENG Jin-Ling-2, SHI Chun-Hai-1

Scientia Agricultura Sinica. 2014, 47(8):  1471-1480.  doi:10.3864/j.issn.0578-1752.2014.08.003

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【Objective】 In present study, BC1F1 1 and BC1F1 2 populations constructed from Brassica napus genetic population TN DH and parents Tapidor and Ningyou7 were used to analyze the distribution and linkage markers of QTLs with embryo genetic effects, maternal genetic main effects, and their QTL × environment interaction effects on oil and protein contents in rapeseed, as well as to study the influence of environmental interaction on QTL mapped on different genetic systems and explore the optimal strategies and methods for improvement of these two quality traits in marker-assisted selection. 【Method】 A total of 202 TN DH lines and parents were planted in accordance with the general method for field experiments, based on randomized block design with two replications for two years. At flowering stage, BC1F1 1 and BC1F1 2 populations were constructed by double cross between 202 TN DH lines and parents, and the mature seeds of backcross population and parents were harvested for determining the oil and protein contents in rapeseed. Near infrared reflectance calibration models and methods were used for the measurement of oil and protein contents. A genetic map and a newly developed QTL mapping software and methods considering two different genetic systems for quality traits of dicotyledonous crop seeds were used to detect QTLs for oil and protein contents with the data from BC1F1 1 and BC1F1 2 populations in different years. 【Result】 A total of seven QTLs relevant to oil and protein contents in rapeseed were distributed in A1, A4, A6, A7, C2 and C5 linkage groups, in which four QTLs associated with oil content were detected as well as three QTLs for protein content, with total phenotypic contributions of 49.1% and 59.6%, respectively. All QTLs showed extremely significant embryo additive main effects and maternal additive main effects, in which four QTLs were found to have significant or extremely significant embryo dominant main effects, and two QTLs for oil content had significant environmental interaction effects. qOC-6-3 and qPC-4-1 were detected as the important QTLs with larger genetic effects, which could explain 36.3% and 37.9% of the phenotypic variation in oil content and protein content, respectively. qOC-4-2 and qPC-4-1 were co-localized in the A4 linkage group between molecular markers HS-K02-2 and HBR094 with the confidence interval of 17.5-19.4 cM. 【Conclusion】 The results showed that oil content and protein content in rapeseed could be controlled by the expression of QTLs located in different genetic systems of seed embryo and maternal plant, and environment interaction effects play more significant roles on the performance of oil content while the QTL expression of protein content was relatively stable in different environmental conditions. In addition, qOC-6-3 detected on A6 linkage group and qPC-4-1 on A4 linkage group are major QTLs for oil and protein contents，respectively, and three QTLs for protein content in present experiment have not been reported ever since.

TILLAGE & CULTIVATION·PHYSIOLOGY & BIOCHEMISTRY·AGRICULTURE INFORMATION TECHNOLOGY

Measurement and Analysis of 3D Wheat Root System Architecture with a Virtual Plant Tool Kit

CHEN Xin-Xin-1, DING Qi-Shuo-1, 2 , DING Wei-Min-1, TIAN Yong-Chao-2, ZHU Yan-2, CAO Wei-Xing-2

Scientia Agricultura Sinica. 2014, 47(8):  1481-1488.  doi:10.3864/j.issn.0578-1752.2014.08.004

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【Objective】 A consistent and useful system for virtual crop root system simulation was constructed with integrated hardware and software, and applied to quantify post-paddy wheat RSA. 【Method】 A crop RSA digitizer was designed and fabricated in a purely mechanical manner. The digitizer was constructed with a leveled rotating beam, a cross slider and a vertical slider. All the beam and sliders were calibrated with rulers. The coordinated movements of the three moving parts allows the measuring tip to access any possible point within the 3D root-soil volume. Root zone soil sample, installed on the platform of the digitizer, was stripped in layers, each in 3-5 mm thickness. The positions of the exposed roots were digitized. This laminated treatment continued until the whole RSA was digitized. Collected RSA data was then transferred to Pro-E, where the virtual RSA was reconstructed. As the data set used for RSA reconstruction came directly from undisturbed field-grown crop, the reconstructed virtual RSA was able to represent the actual RSA state and the dynamical process of RSA in situ. The virtual RSA was then analyzed with tools supplied in the Pro-E, where the 3D RSA parameters were calculated, such as soil exploration ability of root system, root total length and mean root elongation rate, etc. Wheat RSA dynamics was also quantified with these parameters. 【Result】 Being easy to operate, suitable for dusting environment and highly stable under noisy conditions, the proposed RSA digitizer provides a measurement resolution of <1 mm and can be used as a satisfactory tool for RSA measurement. Pro-E provides a means of realizing the virtual RSA, and also an in-depth computation of RSA parameters. Results shown that the proposed virtual RSA method was able to illustrate structural characteristics and soil exploitation abilities of root system in each stage of wheat seedling development, as well as the root-colonized soil depth and circumferential root expanding dynamics. High heterogeneity of RSA was observed in each wheat seedling stage. Wheat root was confined in shallow soil layers in winter, but rapidly expanded to deeper layers in standing and tillering stages. More rapid development of root was observed in the shooting stage. 【Conclusion】 The proposed virtual RSA, integrated with hardware and software, is a technical solution for accurate quantification of root system dynamics, root-soil relation, root topology and the dynamics of soil space exploration. This tool kit provides a solution for RSA quantification in each post-paddy wheat seedling stage, thus illustrating the dynamics of wheat RSAs and satisfying the practical needs for crop RSA study.

Effects of Photo-synthetically Active Radiation on Photosynthetic Characteristics and Yield of Soybean in Different Maize/Soybean Relay Strip Intercropping Systems

CUI Liang, SU Ben-Ying, YANG Feng, YANG Wen-Yu

Scientia Agricultura Sinica. 2014, 47(8):  1489-1501.  doi:10.3864/j.issn.0578-1752.2014.08.005

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【Objective】 Intercropping, an agricultural practice of cultivating two or more crops in the same space at the same time, is an old and commonly used cropping practice which aims to match efficiently crop demands for the available growth resources and labor. Cereal and legume intercropping, as one of the multiple cropping systems, has played a very important role in China. However, because of the different canopies between maize and soybean which influences the PAR distribution within relay strip intercropping system, and it not only decreases the light interception but also bad to development of the intercropped soybean, thus resulting in limited photosynthetic characteristics of intercropped soybean which restricts the productivity of maize-soybean relay strip intercropping systems. Therefore, there is a need for exploration of an optimum spatial-temporal configuration management for this system based on competition so that light resources requirement of soybean can be realized in this system. 【Method】 An experiment with soybean (Glycine max L. Merr.) cultivar Gongxuan1 and maize (Zea mays L.) cultivars Denghai 605, Chuandan 418 and Yayu 13 was conducted. The relay strip intercropping systems were designed as soybean intercropping with Denghai 605 (A), Chuandan 418 (B) and Yayu13 (C), and soybean monoculture was used as control (CK). Distribution characteristics of light, photosynthetic characteristics and yield of soybean were studied in maize/soybean relay strip intercropping systems in order to optimize the reasonable group configuration. 【Result】 The PAR density and transmittance varied considerably by different maize-soybean relay strip intercropping systems, and lower than CK significantly (P＜0.05). During the co-growth stage of maize and soybean, the PAR density occurred in treatment A was higher than that in treatments B and C by 54.4% and 90.7%, respectively. The transmittance occurred in treatment A was higher than that in treatments B and C by 7.4% and 17.7% significantly. This means that the configuration of Denghai 605+Gongxuan 1 could improve the PAR density and transmittance in relay strip intercropping system. The Pn, Gs and Ci of intercropped soybean varied considerably in different maize-soybean relay strip intercropping systems. The Pn of treatment A was higher than treatments B and C significantly. Treatments B and C was lower than that in treatment A by 14.16% and 27.23%, Gs of treatment A was higher than that in treatments B and C by 14.6% and 40.4%. The Ci in treatment A was higher than that in treatments B and C by 8.5% and 16.9%. A positive correlation of Pn and Gs was significantly (0.883**), which means that Gs might be the main factor due to the Pn decreased. The contents of Chla, Chlb and Chl(a+b) of intercropped soybean were improved by different maize-soybean relay strip intercropping systems, and the ratio of Chla/b decreased significantly. During the stages of V3, V5 and R1, the content of Chla of treatment C was higher than that in treatments A and B by 5.42%, 10.2%, 5.9% and 3.08%, 4.9%, 3.3%. The content of Chlb of treatment C was higher than that in treatments A and B by 14.4%, 14.9%, 11.73% and 7.8%, 7%, 5.74%. The content of Chl(a+b) of treatment C was higher than that in treatments A and B by 7.27%, 11.1%, 7% and 4.08%, 5.35%, 3.8%, respectively. However, the changing trend of the Chla/b was inverse. The photosynthetic pigment of soybean in different maize-soybean relay strip intercropping systems increased because of the influence of intensified low light, and it is not only beneficial to capture more light but also have a compassion for the low light stress so as to with improve the light use efficiency. The LAI and dry matter weight of soybean in all intercropping systems were lower than monoculture significantly (P<0.05), and varied considerably in different maize and soybean relay strip intercropping systems. This study shown that the LAI of treatments A was higher than treatments B and C, the treatment C was the lowest, and the dry matter weight of treatments B and C was lower than that in treatment A by 35.4% and 57.1%. A positive correlation of LAI and dry matter weight was significantly (0.977**). The results indicate that the compact maize decreased the effect of low light stress on soybean, and increased the LAI and light interception of intercropped soybean and resulted in the improvement of dry matter weight. The yield and yield component of soybean were varied considerably in all of the maize-soybean relay strip intercropping systems. This study shown that the Pods per plant of treatment A was higher than treatments B and C by 11.28% and 32.75%, the seeds per plant of treatment A was higher than treatments B and C by 5.19% and 13.34%, the seeds per pod of treatment A was higher than treatments B and C by 6.4% and 22.5%, Seeds weight per plant of treatment A was higher than treatments B and C by 2.16% and 6.22%. This means that the yield of intercropped soybean decreased as influenced by the intensified low light stress. 【Conclusion】 Suitable plant type configuration could improve radically light conditions, photosynthetic efficiency and yield of relay strip intercropping soybean.

PLANT PROTECTION

Induced etr1-1 Expression in Petunias is Responsible for Its Tolerance to Botrytis cinerea

WANG Hong-1, LIN Jing-1, LIU Gang-2, LI Chun-Xia-2, LUO Chang-Guo-3, LI Gang-Bo-2, ZHANG Zhen-2, CHANG You-Hong-1

Scientia Agricultura Sinica. 2014, 47(8):  1502-1511.  doi:10.3864/j.issn.0578-1752.2014.08.006

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【Objective】The objective of this study is to clarify the effect of induced etr1-1 expression on petunia response to infection by Botrytis cinerea, the causal agent of gray mold disease. 【Method】Detached leaves of GVG: etr1-1 transgenic petunias treated by DEX were inoculated with B. cinerea, then the regulation mechanism of etr1-1 was investigated by observing disease symptoms, measuring disease severity and analyzing gene expression by quantitative RT-PCR and semi-quantitative RT-PCR. 【Result】Induced etr1-1 expression by dexamethasone resulted in the retarded senescence and reduced disease symptoms on detached leaves. During the period of inoculation, the percentage of increased disease incidence was 0 on DEX-treated leaves, whereas 66.77% on control leaves. The growth rate of the leaf lesions on DEX-treated leaves was 4.69 mm•d-1, whereas 6.29 mm•d-1 on control leaves. qRT-PCR and semi-qRT-PCR provided the following results. The extent of decreased disease incidence was negatively correlated with the etr1-1 expression. Following prolonged induction time, the level of etr1-1 expression was enhanced on DEX-treated leaves and obtained the peak at 3rd day after DEX treatment. 10.71-fold changes were increased on mock-inoculating leaves than that on B. cinerea-inoculating leaves. The maximum expression of Bcact was obtained at 1 dpi on DEX-treated leaves, whereas, at 2 dpi on control leaves. The maximum level of Bcact expression was 20-fold lower on DEX-treated leaves than that on control leaves. Infection of B. cinerea activated CP10 expression and caused the senescence symptom. In comparison, 23.6-fold changes were decreased on DEX-treated leaves than on control leaves. The expression of ACO exhibited a decline after an up on both DEX-treated and control leaves. The peak of expression level was observed at 2 dpi. The peak of expression of ACO was only 6.75 on DEX-treated leaves, which suggested 7.33-fold lower than that on control leaves. On DEX-treated leaves with induced etr1-1 expression, repressed genes expressions in ethylene/JA pathway decreased ERF4 and ERF8 expression and consequently reduced pathogenesis-related gene expression. The expressions of ERS1 and ETR2 were observed and the peak of expression came out at 3 dpi on both DEX-treated and control leaves, however, 51-fold lower expression was observed on DEX-treated leaves than that on control leaves. The expression was suppressed at 1 and 2 dpi, and activated at 3 dpi on DEX-treated leaves. Following the prolonged inoculation time, the level of EIN2 expression was enhanced on control leaves. It was found that the maximum expression on DEX-treated leaves was 31.58-fold lower than that on control leaves. The expression of EIL1 showed the similar patterns as that of EIN2. It was observed that 2-fold lower expression of EIN2 on DEX-treated leaves than that on control leaves. AOC and COI1 played important roles in the biosynthesis and signal pathway of jasmonic acid. The expression of AOC and COI1 was repressed at 1-2 dpi and activated at 3 dpi on DEX-treated leaves, however, the expression level was increased following the prolonged inoculation time on control leaves. It was found that 12.96-fold lower expression on DEX-treated leaves than on control leaves. The expression of COI1 was discovered and obtained the peak at 3 dpi, regardless of DEX-treated leaves and control leaves. It was observed that 6.14-fold lower expression of COI1 on DEX-treated leaves than on control leaves. The expression of ethylene response factors (ERFs), as downstream components, integrated the ethylene and JA signaling pathway. The expression of ERF4 showed up before a decrease pattern on DEX-treated leaves. The expression level of ERF4 showed rising patterns following inoculation. The expressions of ERF8 displayed ascend patterns following the prolonged inoculation time, regardless of DEX-treated and control leaves. It was found that 2.96- and 3.52-fold lower expression of ERF4 and ERF8 on DEX-treated leaves than on control leaves. Little expression of pathogenesis-related genes was monitored on DEX-treated leaves and the peak of all genes was lower than 10, whereas, the expression was enhanced on control leaves, such as, the maximum level of expression of PR1, EX-CHI, OSM, Defense1 and AC-CHI was 10 521.11, 184.95, 184.96, 23.39 and 14.58, respectively.【Conclusion】Induced etr1-1 expression delayed the senescence of leaves of petunia caused by B. cinerea and was consequently responsible for tolerance of GVG: etr1-1 transgenic petunias to B. cinerea.

Effects of Tebufenozide on mRNA Expression of Dopa Decarboxylase Gene in Larvae of Spodoptera litura Fabricius

ZHANG Wen-Hui, HOU Hai-Xia, YANG Qiong, WANG Shu-Hui, MAO Xu-Lian, LIU Yong-Jie

Scientia Agricultura Sinica. 2014, 47(8):  1512-1521.  doi:10.3864/j.issn.0578-1752.2014.08.007

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【Objective】 The objective of this study is to clone the full length cDNA of dopa decarboxylase (DDC) gene from 5th instar larvae of Spodoptera litura Fabricius and to investigate the effects of tebufenozide on mRNA expression of DDC gene in the process of cuticular formation. 【Method】 RT-PCR and RACE techniques were used to clone the complete cDNA of DDC gene from S. litura. Bioinformatics tools were applied to analyze both the DNA sequence and protein sequence. Toxicity of tebufenozide to different instar larvae of S. litura was determined by artificial diet dipping method. The mRNA expression of the gene in 5th instar larvae of S. litura which were continuously treated with tebufenozide for different lengths of time were investigated using real time quantitative PCR. 【Result】 The complete DDC gene from 5th instar larvae of S. litura was cloned. Open reading frame (ORF) of DDC was 1 263 bp in length, encoding 420 amino acids. The 5′ end and 3′ end non-coding regions were 105 and 79 bp in length. The sequence was deposited in GenBank database with accession number of KF620492. Homology and phylogenetic analysis indicated that the DDC of S. litura had very high similarity with that of Lepidoptera. The DDC three-dimensional structure was successfully predicted by means of SWISS-MODEL online tools. The structure was similar to the DDC structure of Drosophila. The function structure domain had good conservative property. The results showed that the toxicity of 3rd, 4th, 5th and 6th instar larvae’s LC50 were 33.32, 40.28, 71.20 and 71.97 mg?L-1, respectively. The DDC gene mRNA expression was significantly higher than CK when the larvae were treated with four tebufenozide sublethal doses (7.50, 16.24, 28.34, 45.63 mg?L-1) for 12, 24, 36 and 48 h, respectively. The activation rates raised and then declined with tebufenozide-treated time increasing. The expression of the larvae treated with 16.24 mg?L-1 sublethal doses was the highest after 24, 36 and 48 h tebufenozide-treated time. Meanwhile, the four tebufenozide sublethal doses had the highest expressions after 36 h. The DDC gene mRNA expression had a sharp decrease and was significantly lower than CK after 60 h treated time. The tebufenozide activated the DDC gene expression and then inhibited it. 【Conclusion】 DDC gene was cloned and characterized from S. litura. The interference in the formation of new larval cuticle by tebufenozide is closely related to the effects on DDC gene mRNA expression by tebufenozide.

Spatio-Temporal Expression and Insecticide Tolerance Analysis of Carboxylesterase Gene LmCesF1 from Locusta migratoria

ZHANG Jian-Qin, GE Ping-Ting, LI Da-Qi, WANG Yan, ZHANG Jian-Zhen, MA EnBo

Scientia Agricultura Sinica. 2014, 47(8):  1522-1530.  doi:10.3864/j.issn.0578-1752.2014.08.008

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【Objective】 The objective of this study is to provide a new candidate gene for pest management through studying the function of pesticide detoxification of carboxylesterase gene LmCesF1 in Locusta migratoria. 【Method】The cDNA sequence of LmCesF1 was got from L. migratoria transcriptome database. Amino acid sequence was deduced by using the ExPASy proteomics website software. The molecular mass (MM) and isoelectric point (pI) were predicted by using ExPASy tools. To identify the catalytic triad and substrate binding pocket presented in deduced amino acid sequence, the BLASTp analyses were undertaken in NCBI conserved domain database (CDD). Signal peptide was predicted by SignalP4.0 web tools. The NetNGlyc1.0 Server was used to predict potential N-glycosylation sites of LmCesF1. Phylogenetic tree of LmCesF1 and carboxylesterases from other insect species was constructed with neighbor-joining method. Stage- (including egg, 1-5 instar nymphs and adult) and tissue-dependent expression patterns of LmCesF1 were conducted by reverse transcription quantitative PCR (RT-qPCR). The efficiency of RNAi was assessed by RT-qPCR. RNA interference followed by bioassay was applied to reveal the roles of LmCesF1 in insecticide detoxification. The insecticide was applied at 24 h after ds LmCesF1 injection, dsGFP was used as control. Each nymph was topically applied with 3 µL droplet of insecticide solution (240 ng for malathion, 15 ng for chlorpyrifos, 0.6 ng for deltamethrin and 51 ng for carbaryl) onto the abdomen between the 2nd and 3rd sterna. The doses of these insecticides were predetermined to be approximately LD30 by bioassay. Mortality was recorded at 24 h after topical application of the insecticides. 【Result】 The full-length cDNAs of LmCesF1 (3 121 bp) was obtained from L. migratoria transcriptome database. The open reading frame (ORF) of LmCesF1 was 2 490 bp, encoded 830 amino acid residues. LmCesF1 had a signal peptide at N-terminus. The deduced amino acid sequence of LmCesF1 had the catalytic triad G-P-N. Phylogenetic tree analysis showed that LmCesF1 was clustered to the F clade (nonlepidopteran juvenile hormone esterases). LmCesF1 showed lower expression at egg and early nymph stages, then reached a peak in the 4th-instar nymphs and kept high expression until the adult day 5. The results of tissue-dependent expression of LmCesF1 suggested that the highest expressions of LmCesF1 were found in the foregut, hindgut and fat bodies, but the lowest expressions were in the gastric caeca and midgut. The remaining transcript levels of LmCesF1 were 22.6% and 30% in the nymphs injected with dsLmCesF1 as compared with those in the control (injected with dsGFP) at 12 h and 24 h, respectively. These results indicated an effective silencing of LmCesF1 by RNAi at 12 h and 24 h. Insecticide bioassay showed that the nymph mortality in response to carbaryl treatment increased by 32.9% after the expression of LmCesF1 was suppressed. In contrast, similar exposures of the locusts to malathion, chlorpyrifos, and deltamethrin after the suppression of LmCesF1 did not show significant effects on the susceptibility of the locusts to these insecticides. These results indicated that LmCesF1 most likely to be involved in detoxification of carbaryl in L. migratoria. 【Conclusion】 A juvenile hormone esterase (JHE) like gene LmCesF1 was identified from L. migratoria. This gene was clustered in clade F. The catalytic triad of LmCesF1 has been changed to G-P-N. The transcripts of LmCesF1 were detectable in all tested tissues. LmCesF1 might play a significant role in detoxification of carbaryl in L. migratoria.

SOIL & FERTILIZER·WATER-SAVING IRRIGATION·AGROECOLOGY & ENVIRONMENT

Effect of Field Border Width for Irrigation on Water Consumption Characteristics, Yield and Water Use Efficiency of Wheat

MA Shang-Yu-1, YU Zhen-Wen-1, ZHANG Yong-Li-1, ZHAO Jun-Ye-2, SHI Yu-1, WANG Dong-1

Scientia Agricultura Sinica. 2014, 47(8):  1531-1540.  doi:10.3864/j.issn.0578-1752.2014.08.009

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【Objective】 The objective of this experiment was to study the effects of different border widths for irrigation on water consumption characteristics, grain yield and water use efficiency of wheat, and to provide a theoretical basis for water-saving and high-yielding cultivation of wheat. 【Method】 With the high-yielding winter wheat cultivar Jimai22 as the test material, a field experiment was conducted under high-fertilized and 2.09‰ surface longitudinal slope conditions in Shiwang village (35°24′N, 116°24′E), Yanzhou, Shandong, by using four field border widths designed as 1.0 m (W10), 1.5 m (W15), 2.0 m (W20) and 2.5 m (W25), respectively, with the same border length of 60 m during the 2010-2011 and 2011-2012 wheat growing seasons. All the treatments were irrigated at jointing stage and the inflow cutoff was designed as 90%. Water was supplied by a pump outlet, directing the water to the plots by using plastic pipes. A flow meter was used to measure the amount of water applied. Water consumption characteristics, yield and water use efficiency of wheat were analyzed by dividing the field border into three regions of 0-20 m, 20-40 m and 40-60 m. 【Result】 In 2010-2011 wheat growing season, the W20 treatment showed that the total water consumption amount, irrigation amount and the ratio of irrigation amount to total water consumption amount were significantly lower than other treatments. However, the ratio of precipitation amount to total water consumption amount was the highest among all treatments. In addition, the ratio of soil water consumption amount to total water consumption amount was significantly higher than that of W10, but no difference with W15, and soil water consumption amount in 100-160 cm soil layers was higher than that of W10 and W15 treatments, but no difference with the W25 treatment. In 2011-2012 growing season, the total water consumption of W20 treatment had no significant difference with other treatments, and the irrigation amount and its ratio to total water consumption amount were significantly lower than those of other treatments, while the ratio of precipitation amount and soil water consumption amount to total water consumption amount had no differences with other treatments, and soil water consumption amount in 100-160 cm soil layers was significantly higher than the W15 treatment, but no difference with the W25 treatment. In both growing seasons, after irrigated at jointing stage, the average relative soil water contents of W20 showed no significant difference with W15 and W25, while it was significantly higher than other treatments at anthesis stage. From anthesis to maturity, the water consumption percentage of W20 was significantly higher than other treatments. After irrigated at jointing and anthesis stages, the soil water of W20 treatment distributed more uniformly in different regions of the same border than other treatments, because the coefficient of variability of the average relative soil water contents in different regions was the lowest. The coefficient of variability of the grain yield in different region was the lowest. As for the average grain yield, the W20 treatment was significantly higher than those of other treatments. Also the W20 treatment got the highest water use efficiency and irrigation water use efficiency. 【Conclusion】Considering the grain yield, water use efficiency and irrigation water use efficiency, the W20 treatment was considered to be the best field border width for water-saving and high-yield in this study.

Canopy Interception of Summer Corn and Its Influencing Factors Under Natural Rainfall

HAN Xue-1, 2 , WANG Li-1, 2 , WANG Yan-Ping-1

Scientia Agricultura Sinica. 2014, 47(8):  1541-1549.  doi:10.3864/j.issn.0578-1752.2014.08.010

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【Objective】 Canopy interception is commonly considered to be the frontier and hot issue of ecological hydrology. In this study, the characteristics of corn canopy interception and its influencing factors were explored. The purpose was to evaluate the water efficiency of natural rainfall of summer corn much reasonably, provide a theoretical basis for scientific planting, improve agricultural crop production, and prevent farmland from soil erosion in the dry farming region.【Method】The method of water balance was used to measure the canopy interception, throughfall, and stemflow of summer corn. Throughfall was collected by rain gauges. Stemflow was assembled by collection tube at the bottom of stem. A transfer hose was connected with stemflow collection tube to drain the water to the plastic drum, and then collect the stemflow until it can be measured. Total rainfall was observed by automatic weather station in real time and corrected by artificial observation.【Result】Under different rainfall levels (0.1-4.9 mm, 5.0-14.9 mm, 15.0-29.9 mm), the canopy interceptions were 1.1 mm, 2.6 mm and 13.0 mm, and the average of canopy interception was 1.7 mm. The canopy interception rates were 12.3%, 12.1%, 15.3%, and the average of canopy interception rate was 13.3%. A linear regression model was developed to express the relationship of canopy interception rate with leaf area index and plant height. A power function between canopy interception and vapor pressure deficit and rainfall duration was founded. An exponential function between canopy interception and the rainfall amount was developed. Finally, a regression model was considered to express the relationship between corn canopy interception and possible influencing factors (R2=0.946). 【Conclusion】 Under natural rainfall, the summer corn canopy interception was not to be neglected, and it was influenced by many factors. In this study, summer corn canopy interception was impacted by planting density, corn morphological index (height, leaf area) and meteorological factors (such as rainfall, wind speed, vapor pressure deficit, and rainfall duration). Establishing a regression model of canopy interception with a variety of factors can provide a theoretical basis for scientific planting and improve the agricultural water use efficiency in the dry farming region.

HORTICULTURE

Molecular Mapping and Candidate Gene Analysis for Heavy Netting Gene (H) of Mature Fruit of Cucumber (Cucumis sativus L.)

WANG Min-1, GU Xing-Fang-1, MIAO Han-1, LIU Shu-Lin-1, WANG Ye-1, ToddC.Wehner2 , ZHANG Sheng-Ping-1

Scientia Agricultura Sinica. 2014, 47(8):  1550-1557.  doi:10.3864/j.issn.0578-1752.2014.08.011

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【Objective】 Heavy netting of mature fruit is one of the important phenotypes of cucumber. Molecular mapping and candidate gene analysis for this trait will provide a theoretical basis for improvement of fruit traits. It also can lay a good foundation for fine mapping and gene cloning. 【Method】 Cucumber inbred lines PI205996(P1) without heavy netting mature fruit and PI263079 (P2) with heavy netting mature fruit were used as the experiment materials for genetic analysis and gene mapping in this study. Bulked segregation analysis (BSA) was performed in the F2 population with 230 individuals using 2 112 SSR markers. The sequence and re-sequencing information of 9 930 and 115 core germplasms were used to develop new SSR markers in the primary mapping region of the heavy netting gene by Primer 6.0. JoinMap 4.0 and MapInspect softwares were employed to construct a SSR linkage map for the gene. Bioinformatics analysis was conducted to detect candidate genes. 【Result】 Genetic analysis showed that a single dominant nuclear gene (H) controlled the heavy netting of mature fruit trait in PI263079. There were 255 of 2 112 SSR primers showed polymorphism in the parent lines and the polymorphic rate was 12.1%. Using the 255 polymorphic markers, the DNA of seven individuals with heavy netting fruit and seven individuals without heavy netting fruit was analyzed. Nine SSR markers on cucumber chromosome 5 were identified to be linked with the H gene. Flanking markers SSR13006 and CSWCT-17 were 3.6 and 8.2 cM away from the H gene, respectively. A total of 97 pairs of new SSR primers were developed based on the sequence information in the primary mapping region of the H gene. And only four polymorphic markers were detected between the parent lines with the polymorphic rate of 4.1%. Finally, 13 SSR markers were identified to be linked with the H gene after analysis of the F2 mapping population using the four new developed molecular markers. Flanking markers SSR13006 and SSR-90 were 3.6 and 1.7 cM away from the H gene, respectively. The physical distance between SSR13006 and SSRH-90 was 297.7 kb containing 29 predicted genes. Csa5G591790 was speculated as a possible candidate gene. 【Conclusion】 The heavy netting of mature fruit trait of PI263079 was controlled by one dominant nuclear gene (H). It was located on the Chr.5 of cucumber delimited in a physical distance of 297.7 Kb. The results in this study will be of great benefit to fine mapping and gene cloning for the H gene and MAS of heavy netting of mature fruit.

Growth Regularity and Bulb Propagation Techniques of Lilies in Open Field

WANG Ying-1, 2 , 3 , WANG Liang-Gui-3, HUANG Cheng-Ming-4, LI Yu-Fan-1, 3 , YUAN Su-Xia-1, LIU Chun-1, MING Jun-1

Scientia Agricultura Sinica. 2014, 47(8):  1558-1566.  doi:10.3864/j.issn.0578-1752.2014.08.012

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【Objective】 The growth regularity and bulb propagation techniques of the 2 lily groups under the open conditions was studied in order to provide reference for highly efficiency, low cost, large-scale production and landscape planting application.【Method】Using Lilium lancifolium, Lilium ‘Yelloween’as materials, the phenophase and the morphology were observed, curve of the measured data of plant height and bud was fitted by logistic equation and exponential equation, the growth model was constructed, meanwhile, the bud picking experience was designed, planting direction and planting density were studied to find out the growth effect of the bulb. 【Result】 The result showed that, there was a big difference between L. lancifolium and ‘Yelloween’on the phenophase. L. lancifolium bloomed later, needed higher active accumulated temperature which was 4 256℃, but ‘Yelloween’ needed only about 4 981.5℃. For L. lancifolium, the plant height equation was y=97.5949/(1+e^-(x-62.6822)/22.8819), the diameter equation was y=0.1578e^(0.0358x), the length equation was y=90.2066/(1+e^-(x-117.1807)/8.7295), which were accorded with logistic equation. For ‘Yelloween’, the plant height equation was y=70.3771/(1+e^-(x-19.1133)/9.5840), the bud’s diameter equation was y=41.2215/(1+e^-(x-65.9236)/17.1028), bud length equation was y=158.9531/(1+e^-(x-62.9011)/9.7562), which were accorded or similar with the exponential equation. The bud picking experience showed that picked the bud on the blooming day achieved the best effect on ‘Yelloween’, the average fresh weight of bulb was 89.71 g, and 18.93 cm in bulb circumference, no bud picking was the best way for L. lancifolium, with the average fresh weight of bulb was 87.59 g. Planted in NS-trending direction increased the height and steam diameter slight. The best planting spacing of ‘Yelloween’was 10 cm×20 cm, then the production was 26 936 kg per 667 m2, which was considered as the best yield, then the bulb circumference could increase by 3 grades if combined with the bud picking result.【Conclusion】Height growth regularity of the 2 lilies and the buds’ diameter growth regularity conformed to logistic curve, while the buds’ diameter regularity of ‘Yelloween’ conformed or similar to the exponential equation. No bud picking was the best for L. lancifolium,while picking buds on the blooming day was best for ‘Yelloween’, combined the appropriate planting density with NS-trending direction planting , the bulb circumference could increase by 2 to 3 grades.

STORAGE·FRESH-KEEPING·PROCESSING

Influence of Brown Rice Flour on Wheat Dough Rheological Properties and Cookie Quality Characteristics

TANG Xiao-Zhi, HU Zhan-Qiang, ZHOU Jian-Min, FANG Yong, SHEN Xin-Chun, HU Qiu-Hui

Scientia Agricultura Sinica. 2014, 47(8):  1567-1576.  doi:10.3864/j.issn.0578-1752.2014.08.013

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【Objective】Effects of brown rice flour on the thermomechanical and dynamic rheological properties of wheat flour dough as well as physical and sensory characteristics of the cookies were investigated. The aim of the research is to provide a basis for further utilization of brown rice in developing new food products.【Method】 Mixolab and dynamic rheometer were employed to study the effects of brown rice flour replacement on rheological properties of wheat flour dough. The dough microstructure was observed by scanning electron microscope (SEM). Cookies were prepared and tested to investigate the effects of brown rice flour replacement on cookie quality characteristics. 【Result】 With the addition of brown rice flour, the water absorption of the flour dough and the degree of protein weakening increased, while dough development time and stability time decreased. The addition of brown rice flour diluted the concentration of gluten and destroyed the continuous state of the flour dough, which resulted in being difficult to form the stable gluten network structure, and subsequently leading to higher degree of protein weakening under double effects of mechanical force and heating. The increase of replacement ratio of brown rice flour decreased the temperature of starch gelatinization, peak and break down viscosity of the mixed flour, and setback value of the starch due to the higher degree of starch hydrolysis. It was shown from dynamic rheological studies that with the addition of brown rice flour, the dough remained viscoelastic system and storage modulus and loss modulus increased, while tan δ of the dough decreased, indicating higher degree of molecular cross-linking and elastic proportion in the mixed system. From SEM, with the addition of 10% brown rice flour, the dough microstructure became loose with small cracks and starch granules distributed unevenly. Irregular shape of rice starch granules could be observed and their adhesion force to dough surface significantly decreased. With further increase of replacement ratio, more and more big cracks could be observed and fibres were exposed to the dough surface. With the addition of brown rice flour, the width and expansion ratio of the cookies increased, whereas the thickness, hardness, score of taste, color, flavor and overall acceptability all decreased, especially score of color and flavor, indicating that existence of rice bran made the processed food hard to be acceptable due to high content of fibres, and especially its unpleasant smell and dark color. 【Conclusion】 The addition of brown rice flour made the wheat flour dough more elastic, but not beneficial to the stability of the dough. The rice bran, especially its unpleasant odor and dark color, is still the key factor affecting the acceptability of processed foods.

ANIMAL SCIENCE·VETERINARY SCIENCERE·SOURCE INSECT

Myostatin and Its Double-Muscling Phenotype in Animals

WANG Jian-Qi, CAO Wen-Guang

Scientia Agricultura Sinica. 2014, 47(8):  1577-1587.  doi:10.3864/j.issn.0578-1752.2014.08.014

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Myostatin (MSTN), also known as growth differentiation factor 8(GDF-8), is a member of the transforming growth factor β(TGF-β) superfamily, which was first obtained from screening a murine skeletal muscle complementary DNA library in 1997. The gene of MSTN consists of three exons and two introns, the gene is very conservative as only three or fewer differences exist among the amino acid sequences of mature peptide in all studied species. High levels of expression of MSTN has been found mainly in skeletal muscle although it is expressed in multiple tissues, including the cardia, adipose, placenta, mammary gland, uterus, olfactory neuron, liver, spleen, lung, and kidney, etc. The mature MSTN is liberated as a homogeneous dimer from prepromyostatin through proteolytic processing of signal peptidase, furin and BMP-1/tolloid family. MSTN acts as a strong autocrine/paracrine negative regulator of muscle growth. It inhibits the proliferation and differentiation of myoblast cells through activating TGF-β, p38 MAPK, ERK1/2 and JNK signal pathway, and inhibiting IGF-AKT and Wnt signal pathway. MSTN also regulates glucose uptake and metabolism in the muscle cell. Myostatin-null animals showed significant increased muscle mass, that is double-muscling phenotype, improved muscle healing through enhancing regeneration and reducing fibrosis, etc; reduced adipogenesis and consequently decreased leptin secretion, and enhanced thermogenesis through driving browning of white adipose tissue; increased bone density and bone mineral content, and increased fracture callus size and strength for fracture healing. Naturally occurred loss-of-function mutations in the MSTN gene have also been shown to underlie the double-muscling phenotype in mammals. Such known mutations are found in several species including p.D273RfsX13(nt821(del11)), p.C313Y, p.F140X(nt419(del7–ins10)), p.Q204X, p.E226X and p.E291X in cattle, c.960delG(p.K320NfsX39) and c.120insA（p.N40MfsX9）in sheep, c.939-940delTG in dogs and c.373+5G＞A in human. In targeting the MSTN gene in sheep, a gain-of-function miRNA mutation, c.2360G＞A(g.6223G＞A), can also show double-muscling phenotype. Double-muscled animals not only have more muscle, but also have less bone, less fat. Therefore, it has been a hot topic to optimize the functions of MSTN and reduce the side effects of double-muscling phenotype with genetic engineering techniques to breed preeminent table-purpose livestock. For example, a conservative missense mutation p.F94L exists in cattle, which does not alter the function of MSTN and has no side effects of double-muscling phenotype, but showed increased muscle mass and decreased fat depth and intramuscular fat content, and had no significant effect in birth and growth traits. In brief, investigation into MSTN will not only give a chance to further elucidate the mechanism involved in muscle growth, but may also help to breed animals. This review summaries the structure, signal pathway, phenotype, mutants, and the mechanism of the MSTN in mammals and their implications in livestock.

Plasma Metabolomics Study of Dairy Cows with Clinical and Subclinical Ketosis

SUN Ling-Wei, BAO Kai, LI Ying, LI Lan, ZHANG Hong-You, XIA Cheng, WU Ling

Scientia Agricultura Sinica. 2014, 47(8):  1588-1599.  doi:10.3864/j.issn.0578-1752.2014.08.015

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【Objective】 This study aims to search the relational metabolites in plasma of cows with clinical and subclinical ketosis by the method of metabolomics based on the technology of gas chromatography (GC) / mass spectrometry (MS) techniques, find out molecular markers for endogenous metabolism for early diagnosis and prognosis of ketosis, and reveal the essence of ketosis. 【Method】 Total 24 dairy cows with clinical ketosis (CK), 33 dairy cows with subclinical ketosis (SK) and 23 healthy controls (C) were selected for the research. Venous blood of cows was collected, for the preparation of plasma samples, and the biochemical criterions such as β-hydroxybutyric acid, glucose and so on were detected. After the plasma samples were pretreated, cows plasma metabolites were detected by GC/MS technique. The detected metabolites were identified by mass spectral database. Principal component analysis (PCA) and partial least squares discriminant analysis (PLS-DA) of multivariate statistical methods were used for recognizing different metabolic patterns among clinical ketosis, subclinical ketosis and healthy controls. After establishing the diagnosis models of disease by partial least squares discriminant analysis, potential biomarkers of disease were selected. 【Result】 A stable and reliable GC/MS analytical method was established to obtain the metabolite profiles of the plasma samples. A commercial mass spectral database (NIST2008) was used for the rapid identification of detected metabolites and totally 267 variables were detected. The metabolomics data were imported into Simca-P 11.0 software for PCA and PLS-DA . Ketosis groups and healthy groups could be clustered and distinguished by the metabolomics data, and 40 types of metabolites without difference among 3 groups were found out. Results showed that compared with the healthy control group, different metabolites obtained in subclinical and clinical ketosis groups were 32, different metabolites obtained between clinical ketosis group and subclinical ketosis group were 13. Through seeking the KEGG database and analysis of metabolites, these metabolites primarily were related to amino acid metabolism, fat metabolism and carbohydrate metabolism.【Conclusion】 The present study is an integrated detection of dairy cattle ketosis based on plasma metabolomic profiling by GC/MS combined with multivariate statistical analysis. It was firstly discovered that 40 metabolites (i.e. fatty acids, amino acids, carbohydrates, et. al) were differentially found among CK, SK and C. It proved that the plasma GC/MS metabolite profiles can effectively distinguish clinical and subclinical ketosis groups from healthy control group. This further proved that metabolomics technology, to some extent, can reveal the development and progression of clinical and subclinical ketosis, while the substances of different content and contributing to classification may be the potential metabolic marker and objective indicators for diagnosing ketosis. To research ketosis, in the process of the occurrence and development of clinical and subclinical ketosis, parts of metabolic patterns and metabolic pathways in plasma changed. Furthermore, new potential metabolites could shed light on new strategies for the diagnosis, prognosis, and prevention of ketosis .

Deposition of Foie Gras Goose Body Fat, Composition of Lipid and Metabolism of Lipid in Overfeeding Period

WANG Bao-Wei-1, SHU Chang-Ping-2, GE Wen-Hua-1, YUE Bin-1, ZHANG Ming-Ai-1, JIANG Yang-3

Scientia Agricultura Sinica. 2014, 47(8):  1600-1610.  doi:10.3864/j.issn.0578-1752.2014.08.016

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【Objective】 This paper aims to investigate the relationship between metabolism of fat , deposition of foie gras goose body fat and liver weight through conducting an experiment to measure and analyze the deposition of foie gras goose body fat, composition of lipid and metabolism of lipid. 【Method】 Two-hundred liver-use ganders of 85 days bred in the same batch incubation, with the same rearing conditions whose weight difference was not significant (P＞0.05) were selected and overfed, and the overfeeding period lasted for 30 days. The overfeeding started at the end of the pre-test, 30 geese were randomly selected, slaughtered and blood was taken every 6 days(0,6,12,18,24,30d). Each is a duplicate to overfeeding 0 d as a control .The related indicators of liver weight, sebum weight, abdominal fat weight, intestinal fat weight, sebum rate, percentage of abdominal fat, intestinal fat rate composition of lipid and metabolism of lipid were measured. Added the same diet and overfed quantify the amount identically for geese. After boiling the screening of corn kernels for 5-10 min in the water pot, removed and drained, hotted goose fat 1%, 0.3% salt and stirred well after cooling spare as overfeeding diet. Double mechanical overfeeding methods were used and overfeeding column rearing was adopted.【Result】 The weight of abdominal fat, sebum and intestinal fat increased with the extension of overfeeding time, abdominal fat, sebum, intestinal fat, foie gras fat deposition reached the peak during 12-18 d. At overfeeding 30 d, the weight of sebum ＞ abdominal fat ＞ intestinal fat. In addition to overfeeding 6 d sebum rate and fatty liver weight were significantly negatively correlated (r = -0.869), in different overfeedings, foie gras goose abdominal fat weight, sebum weight, intestinal fat weight, abdominal fat, subcutaneous fat percentage, intestinal fat rate and liver weight showed a highly significant positive correlation (P＜0.01). Overfeeding changed the levels of non-estesterified fatty acid (NEFA) and Apo-A significantly. Throughout the course of overfeeding, apolipoprotein-B(Apo-B) showed a downward trend with the increasing overfeeding time, but it was not significant (P＞ 0.05) in the differences among various stages of overfeeding. With the extended feeding time, triglyceride (TG), total cholesterol (TC), high density lipoprotein-cholesterol (HDL-C), low density lipoprotein-cholesterol (LDL-C) and very low density lipoprotein-cholesterol (VLDL-C) levels increased. The maximum values of cholinesterase (CHE), lipase (LPS), lipoprotein lipase (LPL), hepatic lipase (HL) and activity of total lipase appeared during 18-24 d, which also demonstrated a trend of rising first and declining then. 【Conclusion】 Overfeeding significantly changed the composition of body fat goose foie gras. Overfeeding significantly changed the composition of blood content of fatty liver for geese, and liver weight and body fat deposition showed a significant positive correlation (P ＜0.01). After overfeeding for 12 to 24 days, the metabolism of body fat and deposition of fat achieved its maximum, foie gras grew fastest.

Bioinformational Analysis and Expression Pattern of V-ATPase in Silkworm (Bombyx mori)

WANG Xin, LI Yi, CHEN Quan-Mei, YI Qi-Ying, XIE Kang, WU Yong, ZHAO Ping

Scientia Agricultura Sinica. 2014, 47(8):  1611-1621.  doi:10.3864/j.issn.0578-1752.2014.08.017

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【Objective】 The objective of this study is to identify the vacuolar-type ATPase (V-ATPase) subunit A and subunit B’s coding genes in silkworm (Bombyx mori) and analyze its expression level in different tissues on the 3rd day of 5th instar larvae and different sections of silk gland on wandering stage. 【Method】 The silkworm V-ATPase subunit A and subunit B’s coding genes were identified by bioinformatics. The domains of the V-ATPase subunit A and subunit B were predicted by online tools. Sequence alignment among other species as well as phylogenetic analysis were done by software. The expression levels in different tissues of silkworm on the 3rd day of 5th instar larvae were analyzed by real-time quantitative polymerase chain reaction (PCR). To understand the pH of different sections of silk gland on wandering stage, a pH indicator, bromphenol blue was injected into the body of silkworm through the pore. After that the silk gland was stained in different colors and the pH was indicated by those colors. At last, real-time quantitative PCR was used to make sure the expression levels of different sections of silk gland on wandering stage.【Result】The coding genes of V-ATPase subunit A and B BGIBMGA008295 (GenBank accession number NM_001098359.1) and BGIBMGA002241 (GenBank accession number NM_001098358.1) were identified. There were 3 highly conserved domains in subunit A and B, which were β barrel domain in N terminal, nucleotide binding domain in the central region, and domain in C terminal. Sequences alignment revealed that the identities of V-ATPases subunit A and B among different species were more than 90%. It indicated that the V-ATPases subunit A and B among different species were highly conserved. The cladogram showed that silkworm V-ATPase subunit A and B were clustered with the other lepidopterous insects. Real-time quantitative PCR results showed that these two genes were expressed mainly in mid-gut, fat body, gonad and silk gland on the 3rd day of 5th instar larvae. The pH for different sections of silk gland was analyzed by bromophenol blue staining. The results indicated that the pH of posterior silk gland (PSG), posterior section (PMSG) and middle section (MMSG) of middle silk gland were larger than 7 while the anterior section of middle silk gland (AMSG) and anterior silk gland (ASG) were acidic. The expression level of V-ATPase subunit A and B in different sections of silk gland on wandering stage were analyzed and it was found that the two genes were highly expressed in ASG and AMSG. The V-ATPase may involve in the generation and maintenance of the acidic environment in these two sections.【Conclusion】This study made clear the expression level of V-ATPases in different tissues in silkworm and the V-ATPases may involve in the acidification of the lumens of ASG and AMSG on wandering stage. This further provides a basis for functional study of V-ATPases.

RESEARCH NOTES

Molecular Cloning and Functional Characterization of Transcription Factor HbERF1 in Hevea brasiliensis

AN Ze-Wei-1, XIE Li-Li-1, 2 , WANG Qi-Tong-1, LI Ya-Chao-1, CHENG Han-1, HU Yan-Shi-1, HUANG Hua-Sun-1

Scientia Agricultura Sinica. 2014, 47(8):  1622-1633.  doi:10.3864/j.issn.0578-1752.2014.08.018

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【Objective】AP2/ERF transcription factors play important roles in response to abiotic stress in plant. The objectives of this study are to clone ERF genes related to abiotic stress in rubber tree, analyze their expression patterns in response to abiotic stress and identify their biological functions by overexpressing in Arabidopsis thaliana so that it can provide candidate genes for rubber breeding in relation to stress tolerance. 【Method】The full cDNA sequence of HbERF1 was cloned from the rubber tree by RACE technology, then the temporal and spatial expression patterns of this gene were investigated under abiotic stress by real-time PCR. The HbERF1-overexpressing Arabidopsis was established by Agrobacterium-mediated transformation. The overexpressing lines were identified by Southern blotting and qRT-PCR. The phenotype and gene expression were analyzed under drought, NaCl and PEG stress in overexpressing lines, and the functions of HbERF1 were characterized preliminarily. 【Result】 HbERF1 (GenBank No. JQ914647) had no intron. The full-length cDNA of HbERF1 was 1 178bp in length, containing a 642-bp open reading frame flanked by a 62-bp 5’-UTR and a 474-bp 3’-UTR. HbERF1 encoded a 213 aa protein, which contained one AP2 domain and one EAR motif. The results of phylogenetic analysis showed that HbERF1 protein was classed into B-1 subgroup of ERF subfamily, and it showed 72%, 62%, and 61% identity with RcERF, PtERF46 and AtERF4, respectively. The real-time PCR analysis showed that HbERF1 was NaCl-, PEG- and drought -responsive in the leaves and bark of rubber tree. The expression of HbERF1 was repressed by abscisic acid (ABA) and methyl jasmonate (MeJA) in leaves and bark, meanwhile also by salicylic acid (SA) in the bark. Under NaCl- and PEG stress, HbERF1 was down-regulated in the first stress phase (≤8 h) in leaves, while HbERF1 was strongly induced in bark when it was stressed for 4h. Under drought stress, the expression of HbERF1 continuously increased in leaves and bark with the increase of drought degree, and the expression was higher in bark than in leaves. The overexpression of HbERF1 enhanced the tolerance to NaCl, PEG and drought in Arabidopsis. The expression of ethylene receptor gene, AtETR1 and AtERS1, were also repressed by HbERF1 overexpression in Arabidopsis, in which the expression of AtETR1 and AtERS1 was about 19-fold and 9-fold lower than those in the control, respectively, and the difference was highly significant(P＜0.01). Under NaCl stress, the expression of AtRD22 and AtRD29A had a 115-fold and 100-fold increase in overexpressing Arabidopsis, respectively, and these two genes were also induced under 20% PEG stress. Nearly all of the leaves became dehydrated and wilted in wild-type Arabidopsis after it was watered with 300 mmol•L-1NaCl for 15 days, while only few leaves became dehydrated and wilted in overexpressing line S1. Line S1 had been in the reproductive growth since it was watered with 20% PEG for 15days, while wild-type Arabidopsis was still in the vegetative growth, and some leaves became dehydrated. After stopping watering for 2 weeks, wild-type Arabidopsis was more wilted than line S1; and the survival rate of wild-type and overexpressing Arabidopsis was 35.0% and 95.0% after re-watered for 1 week, respectively. 【Conclusion】 HbERF1 was induced by ABA, MeJA, SA and ethylene (ETH), and it is a positive regulator of ETH-responsive genes and drought tolerance in the rubber tree.

Impacts of Magnetized Water Irrigation on Soil Infiltration and Soil Salt Leaching

ZHANG Rui-Xi-1, WANG Wei-Bing-2, CHU Gui-Xin-1

Scientia Agricultura Sinica. 2014, 47(8):  1634-1641.  doi:10.3864/j.issn.0578-1752.2014.08.019

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【Objective】 Soil salinization is one of the major obstacle factors affecting crop yield in arid region, improvement of soil by using magnetized water irrigation can be traced back to the 1960s. In this study, a laboratory soil cylinder simulation experiment and a field plot experiment were conducted to investigate the effects of magnetized water irrigation on soil water infiltration and soil salt leaching. The purpose of this research is to find a novel method to reduce soil salt content and prevent soil secondary salinity in water-saving agriculture. 【Method】 In the present study, a laboratory soil cylinder simulation experiment and a field trial were carried out to investigate the impacts of magnetized water irrigation on soil infiltration, soil moisture, and salt movement in irrigation treatments with different magnetic field intensities, under given constant water head of 7.5 cm. The soil cylinder incubation experiment included 0 (Control), 100, 300, and 500 mT of magnetic field intensity, respectively. Infiltration from soil cylinder top to down, reached the entire soil cylinder 2/3, and then samplings were made. The field trial had two treatments: conventional water irrigation (CK) and magnetized water irrigation (T), and the magnetic field intensity was 300 mT. The field experiment was finished in the lysimeter, and the lysimeter area was 6.67 m2. Meanwhile, soil water leakage and salt distribution were also measured under the magnetized water drip irrigation condition. 【Result】 An obvious effect of magnetized water on accelerating soil infiltration and increasing soil moisture were observed. Compared with the control (CK), soil wetting front speed was markedly improved with 300 mT magnetized water treatment. For instance, at soil water infiltration time of 360 minute, soil wetting front reached up to 17.0 cm and 18.5 cm depth, respectively, under CK and 300 mT magnetized water irrigation. At the end of irrigation, soil EC value in 28 cm depth was 10.9 mS•cm-1 in CK and that was 12.7 mS•cm-1 in 300 mT magnetized water treatment, Cl- contents were 17.95 g•kg-1and 25.04 g•kg-1, Na+ contents were 4.61 g•kg-1 and 5.55 g•kg-1, which increased by 16.5%, 39.5%, and 20.4 % compared with the control (0 mT). Magnetized water irrigation significantly increased the amount of soil water leaching in a plot experiment. The weights of soil water leakage which collected in the first times were 18.8 kg and 21.9 kg, respectively, in CK and 300 mT magnetized water treatments, through water magnetized, the amount of soil water leaching increased by 16.9 %. At the end of irrigation, soil salt content in 0-100 cm soil layer decreased significantly, and soil salt content in 0-100 cm layer was significantly lower in magnetized water irrigation treatment than the control. Such as in soil surface layer 0-20 cm , soil salt content decreased by 13.8% in CK and 23.2% in magnetized water treatment; in soil depth layer 80-100 cm , soil salt content reduced by 11.6% and 29.8% in CK and magnetized water irrigation.【Conclusion】From the study a conclusion could be drawn that magnetized water irrigation exerted a significant role in accelerating soil water infiltration and promoting soil salt leaching in arid region, and 300 mT magnetic field intensity was optimal treatment in this experiment. In production practice, magnetized water drip irrigation as a way to improve soil salinization was very simple and efficient. The study will provide a theoretical basis and technical support for the use of magnetized water irrigation in production.

Effects of Returning Farmland to Tea on Soil Organic Carbon Pool of Hilly Region in the Western Sichuan

LI Wei, ZHENG Zi-Cheng, LI Ting-Xuan, WANG Yong-Dong

Scientia Agricultura Sinica. 2014, 47(8):  1642-1651.  doi:10.3864/j.issn.0578-1752.2014.08.020

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【Objective】Soil organic carbon pool can effectively characterize the sequestration capacity of soil carbon, study on dynamics of soil carbon pool in typical area of returning farmland to tea of hilly region in the western Sichuan is of great importance to understand the changes and effect evaluation during the process of ecological restoration.【Method】The research regarded different ages of returning farmland to tea (2-3, 9-10 and 16-17 years old) located in Zhongfeng Township of Mingshan District as a study plot, and the cropland was used as contrasts. Several indexes were analyzed such as the concentration and density of soil total organic carbon (TOC), labile organic carbon (LOC), and non-labile organic carbon (NLOC) to understand the effect of returning farmland to tea, the soil carbon pool management index (CPMI) was calculated, and the relationship between soil organic carbon pool, CPMI and the de-farming years was analyzed.【Result】The mass fraction of TOC firstly decreased and then increased after returning farmland to tea. In 0-10 cm, 10-20 cm and 20-40 cm soil layers, TOC concentration increased by 5.67%, 5.67% and 0.78 % compared with the control after 16 years of returning farmland to tea, TOC density also increased significantly in this period. The mass fraction of LOC after returning farmland to tea increased significantly, and the average increase was 23 times compared with TOC’s. The LOC density in 40 cm soil layer improved from 132.26% to 182.26% in the following order: 2-3 years, 9-10 years, and 16-17 years of returning farmland to tea. The density of soil LOC in the 0-10 cm and 0-20 cm soil layers accounting for 0-40 cm soil depth were more than 30% and 50%, respectively. Mass fraction and density of NLOC were consistent with the trend of TOC after returning farmland. Compared with the control, 2-3 years of returning farmland to tea had the highest efficiency ratios of LOC, and the surface accumulation was relatively obvious but there was no significant difference which shows that the efficiency ratios of LOC were not sensitive to the change of soil depth. With the increase of de-farming year, Soil CPMI fell after the first increase, but was significantly higher than the control, and reached the maximum value in 16-17 years of returning farmland to tea. The CPMI in 0-10 cm and 20-40 cm soil layers were comparatively large, indicating that surface and deep carbon pools have a certain effect on carbon sequestration, and the impact by returning farmland to tea on the surface layer’s carbon pool was particularly evident.【Conclusion】 Soil TOC, LOC and NLOC concentrations increased with the increasing period of retuning farmland to tea. After 16 years of returning farmland to tea, the density of TOC, LOC and NLOC were significantly higher than the control in 0-40 cm soil depth, the carbon accumulation in 0-10 cm soil layer was the best. The efficiency ratios of soil LOC significantly increased by returning farmland to tea, the CPMI in 0-10 cm soil layer of 16-17 years of returning farmland to tea was the highest, indicating that the engineering of returning farmland to tea enhanced soil carbon sink and improved soil quality.

Determination of Carotenoids in the Fruit of Elaeagnus conferta Roxb. by High Performance Liquid Chromatography

HU Hai-Tao-1, JIN Xiao-Qin-1, CHENG Long-Ping-1, YANG Li-1, CHEN Jian-Hua-1, FAN Ru-Chang-2, YANG Ling-1

Scientia Agricultura Sinica. 2014, 47(8):  1652-1656.  doi:10.3864/j.issn.0578-1752.2014.08.021

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【Objective】The berry of Elaeagnus umbellata Thunb. was found to accumulate lycopene up to 54 mg per 100 g of fresh weight, which is about 17 times higher than that of tomato. However, little is known about the carotenoid composition and content in the big and good tasty flesh of E. conferta Roxb., also a member of Elaeagnaceae family. This study aimed to analyze carotenoid components and contents in the fruit of E. conferta Roxb. by high performance liquid chromatograph（HPLC）method.【Method】 Methanol:chloroform (1:2 v/v) was used to extract carotenoids from ripe fruits of E. conferta Roxb.. The separation was carried on a YMC C30 (4.6 mm×250 mm, 5 µm, column temperature 235℃) and an injection volume was 20 μL. Methanol (A), 80% methanol containing 0.2% ammonium acetate (B) and methyl tert-butyl ether (C) were used as the mobile phase at a flow rate of 1.0 mL•min-1 through gradient elution: (1) 0-6 min, 95%A+5%B; (2) 7-11 min, 80%A+5%B+15%C; (3) 12-32 min, 30%A+ 5%B+65%C; (4) 33 min to end, 95%A+5%B. The eluent was detected at 244 nm. 【Result】 Lycopene and β-carotene were two major carotenoids in the flesh of E. conferta Roxb., and their content differed markedly between two genetic genotypes. The β-carotene content in Detianmihuahutuizi 1 was up to (231.84±16.00) µg•g-1DW, which is one of the highest ones in the known species. The flesh of Detianmihuahutuizi 2 contained lycopene of (1 054.10±29.19) µg•g-1DW, which is about seven times higher than that of tomato.【Conclusion】The results showed that the flesh of E. conferta Roxb. accumulated abundant lycopene and β-carotene, which endow E. conferta Roxb. as a wilding fruit resource with great development and application value. Meanwhile, E. conferta Roxb. also is an ideal research material for studying molecular mechanism of high accumulation of lycopene and β-carotene in fruit.