Scientia Agricultura Sinica

Fine Mapping of an Early Senescence Gene in Rice

ZHAO Chun-De-1, 2 , ZHANG Ying-Xin-1, 2 , LIU Qun-恩1, 2 , YU Ning-1, 2 , CHENG Shi-Hua-1, 2 , CAO Li-Yong-1, 2

Scientia Agricultura Sinica. 2014, 47(11): 2069-2077. doi:10.3864/j.issn.0578-1752.2014.11.001

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【Objective】The objective of this study is to conduct genetic analysis and gene mapping for the rice early leaf senescence mutant W330.【Method】An early leaf senescence mutant, designated as W330, was isolated from the progeny of 60Co-γ-treated indica rice cv. Zh8015. Phenotypes of the W330 mutant were observed and its main agronomic traits were analyzed under field conditions in Hangzhou and Sanya. After the W330 mutant was crossed with 02428, leaf phenotypes of the F1 progenies，the segregation ratio of normal and early senescence plants in the F2 populations were investigated. An F2 population derived from the cross of cv. 02428 with W330 was used for genetic analysis and gene fine mapping. Candidate gene assay was conducted by gene sequencing, enzyme digestion, gene expression and phylogenetic analysis.【Result】W330 plants showed leaf senescence phenotype from 3-leaf-stage to maturity stage. Compared with wild-type parent Zhonghui8015, the plant height，tiller and blade width of W330 decreased significantly, and the number of productive panicles per plant, the number of spikelets per panicle and seed setting rate were also significantly reduced. All F1 plants generated by crossing early senescence mutant W330 with 02428 showed normal leaf. The segregation ratios of normal plants and early senescence plants in two F2 populations were both 3:1, indicating that the W330 was controlled by a single recessive nuclear gene which was mapped on the short arm of chromosome 3. With developed SSR and Indel markers, the gene was finally narrowed to an interval of 21.5 kb between markers CD-5 and CD-7. Among four ORFs in this region, the LOC_Os03g0131200 encoding a Catalase (OsCATC) was probably the candidate gene. The sequencing analysis and enzyme digestion revealed the variable splicing in the first intron was probably responsible for the early senescence phenotype, which was confirmed by assay of gene expression and enzyme activity. Compared with wild-type parent Zhonghui8015，the content of catalase activity decreased by 47.8% and the content of H2O2 increased by 2.7 times. It was speculated that the W330 mutant gene was allelic to OsCATC gene. Otherwise, phylogenetic analysis revealed that OsCATC was an independent evolution in rice. Expression analysis of catalase genes by real-time PCR showed that OsCATA and OsCATB have a significant increase in the expression of leaves, while the expression of OsCATC has no significant changes. It was a speculated that the three highly homologous genes have complementary mechanisms in rice plants. 【Conclusion】 The W330 mutant gene was allelic to OsCATC gene. A point mutation in introns of OsCATC gene in the W330 mutant makes catalase inactive, resulting in the leaf senescence phenotype.

Distribution of Powdery Mildew Resistance Gene Pm21 in Chinese Winter Wheat Cultivars and Breeding Lines Based on Gene-Specific Marker

JIANG Zheng-1, 2 , WANG Qi-Lin-1, 3 , WU Jian-Hui-1, 3 , XUE Wen-Bo-1, 2 , ZENG Qing-Dong-1, 3 , HUANG Li-Li-1, 3 , KANG Zhen-Sheng-1, 3 , HAN De-Jun-1, 2

Scientia Agricultura Sinica. 2014, 47(11): 2078-2087. doi:10.3864/j.issn.0578-1752.2014.11.002

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【Objective】Powdery mildew, caused by Blumeria graminis f. sp.tritici (Bgt), is an important disease that causes substantial yield losses in wheat (Triticum aestivum L.) in China, there is a great significance for using resistance genes to control this disease. Powdery mildew resistance gene Pm21, transferred from Haynaldia villosa by a 6VS/6AL translocation, confers durable and broad-spectrum resistance to the disease. This study is to develop gene specific PCR marker, analyze the distribution of Pm21 in China winter wheat regions, and to provide a theoretical basis and technical support for reasonable arrangement and marker-assisted selection for Pm21.【Method】Based on the sequence of Stpk-V gene which was cloned and related to the resistance pathway (GenBank accession number: HQ864471.1), the protein sequences were extracted and the conservative start-stop sites were analyzed by Pfam software, in order to amplify this gene specifically, the marker WS-1 was developed excluding conserved domain. For constructing an F2 populations derived from susceptible cultivar Avocet S and the lines 92R137 carrying Pm21, infection types in F2 plants were evaluated by artificial inoculation with isolate E09 during seedling stage, F2 plants were amplified by WS-1, and the testing results and infection types were analysed to confirm the accuracy of WS-1. A total of 662 wheat cultivars and breeding lines growing in different winter wheat regions in China were detected by WS-1 to analyze the distribution of Pm21, and the materials carrying Pm21 were further tested the resistance to powdery mildew under field conditions. In order to further prove the accuracy of WS-1, the marker NAU/xibao15902 developed by Cao Aizhong and others was used to amplify the lines with Pm21 and without Pm21 (50 samples from each type). 【Result】 As a dominant molecular marker, WS-1 could be amplified a 949 bp fragment in the lines with Pm21 on 8% non-denaturing polyacrylamide gel, but did not in the lines without Pm21. The 377 F2 populations segregated 286 resistant﹕91 susceptible fitting the 3﹕1 ratio, which indicated that Pm21 presents a single dominant gene, and the testing results by WS-1 were consistent with infection types. There were 49 lines showed positive strand in 662 lines, and the average distribution frequency of Pm21 gene was 7.4%; the lines containing Pm21 in Southwestern Winter Wheat Region, Northern China Plain Winter Wheat Region, Yellow and Huai Valley Facultative Wheat Region and Middle & Lower Yangtze Valley Winter Wheat Region were 33, 4, 9, and 3, and their frequencies were 34.4%, 5.3%, 3.1% and 1.5%, respectively.【Conclusion】WS-1 can be detected wheat cultivars and breeding lines which carry Pm21 accurately, also can be applied to the gene pyramiding breeding in the future. The distribution of Pm21 in winter wheat regions is different, the risk of pathogen directional selection will be promoted because of the high frequency of Pm21 gene in wheat cultivars of Sichuan and Guizhou provinces, and we should pay more attention to it in wheat breeding.

Construction of a Genetic Linkage Map in Mungbean

WU Chuan-Shu-1, 2 , WANG Li-Xia-2, WANG Su-Hua-2, CHEN Hong-Lin-2, WU Jian-Xin-2, CHENG Xu-Zhen-2, YANG Xiao-Ming-1, 3

Scientia Agricultura Sinica. 2014, 47(11): 2088-2098. doi:10.3864/j.issn.0578-1752.2014.11.003

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【Objective】On the basis of previous studies, a genetic map of mungbean was constructed by using genome SSR, EST-SSR, STS primers of mungbean and common bean to build a platform for positioning important traits related genes, cloning and molecular marker-assisted breeding of new varieties of mungbean.【Method】A total of 6 686 SSR, EST-SSR, STS primers of mungbean and common bean, including 6 100 genome SSR, 149 EST-SSR, 13 STS primer pairs of mungbean, and 424 genome SSR primers of common bean, were used for PCR amplification to screen polymorphic markers between Australia-imported Berken (highly susceptible cultivar)×ACC41 (highly resistant wild species) and a RIL derived from these two genotypes were tested with the polymorphic markers. Combined with the molecular marker data of previous studies, Mapmarker/Exp 3.0 software was used for map construction and set LOD≥3.0, the maximum figure at 50.00 cM. Finally, Joinmap 4.0 software was used for map integration.【Result】In this study, from the two parents, 6 686 SSR primers were screened, a total of 3 691 pairs of primers were amplified stable products, 588 pairs of polymorphic primers were obtained. Among them, mungbean SSR primers 6 100 pairs, effective amplification 3 459 pairs, the effective rate of 56.7%, obtained 559 pairs of polymorphic primers; Mungbean EST-SSR primers 149 pairs, effective amplification 126 pairs, the effective rate of 84.6%, obtained 21 pairs of polymorphic primers; Common bean SSR primers 424 pairs, effective amplification 97 pairs, the effective rate of 22.9%, obtained 6 pairs of polymorphic primers; Mungbean STS primers 13 pairs, effective amplification 9 pairs, the effective rate of 69.2%, obtained 2 pairs of polymorphic primers; These results indicated that different sources and types of SSR primers to amplify the effective rate of the parent were significantly different. Mungbean EST-SSR primers (84.6%) were the highest, mungbean STS primers (69.2%) and mungbean SSR primers (55.7%) followed, common bean SSR primers (22.9%) were the lowest. An integrated genetic linkage map of mungbean containing 585 markers was constructed (including 499 SSR markers, 74 RAPD markers, 9 STS markers and 3 RAPD markers). The total length of the map was 732.9 cM and covered 11 linkage groups. The average distance between markers was 1.25 cM. The average distance of each linkage group spanned 66.63 cM. The average number of markers was 53.18 for each of 11 chromosomes. The length of each linkage group ranged from 35 to 92 markers was from 45.2 cM to 112.8 cM. LG1 linkage group contained the largest number of 92 markers, the length was 112.8 cM. LG11 linkage group contained the minimum number of 35 markers, the length was 48.7 cM. Of the 585 markers loci mapping conducted χ2 tests under P＜0.05 and P＜0.01 conditions, respectively, 79 and 151 markers showed a segregation distortion, the total number of sites marked 39.3%.【Conclusion】A linkage map of mungbean with a maximum and the highest density genetic markers was constructed compared with that published at home and abroad at present.

Effects of Accurate Fertilizer Model (AF) on Yield and Population Quality of Hybrid Indica Rice Cultivars in Guizhou Highland Area

LUO De-Qiang-1, 2 , WANG Shao-Hua-1, JIANG Xue-Hai-1, 2 , LI Gang-Hua-1, ZHOU Wei-Jia-2, LI Min-2, JI Guang-Mei-2, DING Yan-Feng-1, LING Qi-Hong-1, LIU Zheng-Hui-1

Scientia Agricultura Sinica. 2014, 47(11): 2099-2108. doi:10.3864/j.issn.0578-1752.2014.11.004

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【Objective】Rice yield is largely affected by many factors including environment and cultivation. Therefore, targeted use of cultivation techniques according to the climate characteristics can fully exploit the potential of rice yield and improve grain yield significantly. Experiments were conducted to study the effects of accurate fertilizer model (AF) on population quality and yield formation of hybrid indica rice cultivars in high mountain area. 【Method】 Field experiments were conducted in several rice growing areas(Huangping, Suiyang, Yuqing, Xingyi) of Guizhou province in 2010 and 2011. The local high-yielding potential hybrid indica cultivars were separately grown in every rice area, and the population quality and yield formation were studied through the comparison of accurate fertilizer model (AF) and conventional fertilizer model (CF). AF was designed based on Stanford equation, calculate the total N-fertilizer application based on three parameters including the nitrogen demand for the designed grain yield, nitrogen provided by soil, and N-fertilizer use efficiency(NUE). And the nitrogen applied at different growth stages were determined by nitrogen demand of corresponding stage. The total N fertilizer amount was divided into four sections and was applied 1 day before transplanting (30%), 7 days after transplanting (20%), panicle ignition (35%) and at the 2nd leaf from top stretching (15%), respectively. The total nitrogen application amount was identical for the two nitrogen ferltilizer models, and for the CF, nitrogen was applied 1 day before transplanting (20%), 7 days after transplanting (60%), panicle ignition (20%), respectively. Leaf area index (LAI) and dry matter production were measured at N-n, elongation, heading and maturity. Productive panicles, spikelets per panicle, filled grain ratio, grain weight were also measured at maturity.【Result】Compared with CF, AF increased average grain yield of rice by12.4%-48.0% in Huangping, Suiyang, Yuqing and Xingyi, spikelets per panicle increased by over 8.1%, seed-setting rate increased by over 2.5%, population spikelets amount increased by over 14.3%, but seed setting rate and 1000-grain weight was changed insignificantly. Judging from the panicle size, the percentage of small panicle (spikelets per panicle from 0 to 100) decreased by 36.7%-100%, the percentage of big panicle (spikelets per panicle from 100 to 250) increased by 2.2%-11.4%, and huge panicle (spikelets per panicle above 250) increased by 23.3%-94.9%. As for leaf area index (LAI), LAI at elongation stage of the AF rice was 4.2%-11.8% lower in comparison with CF rice. The LAI at heading stage increased by 4.2%-13.9% when LAI of top three leaves and spiklets per leaf area (total spikelets per leaf area, filled grain per leaf area, grain weight per leaf area) of AF rice compared with CF rice. The ratio of spikelet to leaf increased by 9.3%-132.7%, the ratio of grain to leaf increased by 12.8%-35.7%, and the ratio of grain weight to leaf increased by 10.1%-36.3%. The dry matter accumulation decreased at elongation, and increased by 9.8%-50.8% during the period from elongation to heading, and increased by 26.9%-62.7% during the period from heading to maturity.【Conclusion】 The AF management can increase the hybrid rice yield significantly, resulting from controlling the population growth before jointing, promoting group development after jointing, especially increasing LAI and dry matter accumulation in the period from heading to maturity, also including decreasing small panicle formation and developing large panicle.

Effect of High Temperature Stress and Spraying Exogenous ABA Post-Anthesis on Grain Filling and Grain Yield in Different Types of Stay-Green Wheat

YANG Dong-Qing-1, WANG Zhen-Lin-1, NI Ying-Li-1, 2 , YIN Yan-Ping-1, CAI Tie-1, YANG Wei-Bing-1, PENG Dian-Liang-1, CUI Zheng-Yong-1, JIANG Wen-Wen-1

Scientia Agricultura Sinica. 2014, 47(11): 2109-2125. doi:10.3864/j.issn.0578-1752.2014.11.005

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【Objective】 The purpose of this study was to test the effects of high temperature stress and spraying exogenous absisic acid (ABA) post-anthesis on endosperm cell division, grain filling and changes of endogenous hormones in different types of stay-green wheat cultivars, and provide a theoretical basis for enhancing grain weight and minimizing the loss of grain yield result from high temperature. 【Method】 Both Wennong 6 (a stay-green cultivar) and Jimai 20(a non-stay-green cultivar) were grown in the experimental plots covered with transparent plastic sheds at 1 to 5 days after anthesis (DAA), which were mimicked the high temperature that occurs during the natural heat event in this region, whilst exogenous ABA (10 mg?L-1) to the spikes was sprayed at the rate of 100 mL?m-2 between 5 pm and 6 pm from 1 to 3 DAA. High performance liquid chromatography method (HPLC) and a simple method of endosperm cell number determination were used to determine endogenous hormones contents and endosperm cell number, respectively. Both grain filling process and endosperm cell division process were simulated by Richard equation, and then the related parameters were calculated. 【Result】 The endosperm cell number of superior and inferior grains in both cultivars were significantly decreased by high temperature stress, and the endosperm cell division rate was markedly decreased. However, high temperature extended the active endosperm cell division period and the division duration. The grain filling rate of inferior grains in both cultivars was decreased in the high temperature treatment. Grain-filling duration and the active growth phase were also reduced in the high temperature treatment. High temperature after anthesis significantly decreased the number of grains per spike and thousand-grain weight. In contrast, Wennong 6 showed 3.7 and 8.2 grain/spike reduction in superior and inferior grains, respectively. Similarly, Jimai 20 was reduced by 1.3 and 4.3 grain/spike in superior and inferior grains, respectively. The final grain yield of Jimai 20 decreased by 26.22% due to high temperature treatment, and that of Wennong6 decreased only by 19.65%. Spraying exogenous ABA obviously improved grain filling rate, endosperm division rate and increased endosperm cell number in both normal and high temperature treatments. Although the zeatin riboside (ZR) contents in superior and inferior grains were significantly decreased in the high temperature treatment, the gibberellin (GA3) contents in Jimai 20 were markedly enhanced at 3 to 27 DAA, and those in Wennong 6 were also increased at 12 to 27 DAA. The auxin (IAA) content of inferior grains in Wennong 6 was significantly decreased at 15 to 27 DAA in high temperature treatment. The ZR content of superior grains in Jimai 20 was decreased at 3 to 9 DAA by spraying exogenous ABA in the high temperature treatment, but endogenous ABA content of superior grains in Jimai 20 was obviously increased at 3 to 28 DAA and that in Wennong 6 was also increased at 3 to 18 DAA. In the normal temperature treatment, spraying ABA significantly decreased the GA3 contents in both Wennong 6 and Jimai 20. Together, spraying exogenous ABA decreased the GA3 contents of superior and inferior grains in Wennng 6 at 3 to 27 DAA in high temperature treatment, and decreased those in Jimai 20 at 3 to12 DAA and 6 to 15 DAA, respectively. In the normal temperature treatment spraying ABA increased IAA content of superior grains in Jimai 20 at 12 to 18 DAA, and also increased that in Wennong 6 at 6 to 18 DAA. Meanwhile, IAA contents of inferior grains in both cultivars were significantly improved at 6 to 27 DAA. In addition, compared with Jimai 20, Wennong 6 had longer grain-filling duration and the active growth phase, higher mean grain-filling rate and maximum grain-filling rate, larger thousand-grain weight, and greater grain yield.【Conclusion】Both no-stay-green cultivar and inferior grains of the two cultivars were much more influenced by high temperature stress. Grain weight and yield reduction result from high temperature decreased kernel sink and grain filling rate. Exogenous ABA improved grain yield due to increasing the grain sink capacity and grain filling rate through regulating endogenous hormones contents to promote endosperm division and photosynthate accumulation in normal treatment and high temperature stress.

Alleviation of Boron Toxicity on Rape Seedlings by Selenium

DUAN Bi-Hui, LIU Xin-Wei, JIAO Wei, ZHAO Zhu-Qing, HU Cheng-Xiao

Scientia Agricultura Sinica. 2014, 47(11): 2126-2134. doi:10.3864/j.issn.0578-1752.2014.11.006

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【Objective】 Boron (B) is an essential trace element for plant growth, however, excessive boron in soil may exert toxic effects on plants. In boron-deficiency soil, excessive use or uneven application of boron can easily lead to boron toxicity, if such levels of boron are accompanied by excessive salinity, the consequences can be drastic for plants. Boron toxicity is a significant constraint to cereal production in regions worldwide, including parts of southern Australia. Raising the suitable boron range of crops and reduce the toxicity of excessive boron are technical problems to be solved for agricultural production. An appropriate amount of selenium can improve crop resistance. This paper aims to investigate the effect of appropriate amount of selenium on rapeseed biomass and active oxygen metabolism under the condition of excessive boron, and explain the alleviation mechanism of selenium on boron inhabitation of rape growth. 【Method】In the present study, solution culture experiments were carried out using rape seedlings (Brassica napus L. cv. Xiangnongyou 571) as the test material. The interactive effects of boron and selenium especially with a purpose to test whether selenium application is effective in countering the boron toxicity were tested. Boron fertilizer was applied at two levels (50, 500 µmol?L-1), selenium fertilizer was applied at two levels (0, 1 µmol?L-1). The effects of these treatments on rape seedling growth, boron uptake and accumulation, enzymatic and non-enzymatic antioxidant, stress injury, oxidative damage along with defense mechanisms under boron (500 µmol?L-1) stress were assessed. 【Result】 Whilst boron application alone at 50 µmol?L-1 level was beneficial for the growth, treatment with boron severely inhibited the growth at 500 µmol?L-1 level. Under boron stress conditions, the dry weight of rapeseed seedling shoots and roots decreased significantly, rape seedling shoots and roots dry weight severely decreased by 20.1% and 32.0%, respectively, over 50 µmol?L-1 boron, and a lot of boron took up and accumulated over normal boron concentration (50 µmol?L-1). The enzymatic activity of scavenging reactive oxygen species, including catalase (CAT), peroxidase (POD) and ascorbate peroxidase (APX) in rape seedling leaves decreased, CAT, POD and APX activity decreased by 19.7%, 11.0% and 15.0%, respectively, compared to normal boron concentration, and the endogenous reactive oxygen substances (glutathione (GSH)) in leaves were significantly reduced, while hydrogen peroxide (H2O2) and malondialdehyde (MDA) increased significantly by 19.0% and 18.5% over 50 µmol?L-1 boron treatment. The boron-treated (500 µmol?L-1) plants supplemented with 1 µmol?L-1 selenium showed increased shoot and root dry weight, it significantly improved by 22.8% and 28.6% over boron-treated (500 µmol?L-1) plants, respectively, it also indicated that an antagonistic interaction between the two elements. Selenium at 1 µmol?L-1 concentration significantly affected the boron uptake and accumulation, it decreased boron content and accumulation in shoot by 38.6% and 31.9% respectively, over 500 µmol?L-1 boron treatment. Oxidative damage due to boron was lowered, which could be related to the reduction of active oxygen metabolism enzyme and endogenous scavengers in seedling leaves caused by boron toxicity were significantly alleviated. The boron-treated (500 µmol?L-1) plants supplemented with 1 µmol?L-1 selenium showed improved CAT, POD and APX activities, they increased by 12.8%, 15.1% and 15.3%, respectively, GSH and ASA contents increased by 9.7% and 21.0% over 500 µmol?L-1 boron treatment, while the accumulation of hydrogen peroxide and the increase of membrane lipid product malondialdehyde were effectively reduced, selenium application at 1µmol?L-1 to boron treated (500 µmol?L-1) plants, H2O2 and MDA content decreased by 25.1% and 22.1% over 500 µmol?L-1 boron treatment, respectively. 【Conclusion】 It was suggested that an appropriate amount of selenium can increase reactive oxygen metabolism of rapeseed and reduce the accumulation of reactive oxygen, thereby significantly decreasing the impact of excessive boron on rapeseed growth and improving the adaptability of rape seedlings to excessive boron.

Uncertainty Research of Remote Sensing Image Classification Using the Boundary Region-Based Modified Rough Entropy Model

JING Xia-1, 2 , WEI Man-3, WANG Ji-Hua-1, SONG Xiao-Yu-1, HU Rong-Ming-2

Scientia Agricultura Sinica. 2014, 47(11): 2135-2141. doi:10.3864/j.issn.0578-1752.2014.11.007

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【Objective】It is an effective technical method to obtain land cover information by remote sensing classification. Objective and reasonable evaluation of the uncertainty in remote sensing classification maps is significant to agricultural resources survey, crop yield assessment and other remote sensing applications. This study focuses on the problem of uncertainty evaluation for remote sensing image classification at the land cover scale. A new uncertainty evaluation criteria, boundary region-based modified rough entropy model (BMREM) is proposed based on the improvement of modified rough entropy model (MREM). 【Method】 The traditional MREM model measures the overall average uncertainty of the same class feature. But the classification uncertainty is mainly caused by the boundary between different feature classes in RS image. So, in this study, all pixels value range for class feature is replaced by the boundary range of one feature class in image. The rationality of the BMREM model is proved based on rough set theory. And then the BMREM model was established under the impact of boundary region on the uncertainty assessment in remote sensing image classification. Afterwards, the MREM model and the BMREM model are applied to evaluate the uncertainty of remote sensing classification results for one Landsat TM image and one IKONOS image, respectively. The theoretical conclusion was confirmed by uncertainty evaluation result using experimental data of different spatial resolution and different research areas.【Result】 Compared with the MREM model，the uncertainty caused by the classification knowledge can be better described using the BMREM model. So the uncertainty of remote sensing image classification is more reasonable and objective. The empirical results reveal that the irrationality of uncertainty assessment is not obvious using MREM model. It also can objectively measure the uncertainty problem of remote sensing image classification if the mixed pixel is more serious. But it is difficult to objectively evaluate the uncertainty of remote sensing image classification by MREM model if the ground objects distribution is more concentrated in study area and the area is relatively large.【Conclusion】This study proved that MREM model magnified the value of uncertainty caused by existence of boundary region from the theory analysis and model application. Yet the uncertainty generated by classification knowledge can be better depicted using the BMREM model. So the BMREM model measured the classification uncertainty at the scale of land cover classes more reasonably and accurately.

Construction of Enhanced Gene Knockout Frequency Recipient Strain by Deletion of Vdku80 in Verticillium dahliae

TIAN Li-1, LIU Na-1, XU Rong-Qi-2, QU Zhi-Cai-1, LIU Qian-1

Scientia Agricultura Sinica. 2014, 47(11): 2142-2150. doi:10.3864/j.issn.0578-1752.2014.11.008

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【Objective】 The objective of this study is to characterize Vdku80 of Verticillium dahliae, which encodes the protein involved in the nonhomologous end-joining double DNA break repair pathway, and to provide a recipient strain with enhanced gene knockout frequency. 【Method】 V. dahliae Vdku80 deletion mutant ΔVdku80 was constructed as previously reported. Briefly, Vdku80 gene knockout was accomplished through a combination of techniques, beginning in Vdku80 homologous DNA fragment construction. Individual V. dahliae cells were genetically transformed via Agrobacterium tumefaciens mediated transformation. Recombination then occurred in the region of that sequence within the gene of Vdku80, resulting in the insertion of hygromycin resistance gene to disrupt the gene of Vdku80. Vegetable growth, sporulation and pathogenicity of ΔVdku80 were studied compared with those of wild type strain. The effect of the absence of Vdku80 on gene knockout frequency was tested by disruption of ChsV and ChsVI, which encoded V. dahliae chitin synthases.【Result】Vdku80 deletion mutant strain (ΔVdku80) was successfully constructed. Deletion of Vdku80 had no effect on colony morphology and mycelial growth, as deduced from the monitoring of radial growth of wild type strain and ΔVdku80 on PDA media 8 days after inoculation. Both wild type strain and ΔVdku80 reached highest level of conidia production after growing for 5 days on Czapek media and showed no significant differences with respect to conidia number in this moment. Growth of wild-type strain and ΔVdku80 was inhibited by 0.02% MMS, and the inhibition rate was also similar. Finally, the Vdku80 deletion mutant ΔVdku80 was qualitatively and quantitatively as pathogenic as its corresponding wild-type strains. Altogether, these results showed that the deletion of Vdku80 did not affect mycelial growth, conidia production and pathogenicity of V. dahliae. Using wild-type strain as a gene knockout recipient, gene knockout frequency was 44% and 31% for ChsV and ChsVI, respectively, while using ΔVdku80 as gene knockout recipient, gene knockout frequency was 94% and 87% for ChsV and ChsVI, respectively.【Conclusion】Vdku80 deletion mutant ΔVdku80 displayed wild-type phenotypes regarding growth, sporulation and pathogenicity, but clearly improved gene knockout frequency of other genes. These results support that ΔVdku80 can be a recipient strain with enhanced gene knockout frequency in V. dahliae.

Correlation of Candidatus Liberibacter asiaticus and the Endophytic Community in Diaphorina citri

SUN Li-Qin, YIN You-Ping, WANG Fang, WU Xiao-Fang, WANG Zhong-Kang

Scientia Agricultura Sinica. 2014, 47(11): 2151-2161. doi:10.3864/j.issn.0578-1752.2014.11.009

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【Objective】The objective of this study is to analyze the endophytic bacteria in Diaphorina citri which may affect the pathogenesis of Candidatus Liberibacter asiaticus, and to provide a basis for artificial culture of Ca. L. asiaticus, prevent and control of HLB. 【Method】 The diversity of cultivable endophytic bacteria in different samples of D. citri with or without Ca. L. Asiaticus from different geographical origins were compared by traditional isolation and culture methods. D. citri with and without Ca. L. asiaticus were divided into three parts: the head, thorax and abdomen, respectively. Then V6-V8 area of 16S rDNA were amplified after extracting DNA, the endophytic bacteria in D. citri of with or without HLB pathogen and in different parts of the same carrier state were compared by 16S rDNA-PCR-DGGE. The relative amount in 8 D. citri samples of Bacillus sp., Salmonella sp., Enterobacter sp. and Ca. L. asiaticus was determined, which have different results in traditional isolation and culture methods and PCR-DGGE, using total amount of bacteria as an amount of contrast. Relative amounts of the 3 chosen bacteria and Ca. L. asiaticus, respectively, in every D. citri sample were compared before comparing similarity of the same bacteria in different samples in order to analyze the interaction of the 3 chosen bacteria and Ca. L. asiaticus. 【Result】 Richness of the colony and colony-forming unit of cultivable endophytic bacteria in D. citri with Ca. L. asiaticus were more than in D. citri without Ca. L. asiaticus. Fourteen kinds of bacteria were isolated from D. citri without Ca. L. asiaticus, belonging to 10 genera, such as Bacillus sp.(3), Erwinia sp.(1), Klebsiella sp.(1), Staphylococcus sp.(2), Arthrobacter sp.(1), Pantoea sp.(2), Pectobacterium sp.(1), Salmonella sp.(1), Streptomyces sp.(1), and Massilia brevitalea sp.(1). While only 4 kinds of bacteria were isolated from D. citri with Ca. L. asiaticus, from 3 genera the same as healthy psyllid, such as Bacillus sp., Klebsiella sp., Pectobacterium sp.. B. aerophilus was isolated frequently from D. citri, as a resident flora in D. citri. As the electrophoretogram of 16S rDNA PCR-DGGE revealed, there were significant differences between D. citri with and without Ca. L. asiaticus in the same tissues, but no significant differences were found in different tissues from the same D. citri. Robotic bands No. 10 (Wolbachia sp.), No. 12 (Wolbachia pipientis), No. 13 (Syncytium endosymbiont of Diaphorina citri), No. 14 (Uncultured bacterium), No. 19 (Serratia marcescens), No. 21 and No. 22 (both are Stenotrophomonas maltophilia) were stable in D. citri with and without Ca. L. asiaticus. Enterobacter sp. was one of the dominant endosymbiotic bacteria of D. citri with Ca. L. asiaticus. Wolbachia also was found in D. citri. The results of q-PCR proved that the results of traditional isolation and culture methods and 16S rDNA-PCR-DGGE were correct, and demonstrated that the relative amount of Enterobacter sp. and Ca. L. asiaticus were positive correlation in the 8 chosen samples. 【Conclusion】 Suspected resident flora B. aerophilus was isolated frequently in D. citri. There may be some interactions between the Enterobacter sp. and Ca. L. asiaticus according to the result of q-PCR, which reveals the relative amount of Enterobacter sp. and Ca. L. asiaticus are positive correlation.

Cloning, Expression and Purification of Ultraspiracle Protein Gene from Apolygus lucorum

TAN Yong-An, XIAO Liu-Bin, SUN Yang, ZHAO Jing, BAI Li-Xin

Scientia Agricultura Sinica. 2014, 47(11): 2162-2172. doi:10.3864/j.issn.0578-1752.2014.11.010

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【Objective】 The objectives of this study are to clone the ultraspiracle protein gene of Apolygus lucorum (ALUSP) and obtain the recombinant protein of prokaryotic expression, and to reveal the function of ALUSP and offer a basis for research of the ecdysis and metamorphosis of A. lucorum. 【Method】 The conserved sequence of A. lucorum was obtained by using the degenerate primers from the known USP genes of the other insects. A new set of gene-specific primers matching the primers in the 3′- and 5′-full RACE kit were designed and the original sequence files were assembled to obtain the complete cDNA sequence of the ALUSP. The characteristics of ALUSP gene sequences and which coding proteins were analyzed by using the bioinformatics methods, and the phylogenetic tree was also constructed. To construct its prokaryotic expression vector, T-vector containing ALUSP was dual-enzyme digested by EcoR I and Xho I. The expression vector was induced to express at 15, 25, 30 and 37℃. ALUSP purified protein gene in functional areas was gotten by the GST agarose affinity chromatography and molecular sieve chromatography.【Result】The open reading frame of ALUSP was 1 005 bp in length, encoding a 334 amino acid polypeptide with a predicted molecular weight of 56.63 kD and the theoretical isoelectric point of 8.42. Protein signature analysis revealed that the protein encoded by ALUSP shared typical structural features of ultraspiracle proteins with other insects, including A/B domain (249 bp), C domain (198 bp), D domain (69 bp) and E domain (489 bp), respectively. C domain was highly conserved, including two zinc fingers, a P-box, a D-box and a nuclear location signal that was constituted of eight amino acids. D domain included a T-box which could identify the DNA components and E domain, including a bag structure, was constituted of eight α-helix and β-strand. The blast results showed that the amino acids sequence of ALUSP was the highest in similarity (57.82%) with Nezada viridula, and low in similarity with other insects, such as Melipona scutellaris (46.05%), Scaptotrigona depilis (45.94%), respectively. Phylogenetic tree analysis results showed that the USP genes from Hemiptera were more differentiation between Hymenoptera, which were located in different branches, ALUSP and N. viridula USP was the nearest in genetic evolution, which may be derived from common ancestors. The recombinant plasmid pEGX6P1-ALUSP was high-efficient expression in Rosetta gami B when induced by 37℃ and 1.0 mmol•L-1 IPTG. The 65 kD target protein from the strain containing ALUSP was obtained by the GST agarose affinity chromatography and the molecular sieve chromatography.【Conclusion】The full-length cDNA of ALUSP from A. lucorum was obtained and also got the recombinant protein. The protein had typical characteristics of insect ultraspiracle protein.

Multifractal Characterization of Soil Particle Size Distribution Under Long-Term Different Fertilizations in Upland Red Soil

SUN Mei-1, 2 , SUN Nan-2, HUANG Yun-Xiang-1, XU Ming-Gang-2, WANG Bo-Ren-2, ZHANG Xu-Bo-2

Scientia Agricultura Sinica. 2014, 47(11): 2173-2181. doi:10.3864/j.issn.0578-1752.2014.11.011

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【Objective】 The aim of the study are to analyze the impact of long-term fertilization on the heterogeneity of the soil particle-size distribution of red soils, to investigate the mechanisms of soil development process affected by different fertilization strategies and to explore the ways to promote sustainable development of agriculture while maintaining soil quality. 【Method】 A laser particle size analyzer was used to measure the particle size distribution of the soil samples. The soils were collected from the top 20 cm soil depth in four treatments with a wheat-maize rotation system at two time points in the long-term fertilization experiment on red soil in Qiyang, Hunan province in 1990 and 2012. The treatments include long-term continuously non-fertilizer (CK), chemical fertilizer (NPK), NPK plus manure (NPKM), and manure alone (M). The changes of the multi-fractal parameters of particle size distribution were analysed by the box dimensions method. Furthermore, the correlation analysis between the multi-fractal parameter, soil particle and organic matter was also carried out. 【Result】 The results showed that the multi-fractal parameter represented the heterogeneity of soil particle-size distribution much sensitively and accurately and it was significantly correlated to the contents of soil clay or silt particle and soil organic matter. Generalized dimensions (D(q＜0)) were more sensitive than that of D(q＞0) in the range of -10≤q≤10, indicating that the scaling property of D(q) in the sparse range was less than that in the dense range. Compared to the values of Ds from initial soil samples, long-term fertilization did not significantly influence the capacity dimension (D0) or information dimension (D1), but significantly increased the correlation dimension D2 (0.864-0.883), D1/D0 (0.921-0.932), the singular strength (1.129-1.177) and the singular spectrum width of (1.966-2.707). 【Conclusion】 Long-term application of manure with or without chemical fertilizer significantly improved the local intensity of soil particle-size distribution, promoted soil refinement and accelerated the development of soil inhomogeneity under the similar soil-forming conditions, cropping system and agricultural management. The multi-fractal parameter was much more sensitive to the changes of soil particle-size distribution, which could provide a technical support for the research on precision of soil particle size distribution.

Response of Hot Pepper Growth to Soil Constitution and Water Regimes

JI Ya-Lin-1, SHAO Ming-An-2, WEI Xiao-Rong-3

Scientia Agricultura Sinica. 2014, 47(11): 2182-2192. doi:10.3864/j.issn.0578-1752.2014.11.012

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【Objective】Soil constitution and water regimes directly affect the availability of soil water, which further influence plant growth and physiological processes. Hot pepper is one of the important economic crops in China. In this paper, the specific leaf area, photosynthesis and total evaporation of hot pepper at various soil constitution patterns and water regimes were measured. The objective of this experiment was to reveal the physiological mechanism and soil physics mechanism of hot pepper in response to soil constitution patterns and water regimes, thus providing a scientific basis for soil improvement in sandy region.【Method】A pot experiment was conducted and the hot pepper (Xiangyan 5) was used in this study. This experiment was composed of 4 soil constitution patterns (lower sand, lower soil, soil sand mixture, soil) and 3 water regimes (well-watered supply: 75%-90% field capacity (FC); moderate water stress: 60%-75% FC; severe water stress: 45%-60% FC). The specific leaf area (SLA), net photosynthetic rate (Pn), stomatal conductance (Gs), transpiration rate (Tr) and total evaporation were measured at initial stage (Jun 28), mid stage (Jul 10), late stage (Jul 28) and the end stage (Aug 10) of flowering and fruiting stage.【Result】Through the flowering and fruiting stages, SLA was significantly higher in the lower sand treatment compared with other treatments. Net photosynthetic rate was increased by 35% and 27% higher in the soil and soil sand mixture treatments compared to lower soil treatment. Transpiration rates in the soil, sand soil mixture and lower sand treatments were 53%, 34% and 31% higher than that in the lower soil treatment. However there was no significant difference in pepper growth among the three water regime treatments. Stomatal conductance in the soil, mixture and lower sand treatments were 64%, 41% and 40% higher compared with lower soil treatment. Stomatal conductance was 29% and 36% higher in the well-watered supply and moderate water stress treatments than that in the severe water stress treatment, respectively. These results suggested that the effect of soil constitution patterns on net photosynthetic rate and transpiration rate were much greater than water regimes treatments. In addition, the effect of soil constitution on photosynthesis was related to the measuring periods and water regimes. Soil constitution had effects on the photosynthetic rate at the initial, mid and late stages, on the stomatal conductance and transpiration rate only at the initial stage. Under well-watered supply and moderate water stress regimes, the net photosynthetic rate, stomatal conductance and transpiration rate were higher in the soil and mixture treatments than lower soil treatment, but there was no significant difference between soil constitution treatments under severe water stress condition. Meanwhile, the measuring periods obviously affected the growth of hot pepper. At flowering and fruiting stages, the specific leaf area was the highest at the end stage, photosynthetic and transpiration rates were the highest at the late stage; stomatal conductance was higher at initial, mid and late stages than at end stage. The total evaporation during the four stages was affected by soil constitution and water regimes. Total evaporation was the highest in the soil treatment and the lowest in the lower soil treatment.【Conclusion】The results from this study indicated that the effect of soil constitution patterns on hot pepper growth was much greater than water regimes. Additionally, the effect of soil constitution on photosynthesis was relevant to growing stages. Therefore, for the improvement of sandy soils in the semi-arid sand region, the lower sand and soil sand mixture soil constitution patterns and the soil moisture of 60%-75% field capacity were recommended.

Genetic Variation of F1 Population Between Malus sieversii f. neidzwetzkyana and Apple Varieties and Evaluation on Fruit Characters of Functional Apple Excellent Strains

CHEN Xue-Sen, ZHANG Jing, LIU Da-Liang, JI Xiao-Hao, ZHANG Zong-Ying, ZHANG Rui, MAO Zhi-Quan, ZHANG Yan-Min, WANG Li-Xia, LI Min

Scientia Agricultura Sinica. 2014, 47(11): 2193-2204. doi:10.3864/j.issn.0578-1752.2014.11.013

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【Objective】 To provide basic date for the conservation and sustainable use of Malus sieversii f. neidzwetzkyana. resources and create functional apple breeding theory and technology system, the fruit juvenile period, genetic variation characteristics of fruit and the evaluation of excellent strains for functional apple were studied. 【Method】 Six F1 hybrid groups were developed by Taeraerma and Xiahongrou as male parents and Hanfu, Starkrimson, Golden Delicious and Fuji as female parents. The fruit juvenile period, single fruit weight, hard crispness, soluble solids, Vc, titratable acidity, total phenolic and anthocyanin content, antioxidant ability, the content and components of flavonoids of 868 seedlings and 4 excellent strains from the six F1 hybrid groups were detected and compared with ‘Dehongcui’, which was introduced from foreign countries, and ‘Golden Delicious’. Furthermore, functional apple breeding techniques were discussed.【Result】The flowering rates of 5 groups with M. sieversii f. neidzwetzkyana as female parent were all more than 15% in the third year and the juvenile phases were only 2.33-4.33 years, while the flowering rate of Golden Delicious×Hanfu was only 8.0% in the 4th year and the juvenile phase was 3.33-5.33 years. Variations in most characters of F1 were wide and the variation coefficients were all more than 20%. It indicated that they have a large potential for further selection. The broad-sense heritabilities of total phenols in peel and flesh, acid, anthocyanin and Fe were more than 85%, which had powerful hereditary abilities. The 5 groups with M. sieversii f. neidzwetzkyana as female parent all existed red-soft, red-brittle, white-soft and white-brittle individuals. The anthocyanin and flavonoids content, antioxidant ability and flavonoids species of red strains ‘Hongcui 1’ and ‘Hongcui 10’, whose red area were nearly 1/3-1/2 of full apple area, were extremely significantly higher than reddish apple, such as ‘Hongcui 8’ and ‘Baicui 1’, and the control.【Conclusion】The F1 of M. sieversii f. neidzwetzkyana×apple varieties had the characters of short juvenile phase, powerful heritabilities of total phenols in peel and flesh and wide variation. Some excellent red-brittle functional apple strains with antioxidant ability and higher content and components of flavonoids can be selected from F1 groups.

Establishment and Application of Double Color DNA Fiber Fluorescence in situ Hybridization Using Bacterium Artificial Chromosomes in Apple

WANG San-Hong, ZHANG Zhen, CAI Bin-Hua, QU Shen-Chun

Scientia Agricultura Sinica. 2014, 47(11): 2205-2213. doi:10.3864/j.issn.0578-1752.2014.11.014

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【Objective】The fluorescence in situ hybridization (FISH) on extended DNA fibers is a powerful tool in physical mapping construction, gene site determination in the genome, and so on. However, the technique is less reported in apple for its difficulties in nuclei extraction and fiber extension. To apply it widely in the apple genome research, the double color DNA fiber fluorescence in situ hybridization using apple bacterium artificial chromosomes (BAC) as probes was established in the experiment.【Method】The young leaves from apple ‘Florina’ were well ground in liquid nitrogen. The nuclear suspension was filtrated through nylon mesh of pore size from 161, 100, to 48 μm in turn, and subsequently triton X-100 was added into the suspension for removing chlorophyll. The apple nuclei were broken in STE buffer and DNA fibers were dragged and extended with a clean coverslip. The effects of different nuclear lysis times and five coating-type slides on DNA fibers extension were compared. The bacterium artificial chromosomes BAC 34G16, BAC 45M19, BAC 70J19 and BAC 69A4 from ‘Florina’ self incompatibility S9 locus were extracted by alkaline lysis method. The BAC plasmids were purified by PEG and then labeled with either digoxigenin-11-UTP or biotin-16-UTP using standard nick translation method. The probes and DNA fibers were denatured at 80℃, followed by incubation at 37℃ in a hybridization chamber for 2-3 days. FISH signals were amplified by ‘sandwich’ method and detected under fluorescent microscope.【Result】The experimental system of nuclei extraction from apple young leaves, DNA fiber extension, in situ hybridization and signal amplification was established. The purity and structural integrity of nuclei were obtained and the concentration exceeds 5×103 per microlitre. When the nuclei lyzed in STE buffer for 4 min and fiber extended on the polylysine coating slide, a great many of the ideal DNA fibers exhibiting long, slight, even and straight were obtained. The clear and typical “beads-on-a-string” nature of the DNA fiber FISH signals were observed after in situ hybridization, signal detection and amplification. The two BAC clones 45M19 and 34G16 with the known insert size and site relationship were hybridized on extended DNA fibers to calibrate and standardize the resolution of fiber FISH in apple. The results indicate that a regression equation of Y=3.47X for the best fit to the experimental data, where Y is the molecular probe length in kb and X is the fluorescence signal in μm. The correlation coefficient is R2=0.9215. The experiment also successfully identified two BAC clones 70J19 and 69A4 with unknown insert size and site relationship. The size of BAC70J19 and BAC 69A4 was (112.1±18.4)kb and (133.2±16.3)kb, respectively. There was an overlap region of size (90.2±7.3)kb between the two BAC clones. 【Conclusion】A method of nuclear extraction, DNA fiber extension and in situ hybridization was established using apple young leaves as material and the high resolution of apple fiber FISH was achieved.

Effect of 60Co Gamma Irradiation on Quality and Microstructure of Storage Brown Rice

CHEN Yin-Ji-1, CHEN Xia-1, JIANG Wei-Xin-1, DONG Wen-2, CHEN Zhao-Bo-2, DAI Bing-Ye-2

Scientia Agricultura Sinica. 2014, 47(11): 2214-2223. doi:10.3864/j.issn.0578-1752.2014.11.015

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【Objective】 China is a big consumer of grain, grain storage relates to national security and social stability. Irradiation is used in many areas, such as food storage and processing and so on. This paper conducted with clarify the change mechanisms of rice quality dealt with 60Co gamma irradiation by cooking and texture quality changes of storaging brown rice. The purpose of this study is to determine the suitable irradiation dose, provide a theoretical basis for determining applicable gamma irradiation dose and technical methods of storaging brown rice. 【Method】 After harvest, the grains were peeled, filled into the plastic film bags, and then irradiated with 60Co gamma at irradiation doses of 0.2 kGy, 0.5 kGy, 1.0 kGy and 2.0 kGy. After measuring the initial index of brown rice, the rice samples were stored at (15 ± 0.5 ℃) for one year, the quality of brown rice was measured every three months: amylose content was determined by amylose instrument, gelatinization parameters were determined by using the RNA viscosity instrument, construction quality of rice was measured by properties instrument, microstructure of irradiated rice starch granules were observed with scanning electron microscope.【Result】 In this study, we can conclude the following results. It was found that rice pasting peak viscosity increased with prolonged storage time, the increasing trends were different among different doses, through viscosity and breakdown value increased with prolonged storage time. Gamma irradiation reduced through viscosity, final viscosity, setback value and breakdown value of brown rice. Pasting temperature increased with the irradiation dose, the higher the pasting temperature, the more the brown rice resists to cooking, the higher the quality obtained. Gamma irradiation affected the setback value and final viscosity of brown rice mostly, and 0.2 kGy dose irradiation could increase the stability of cold rice paste and improve cooking quality significantly (P＜0.05), the change trend of final viscosity was similar to setback value. Amylose content of brown rice increased with storage time, after gamma irradiation ,the amylose content increased. For brown rice of low apparent amylose content ,the cooking hardness and amylose content showed no obvious correlation (P＞0.05). During the one year storage, the cooking hardness increased with time, however, gamma irradiation decreased the hardness of brown rice, the hardness of brown rice irradiated at 0.5 kGy and above showed a difference with non-irradiation rice significantly (P＜0.05). Irradiation coule prolong the decline of adhesive property of cooking rice, as a result, 0.5 kGy of irradiation improved the quality of stored cooking brown rice. Irradiation modified the structure and morphology of starch granules, the space between starch granules became larger after irradiation, more water could be penetrated when cooking, so the quality of the cooking brown rice improved.【Conclusion】 Gamma-irradiation changed the starch granule morphology and structure of stored brown rice, improved the quality and texture of cooked rice irradiated during storage at the doses from 0.5 kGy to 2.0 kGy.

Rapid Determination of Vanillin in Infant Formulas by Surface Enhanced Raman Spectroscopy

WANG Shi, CHENG Jie, SU Xiao-ou

Scientia Agricultura Sinica. 2014, 47(11): 2224-2232. doi:10.3864/j.issn.0578-1752.2014.11.016

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【Objective】Vanillin (3-Methoxy-4-hydroxybenzaldehyde) is a kind of food additives with rich milk flavor that is commonly used in infant food. Excessive use of vanillin may cause liver and kidney damage in infants. Therefore the detection method and the control of vanillin in infant milk powder has an important practical significance. At present, the government has not released relevant inspection standards, also have no on-site test methods. In this paper, a rapid qualitative and quantitative analysis of vanillin in infant formulas milk powder was set up by surface enhanced Raman spectroscopy, which is stable, reliable and suitable for the on-site detection, thus providing technical support for government regulations.【Method】In this study, the optimization of the pretreatment, the synthesis of enhanced substrates and the choice of operating parameters were studied, respectively. By comparing the effect of different extraction solvents on extraction, the optimum conditions were established. The effect of different sizes of Au nanoparticles on SERS intensity and the influence of pH or the ionic strength on the testing solutions were investigated in order to obtain the optimum enhanced substrates and detection conditions 【Result】The optimum pretreatment method was described as follows: Firstly, the proteins in the matrix were removed by using lead acetate solution. Secondly, the vanillin was extracted by using methylene chloride. Finally, the extraction was purified by sodium hydroxide solution with pH of 9. With approximate 58 nm of gold colloidal solution as enhanced substrates, 200 μL 1% salpeter solution and 50 μL 1% potassium nitrate solution were used in order to make the characteristic Raman signal of vanillin stronger and the qualitative and quantitative analysis available. The 1 149, 1 497 and 1 542 cm-1 Raman shifts were chosen as the qualitative basis. The intensity of 1 531 cm-1 peak was used as a normalization standard. The optimized method has a good linear correlation (calibration coefficient=0.9913) between the intensity of quantitative Raman peak and the concentration of vanillin over a large working range (30-300 μg•mL-1). The limit of detection (LOD) was 10 μg•mL-1. The recoveries for vanillin spikes (50, 100, 200 μg•mL-1) in milk powder ranged from 80.5% to 86.9% and the relative standard deviations (RSD) were lower than 8.6%.【Conclusion】The actual samples purchased from the local markets were tested by the method of this study and the latest reported methods. The established method was true and reliable compared with the latest reported methods, which is suitable to the on-site and rapid detection of vanillin in infant formulas milk powder with the advantages of simple sample preparation and fast analysis of 5 min per sample.

Influence of Dietary Rapeseed Levels on Growth Performance, Rumen Fermentation Characteristics and Blood Biochemical Indices in Beef Cattle

ZHANG Jie-Jie-1, 2 , ZHAO Hong-Bo-1, YOU Wei-1, CHENG Hai-Jian-1, LIU Xiao-Mu-1, WAN Fa-Chun-1, SUN Guo-Qiang-2, SONG 恩Liang-1, LIU Yi-Fan-1

Scientia Agricultura Sinica. 2014, 47(11): 2233-2241. doi:10.3864/j.issn.0578-1752.2014.11.017

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【Objective】The study was conducted to investigate the effects of different dietary rapeseed levels on growth performance, rumen fermentation characteristics and blood biochemical indices in beef cattle to provide a basis for the rational use of rapeseed during beef cattle fattening. 【Method】 The trial has adopted one-factor completely random design. Forty healthy crossbred Simmental bulls at (447±35.67) kg initial live weight and approximately 18-month old were randomly allocated into 4 experimental diets containing varying proportions of rapeseeds in a completely randomized design. Four treatment diets with different inclusion levels of rapeseeds were: control diet with no rapeseed added, 8.7% rapeseed (low rapeseed, LR), 17.4% rapeseed (middle rapeseed, MR), and 26% rapeseed (high rapeseed, HR), respectively. The animals were allowed 10 days to adapt to the diets and experimental measurements were taken in 90 days. Cattle were weighed at the end of the experiment and feed cost was calculated individually. Blood and ruminal content samples were collected and volatile fatty acid (VFA), ammoniacal nitrogen (NH3-N), triiodothyronine (T3), thyroxine (T4) and thyrotrophin (TSH) were estimated in the four treatments using gas chromatography, colorimetric method and radioimmunoassay. Variance analysis was conducted using the general linear model of SAS 9.2. 【Result】The results showed that, no difference in liveweight before slaughtering among treatments (P＞0.05) was observed, the average daily gain (ADG) in MR group was the highest (1.33 kg•d-1), which was higher than that in HR group (P＜0.05), and 6.40% and 7.25% higher than that in control grounp and LR grounp (P＞0.05). Cattle in the HR group consumed the highest dry matter and LR group consumed the lowest, however there was no significant difference among four groups (P＞0.05). With the increase of rapeseed level, feed cost decreased (P＞0.05). Feed to gain ratio (F/G) was the lowest in MR group and the highest in HR group, however no significant difference among the four groups. Rumen pH, NH3-N and volatile fatty acid concentration did not get significant influence by the increased dietary rapeseed levels (P＞0.05), but NH3-N, butyric acid concentration and the ratio of acetic acid and propionic acid tended to decrease, propionic acid concentration tended to increase, and total VFA tended to increase first and then decrease. Serum TSH was significantly decreased when cattle received MR and HR diets, compared with control diet (P＜0.05). Serum AST was significantly increased when cattle received MR and HR diets, compared with control and LR diet (P＜0.05). UN was markedly decreased when cattle received MR diet, compared with control diet, whereas no differences with other groups. No significant differences were observed among all the groups in blood T3 and T4, ALT, creatinine and cholesterol (P＞0.05).【Conclusion】The data indicate that there is no significant effect on rumen fermentation when adding different diet rapeseed levels. Adding 17.4% rapeseed to beef cattle diets had no effect on ADG during fattening period. The metabolism of thyroid and liver could be aggravated when cattle received dietary with rapeseed.

Effect of Kuqin Compound on mRNA Transcription of Bcl-2/BAX in PK-15 Cells Infected by TGEV

SHAO Qiu-Hong, LING Rong-Bin, ZHU Zhao-Rong, LIU Juan, GUO Zhi-Xing, WANG Peng

Scientia Agricultura Sinica. 2014, 47(11): 2242-2250. doi:10.3864/j.issn.0578-1752.2014.11.018

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【Objective】 The objective of this study is to investigate the effect of Kuqin compound on the transcription of Bcl-2/BAX mRNA in porcine kidney cells (PK-15) infected with transmissible gastroenteritis virus (TGEV) . 【Method】 To study the drug (Kuqin compound) test in vitro by TGEV infected PK-15 cell model, the test was divided into Kuqin compound group, blank control group, model group, and astragalus polysaccharide group, respectively. The morphology change was observed in PK-15 cells by the Swiss staining method and the transcription of Bcl-2 and BAX mRNA was tested by RT-PCR method after determination of tissue culture infective dose (TCID50) of TGEV and the maximum of no toxic concentration of Kuqin compound and astragalus polysaccharide on PK-15 cells. 【Result】The following results were obtained from the observation. Compared with blank control group, the PK-15 cells in model group had obvious cytopathic effects (CPE) including elongated cell shape, intercellular drawing into mesh, and lighter staining in nucleus. The nucleus enrichment, pyknosis and fragmentation were also observed. On the other hand, compared with the model group, CPE in Kuqin compound and astragalus polysaccharide group was improved and the Kuqin compound had better effect with most nucleus structure clear, uniform staining in chromatin and fewer nucleuses became larger or fragmentized. The transcription amounts were also considered as an effect of compound Kuqin on infected PK-15 cells. Compared with blank, the transcription amount of Bcl-2 mRNA in model group was less (P＜0.01). However, the transcription amount of Bcl-2 mRNA in compound Kuqin group was increased by 1.403 times compared with the model group (P＜0.01), by 1.078 times with the Astragalus polysaccharide group (P＞0.05). For the BAX mRNA transcription, the model group had an obvious increase (increased by 1.440 times, 1.290 times and 1.490 times compared with the blank group, compound Kuqing group and the astragalus polysaccharide group) while no obvious changes among the other three groups (compound Kuqin was 1.116 larger than blank and astragalus polysaccharide was 0.966 of the blank). Moreover, the transcription ratio of Bcl-2/BAX in model group had a huge decrease compared with the blank group (P＜0.01). This ratio in compound Kuqin and astragalus polysaccharide groups was much larger compared with the model group (P＜0.01) with the number of 1.809 and 1.904 times, respectively; this number was 0.749 (P＜0.01) and 0.933 times (P＜0.05) of blank, respectively.【Conclusion】Kunqin compound could inhibit apoptosis in PK-15 cells infected by TGEV by up-regulating transcription of Bcl-2 mRNA and down-regulating transcription of BAX mRNA.

Study on Rapid Detection Kit of Fleroxacin by icELISA

JIA Guo-Chao-1, 2 , ZHI Ai-Min-1, LI Meng-Qin-2, SONG Chun-Mei-1, WANG Ling-Ling-1, LIU Ru-Biao-1, HU Xiao-Fei-1, WANG Fang-Yu-1, ZHANG Gai-Ping-1

Scientia Agricultura Sinica. 2014, 47(11): 2251-2261. doi:10.3864/j.issn.0578-1752.2014.11.019

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【Objective】The goals of this study were to obtain immunogen and coating antigen, generate its mice polyclonal antiserum and develop FLE-Kit by its competitive indirect enzyme-linked immunosorbent assay (ciELISA).【Method】Artificial antigen BSA-FLE and OVA-FLE were synthesized using DCC and the mixed anhydride reaction methods by linking carrier proteins BSA and OVA to FLE. The antigens BSA-FLE and OVA-FLE were identified by ultraviolet scanning and SDS-PAGE, then five female white rats were subcutaneously immunized with the immunogen Fle-BSA at multiple sites in the back. The initial immunization was subcutaneously injected with 189 μL of conjugate in 311 μL of PBS (0.01 mol•L-1, pH 7.4) and 0.5 mL of Freund’s complete adjuvant. The rest of five booster immunizations were conducted by injecting 189 μL of FLE-BSA in 311 μL of PBS (0.01 mol•L-1, pH 7.4) and 0.5 mL of iFA at 20-day intervals. After obtained mice polyclonal antiserum, the ciELISA method and rapid detection kit of FLE were developed. The kit was compared with HPLC method to ensure its quality.【Result】The ultraviolet scanning and SDS-PAGE showed that FLE artificial antigen was synthesized successfully. Five BALB/c mice indirect ELISA titer against FLE were all above 2.5×104 and the IC50 of No.4 mice was the lowest (162.18 ng•mL-1). After optimized the ELISA method, the icELISA revealed that the optimal concentration of mice serum was 1:6400, the optimal concentration of coated antigen was 5 µg•mL-1, and the optimal concentration of sheep anti-rabbit IgG was 1:1000. The regression equation was Y =-0.3627x + 1.3517（R2 = 0.9956), the lowest detection limit of the kit was 16.22 ng•mL-1, the assay measured drug residue in pork liver spiked with FLE with coefficient of variation between 9.34%-10.7%, and the average recovery rates between 67.5%-87.9%, respectively. The assay measured drug residue in pork meats spiked with FLE with coefficient of variation between 8.3%-10.4%, the average recovery rates were between 74%-88.2%. Good agreement of the results obtained by ELISA and high performance liquid chromatography (HPLC) further confirmed the reliability and accuracy of the icELISA for rapid detection of SPFX in pork meats. The coefficients of variation of intra-assay and inter-assay were 5.07% and 7.44% during five standard concentrations. The antiserum of FLE had no cross reactivity with other competitors.【Conclusion】The FLE polyclonal antiserum has been generated. The icELISA method and rapid detection kit were developed for the detection of FLE residues with the characters of sensitivity, accuracy, convenience and rapid in this study and they laid a foundation for establishing immunoassay of FLE residues.

Cloning and Expression Analysis of Two Novel PPR Genes in Gossypium hirsutum L.

HE Peng, HUANG Peng, HUANG Sheng, QIAN Hui, YU Jia-Ning

Scientia Agricultura Sinica. 2014, 47(11): 2271. doi:10.3864/j.issn.0578-1752.2014.11.020

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【Objective】 The aim of this study was to clone two novel PPR genes in cotton. Their sequence characteristics were investigated, and their expression in different tissues was analyzed and subcellular localization was identified. All these results could provide a support for further studying PPR genes function of cotton. 【Method】 Two PPR genes were cloned on the basis of ESTs from cotton using RT-PCR techniques. The bioinformatics method was used to analyze the putative amino acid sequence, semi- quantitative RT-PCR and real-time PCR were used to analyze the expression of target genes in tissues and different development periods of fiber. Transient expression system of cotton cotyledons was used to analyze subcellular localization. 【Result】 GhPPRH1 and GhPPRH2 belonged to PLS subfamily of PPR gene family. The full length ORF of both genes is 1 917 and 2 556 bp, encoding 638 and 851 amino acids, respectively. GhPPRH1 has 18 PPR motifs, including one E+ domain and DYW structure. GhPPRH2 has 17 PPR motifs with one E domain, E+ domain and DYW structure. Phylogenetic analysis of homologue PPR proteins of other plants and five cotton PPR proteins showed that GhPPRH1 has a close relationship with OsRF1b of rice. It implied that GhPPRH1 may involve in fertility restoration. Whereas GhPPRH2 might relate to RNA editing because of clustered together with ZmPPR5 of Zea mays. The expression pattern of GhPPRH1 and GhPPRH2 can be found in root, stem, leaf, petal and different development periods of fibers, and GhPPRH1 has relatively higher expression level in root, however high expression level of GhPPRH2 was observed in root, leaf and 15 DPA fibers. Subcellular localization showed that green fluorescence of GhPPRH1 was merged well with red fluorescence of mitochondria marker; green fluorescence of GhPPRH2 was merged very well with chloroplast autofluorescence, which indicated GhPPRH1 and GhPPRH2 might locate in mitochondria and chloroplast, respectively.【Conclusion】Two novel PPR genes were cloned and characterized from Gossypium hirsutum, which belonged to the typical PLS subclass family of PPR. The results of gene expression pattern and subcellular localization implied that two novel genes might participate in organelles RNA processing and modification.

Construction and Analysis of a Full-Length cDNA Library of Peanut Embryos at Different Developmental Stages

CHEN Hua-1, 2 , DENG Ye-1, 2 , ZHANG Chong-1, CAI Tie-Cheng-1, 2 , ZHENG Yi-Xiong-1, 2 , 3 , ZHUANG Wei-Jian-1, 2

Scientia Agricultura Sinica. 2014, 47(11): 2272-2280. doi:10.3864/j.issn.0578-1752.2014.11.021

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【Objective】 The objective of this study is to understand the molecular mechanism of peanut embryo development and obtain important genes related to peanut embryo development. 【Method】 Using peanut variety Minhua 6 as the experimental material, embryos on 10, 20, 30, 40, 50, and 60th day after pegging were sampled. Total RNA was extracted by improved CTAB method. Double strand cDNA was synthesized based on SMART technique. The purified dscDNA was ligated to pDNR-LIB vector digested by SfiⅠ and transformed into DH5α by electroporation to construct a full-length cDNA library of peanut embryos at different developmental stages. Bioinformatics analysis was performed following small-scale EST sequencing.【Result】A successful full-length cDNA library of peanut embryos at different development stages was constructed. The titer of unamplified cDNA library was about 3.5×106cfu/mL. The average cDNA inserts were more than 1 000 bp with a recombination frequency of 95.8%. Small-scale plasmid extraction and subsequent sequencing resulted in 60 ESTs, which were used for further analysis. BLASTX analysis showed that 39 sequences (65% of total sequences) had high similarity with reported genes in Glycine max, Arachis hypogaea, Medicago truncatula, etc. on NCBI with 32 sequences having known or putative functions and functions of other 7 sequences were unclear. The other 21 (35% of total sequences) could not find similarity with known genes in NCBI, which may be novel genes for peanut. GO annotation was performed with BLAST2GO software and the results revealed that the ESTs generated in this study mainly included responsive to stresses and defenses, protein synthesis and transport, lipid synthesis and metabolism, transcription and regulation, seed germination, dormancy and embryo development related genes. Besides, some genes were involved in signal transduction and light morphogenesis process. KEGG pathway analysis showed that the ESTs generated by randomly sequencing in this study mainly involved in alpha-linolenic acid metabolism and linoleic acid metabolism.【Conclusion】A high-quality full-length cDNA library of peanut embryos was constructed successfully using SMART method and some genes related to peanut embryo development were got, such as linoleate 9S-lipoxygenase-4-like, oleosin, zinc finger protein, heat shock protein 90, late embryogenesis abundant protein group 2, lipoxygenase-10, and putative DREB transcription factor, etc.

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