Scientia Agricultura Sinica

Isolation and Functional Analysis of Phosphate-Responsive Gene OsRCI2-9 in Oryza sativa

REN Hong-yan, WEI Qiang

Scientia Agricultura Sinica. 2015, 48(5): 831-840. doi:10.3864/j.issn.0578-1752.2015.05.01

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【Objective】 The objective of this study is to analyze the function of OsRCI2-9 gene and reveal its mechanism of tolerance to low phosphorus stress.【Method】By comparative analysis of transcription profiles in rice transgenic plants with over expression (OsPHR2 (O)) and repression (OsPHR2 (Ri)) of OsPHR2 (the Pi-signaling regulator), OsRCI2-9 gene which belongs to RCI2 family induced under the OsPHR2 (O) background, was isolated. Firstly, the AffyMatrix data were validated by real-time PCR. The primers were designed according to the sequence on the TIGR web and its full length cDNA was amplified and the DNAStar was used to find its open reading frame. The transmembrane domain were predicted using the TMHMM2.0 software. Then the protein sequence was used to search the homologs in Arabidopsis thaliana and Zea mays from Phytozome 9 database by BLASTP program. Multiple alignment and homology analysis were performed among OsRCI2-9 and RCI2s in A. thaliana and Z. mays by usingClustal X 1.8 software and these sequences were used to construct the maximum likelihood tree by using MEGA 5.2.1. The cis-regulatory elements in promoter were searched in PLACE database. Also the expression patterns of OsRCI2-9 under various abiotic stresses were analyzed using the RT-PCR and the real-time PCR. Finally, the transgenic plants with overexpression of OsRCI2-9 driven by CaMV35S promoter was developed for phosphate starvation resistant experiment and the available phosphate were detected.【Result】OsRCI2-9 is located on chromosome 6 of O. sativa. The full-length of OsRCI2-9 was 237 bp with 2 exons and 1 intron and consisted of 78 amino acids. It was observed that the OsRCI2-9 protein has two transmembrane domains. Multiple alignments and homology analysis showed that it has a high sequence similarity with ZmRCI2-2, ZmRCI2-7 and ZmRCI2-8. It was also found that the OsRCI2-9 promoter has many cis-acting elements including the dehydration, cold, ABA, cytokinins, gibberellin signaling and phosphate starvation responsive elements. It was further revealed that the expression of OsRCI2-9 is up-regulated by phosphate starvation and also slightly up-regulated by potassium and iron starvation but down-regulated by nitrogen starvation in roots. Further analysis showed that OsRCI2-9 overexpressed transgenic plants were growing slowly and its tiller number was reduced compared to wild type under the full phosphate conditions, but no significant difference between them were detected under the phosphate starvation conditions. Measurements of the available phosphate showed that the OsRCI2-9 overexpressed transgenic plants significantly increased the soluble phosphate concentration both under the full phosphate conditions and the phosphate starvation conditions.【Conclusion】 The OsRCI2-9 gene significantly participates in the regulation of phosphorus homeostasis in O. sativa.

The Response of a Putative Maize Zinc-Finger Protein Gene ZmAN14 in Transgenic Tabacco to Abiotic Stress

XUAN Ning, LIU Xu, ZHANG Hua, CHEN Gao, LIU Guo-xia, BIAN Fei, YAO Fang-yin

Scientia Agricultura Sinica. 2015, 48(5): 841-850. doi:10.3864/j.issn.0578-1752.2015.05.02

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【Objective】 ZmAN14 is a member of the maize A20/AN1 zinc finger protein gene family. This gene family of rice is involved in the response to abiotic stress. The expression of ZmAN14 in the maize inbred line H21 and transgenic ZmAN14 tobacco under abiotic stress was analyzed. The results will provide novel information for the comprehensive analysis of the functional and molecular mechanisms of maize ZmAN14 and the entire gene family. 【Method】 Sequence analysis of ZmAN14 was performed to confirm that this gene is a member of the maize A20/AN1 zinc finger protein gene family. The experiment was conducted by using maize inbred line H2. At the three-leaf stage, the seedlings were treated with multiple abiotic stress or induced by ABA. After treatment for 0, 1, 3, 6, 12 and 24 h, the whole plants were harvested. In the meantime, the roots, stems, leaves, coleptiles, pistil, stamen, silks and bract leaves were harvested from different growing periods of maize. Real-time quantitative fluorescence PCR was used to analyze the expression profile in different tissues and abiotic stress response of ZmAN14 and its ABA-induced expression profile. The cis-acting element of the promoter area was cloned to perform the comparative analysis. The ZmAN14 coding sequence was cloned onto the GFP expressing vector pMDC85. A subcellular localization method was used to verify the localization of the ZmAN14 protein in the cell. The ZmAN14 coding sequence was cloned onto the GAL4 DNA-binding domain vector. This vector was used to transform yeast. The transformed yeast was spotted onto the defect medium, and the resulted transcriptional activation activity was analyzed. The ZmAN14 coding region was ligated to the plant expression vector p1300-221 to create an overexpression vector. The overexpression vector was transformed into tobacco. T2 homozygous transgenic lines with high ZmAN14 mRNA expression level were used to conduct salt-, drought- and ABA induced experiments, and its response to abiotic stress was observed. 【Result】Sequence analysis showed that the ORF was 516 bp in length, encoding a polypeptide of 171 amino acid residues with an estimated molecular mass of 18.3 kD and a pI of 8.28. The ZmAN14 gene, which contains the A20 and AN1 domains, belongs to the maize A20/AN1 zinc finger protein gene family. Results of the real-time quantitative fluorescence PCR showed that ZmAN14 was abundantly expressed in leaves, and its expression increased under NaCl-, drought-, and ABA-induced stresses. The ZmAN14-GFP fusion proteins were localized in the cytoplasm and nucleus, which was similar to the localization of ZNF216 and ZmAN13, but the ZmAN14 gene itself did not produce a nuclear localization signal. Therefore, ZmAN14 performed its biological function by entering the nucleus together with other proteins. The ZmAN14 transgenic yeast was not grown on the SD/-Leu-His defect medium. No transcriptional activation activity was found when ZmAN14 was expressed in yeast. The over-expression of ZmAN14 in tobacco increased plant resistance to salt and drought at seedling stage. 【Conclusion】The ZmAN14 gene belongs to the maize A20/AN1 zinc finger protein gene family. The ZmAN14 was abundantly expressed in leaves and the increase of its expression level was induced by abiotic stress and ABA. The overexpression of ZmAN14 in tobacco increased plant resistance to salt and drought stresses under abiotic stress and ABA induction at the seedling stage. ZmAN14 might function via its interaction with other proteins. Compared with other A20/AN1 zinc finger proteins, ZmAN14 participated in the response to abiotic stress. ZmAN14 and ZmAN13 showed high homology, but these genes had different control mechanisms (in the opposite directions) in terms of response to salt and drought stresses.

Molecular Characteristics and Functional Identification of Foxtail Millet Transcription Factor WRKY36

ZU Qian-li, YIN Li-juan, XU Zhao-shi, CHEN Ming, ZHOU Yong-bin, LI Lian-cheng, MA You-zhi, MIN Dong-hong, ZHANG Xiao-hong

Scientia Agricultura Sinica. 2015, 48(5): 851-860. doi:10.3864/j.issn.0578-1752.2015.05.03

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【Objective】 Abiotic stresses seriously affect the plant growth development and the crop yield. WRKY transcription factors play a key role in the regulatory mechanism. This study provided experimental data for further study of the molecular characteristics and the function of SiWRKY36 gene. 【Method】 SiWRKY36 gene was isolated from the drought-treated foxtail millet transcriptome profile. Bioinformatics methods were used to analyze the molecular properties of the SiWRKY36 gene. Homologous sequences of SiWRKY36 were selected from the Phytozome program. Homologous analysis and multiple alignments were performed with MEGA 5. MEME and SMART online tools were used for protein sequence analysis. GSDS and PHYRE2 online tools were used to analyze gene structure and tertiary structure of SiWRKY36, respectively. Quantitative real-time PCR (qRT-PCR) was used to examine the expression pattern of SiWRKY36 gene in different abiotic stresses and phytohormone treatments. Full length SiWRKY36 cDNA was ligated into the vector to construct an expression vector for wild-type Arabidopsis plant. The pBI121-SiWRKY36 expression vector was introduced into Agrobacterium tumefaciens strain cells. Transgenic plants were used for resistance identification experiment. 【Result】 SiWRKY36 shared a high similarity with switchgrass and belonged to Group Ι of WRKY family including two WRKYGQK conserved domains. The predicted tertiary structure of SiWRKY36 contained two alpha helix and three beta folds. SiWRKY36 was mainly located in nucleus. The expression pattern showed that SiWRKY36 was involved in responses to various abiotic stresses and exogenous hormones, which was similar with the promoter analysis. The total root length, root surface area and root volume were slightly different between three SiWRKY36 lines and wild-type plants in drought treatment (2% PEG) in Arabidopsis plants. 【Conclusion】 SiWRKY36 transgenic plants have a certain resistance under mild drought conditions.

Effects of Temperature-Light Factor on Cotton Fiber Qualities at Different Spatial Fruiting Branch Positions

ZHANG Xin-xin, CHEN Ji, LIU Jing-ran, Lü Feng-juan, MA Yi-na, WANG You-hua, ZHOU Zhi-guo, CHEN Bing-lin

Scientia Agricultura Sinica. 2015, 48(5): 861-871. doi:10.3864/j.issn.0578-1752.2015.05.04

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【Objective】The objective of this study is to provide a theoretical basis for taking suitable cultivation measures to reduce the decrease extent of fiber quality caused by inappropriate temperature and solar radiation during flowering and boll-forming stage.【Method】The field experiment was carried out in Nanjing with two cotton cultivars (temperature-insensitive cultivar Kemian 1 and temperature-sensitive cultivar Sumian 15). Three sowing dates (25-Apr, 25-May and 10-Jun) and three relative light rates (CRLR100%, 80%, and 60%) were set, thus cotton fiber development and the quality formation can be arranged at different temperature-light (PTP) conditions and different fruiting branch positions.【Result】During the flowering and boll-forming stages, difference in cotton fiber quality was mainly caused by mean daily temperature, mean daily maximum temperature, mean daily minimum temperature and photosynthetic active radiation, which were all reduced with the increase of fruiting branch or fruiting position. Micronaire was the most sensitive fiber quality index to the change of meteorological conditions, followed by fiber strength and fiber length. Cotton fiber length, strength and micronaire were quadratic with PTP. The theoretical maximum value of fiber quality at the 1st and 2nd fruiting positions was highest on the middle fruiting branches (6th to 8th node), followed by the low fruiting branches (2nd to 4th node) and the upper fruiting branches (10th to 12th node); and they were higher than those at the 3rd or greater fruiting positions. Range of optimum PTP for greater fiber quality (reach A grade and AA grade) was broader on the 1st and 2nd fruiting positions of the 6th to 8th fruiting branches. And the range of optimum PTP for fiber quality of temperature-sensitive cultivar was smaller than temperature-insensitive cultivar. Fiber strength was the main restrictive factor to achieving fiber quality with A grade for a smaller range of optimum PTP. Fiber strength and micronaire were the restrictive factors to achieving fiber quality with AA grade because that ranges of optimum PTP for fiber strength and micronaire with AA grade were different. The fiber quality reached A gradewhen PTP was in183.5-633.7 MJ?m^-2 (Kemian 1) and 229.0-589.6 MJ?m^-2 (Sumian 15) and reached AA gradewhen PTP was in 304.7-452.9 MJ?m^-2 (Kemian 1) and 346.6-357.8 MJ?m^-2 (Sumian 15). The CRLR60% treatment of 10-Jun did not reach A grade due to the lack of PTP. And among all of the treatments, only CRLR80% and CRLR60% treatments of 25-Apr and CRLR80% treatment of 25-May reached AA grade.【Conclusion】 Fiber quality indexes have different optimum PTP ranges, and fiber strength and micronaire are the restrictive factors to achieving quality cotton with A grade and AA grade. Delayed the sowing date (25-May) and lowed the light appropriately are benefit to formation high fiber quality.

Effects of Water and Nitrogen Interaction on Peanut Root Growth and Yield

DING Hong, ZHANG Zhi-meng, DAI Liang-xiang, YANG Ji-shun, CI Dun-wei, QIN Fei-fei, SONG Wen-wu, WAN Shu-bo

Scientia Agricultura Sinica. 2015, 48(5): 872-881. doi:10.3864/j.issn.0578-1752.2015.05.05

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【【Objective】The aim of this study was to clarify the effect of nitrogen fertilizer on root growth and yield of peanut varieties differing in drought tolerance under different water conditions and to provide a theoretical basis for the management of water and fertilizer in peanut.【Method】The drought-resistant variety Huayu 22 and drought-sensitive variety Huayu 23 were planted in the anti-canopy tanks using the soil column. The soil water condition had two levels: well-watered conditions (W1) and medium drought (W0) (corresponding soil water contents are 70%-75% and 45%-50% of field capacity, respectively). The nitrogen had three levels: no nitrogen (N0), moderate nitrogen (N1, 90 kg×hm^-2) and high nitrogen ((N2, 180 kg×hm^-2). The root morphological characters, root exudates and yield of peanut were investigated under different treatments. Root samples were collected from 0-20 cm, 20-40 cm and below 40 cm soil layers, respectively. Root length, root surface area and volume were determined by a scanner and analyzed by WinRhizo Pro Vision 5.0a software. 【Result】 The effects of nitrogen application on root development of peanut varieties with different drought resistance were different under different water conditions. Compared with no nitrogen fertilizer, the total root length, total root surface area and the root length and root surface area in 0-20 cm soil layer of Huayu 22 (drought resistant peanut variety) were decreased by application of nitrogen fertilizer under drought stress, while the root biomass, root length and root surface area in the soil layers below 40 cm were increased. The nitrogen fertilizer treatment decreased root biomass, root length and root surface area in 0-20 cm soil layer of Huayu 22 under well-watered conditions, and the root system traits in the soil layer below 40 cm were increased. The root response of Huayu 23 (drought sensitive peanut variety) to water and nitrogen treatment was different from Huayu 22. The total root biomass, total root length, root length and root surface area in the soil layer below 40 cm of Huayu 23 were increased by application of nitrogen fertilizer under drought stress, while the root length and root surface area in the soil layer below 40 cm were decreased under well-watered conditions. The response of root exudates to water and nitrogen in different peanut varieties were consistent. Furthermore, the root exudates of two peanut varieties were decreased under drought stress, and the decreased amplitude of Huayu 23 was larger. Compared with no nitrogen fertilizer, application of nitrogen fertilizer increased root exudates in both peanut varieties under drought stress. The peanut yield of both varieties were increased by application of nitrogen fertilizer under drought stress. The yield of Huayu 22 was increased by application of nitrogen under well-watered condition, however, no significant difference was observed for Huayu 23. And the effects of water and nitrogen interaction on peanut yield were at the significance level in two years. The correlation coefficient between root length and root surface area in the soil layer below 40 cm with yield reached the significance or extremely significant level under drought stress. Under well-watered condition the yield was significantly correlated with the root surface area in 20-40 cm soil layer. The correlation coefficient between root exudates and yield were significant under drought stress and well-watered conditions.【Conclusion】Under drought stress, the root biomass, root length and root surface area of peanut in the soil layer below 40 cm, and root exudates increased by applying nitrogen fertilization. Moreover, root growth was well improved, and that the peanut yield increased simultaneously.

Isolation and Purification of Active Compound from Trichoderma viridescens and Its Inhibitory Activities Against Phytopathogens

ZHANG Liang, ZHANG Jing-ze

Scientia Agricultura Sinica. 2015, 48(5): 882-888. doi:10.3864/j.issn.0578-1752.2015.05.06

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【Objective】An antagonistic compound against Phytophthora spp. was obtained from Trichoderma spp. and its biocontrol potential was evaluated for controlling phytopathogens, thus providing a scientific basis for utilization of biocontrol strains and their antagonistic compounds. 【Method】 Trichoderma strains producing antagonistic compounds were screened by the cellophane method and grew on PDA as inoculants, and then were transfered into rice media for isolation of antagonistic compounds. The cultures of the Trichoderma strains from the rice media were extracted, filtered and concentrated, and initial crude extract was obtained. The crude extract was further purified by column and thin-layer chromatography and the active fractions were ascertained by the biological activity determination. Based on the chemical property of the active fraction obtained, their chemical structure was identified by analysis of gas chromatography-mass spectrometry (GC-MS). The biological activity of different types of plant pathogens was determined with the antagonistic compound obtained, including the oomycete P. capsici and P. melonis, ascomycete Fusarium oxysporum and basidiomycete Rhizoctonia solani. 【Result】 The initial screening assays showed that the strain TS0404 of T. viridescens produced an antagonistic compound with strong inhibitory activity against P. capsici. The results of isolation, purification and bioactivity determination displayed that the active fraction obtained was yellow oily liquid. Mass spectra revealed the biggest ion peak of active fraction was 166, which was identified as 6-pentyl-2H-pyran-2-one (6-PP). The bioactive determination demonstrated that the 6-PP had significant inhibitory activity against hyphal growth of P. capsici, P. melonis, R. solani and F. oxysporum (EC50 115.26, 99.58, 126.46 and 315.75 μg?mL^-1, respectively) but the inhibitory effect on P. melonis was the best among them and hyphal growth was completely inhibited when its concentration reached 300 μg?mL^-1. Similarly, the 6-PP had a conspicuous inhibitory activity against zoosporangial germination of P. capsici and P. melonis but the inhibitory effect on P. melonis was the best and zoosporangial germination of P. melonis was completely inhibited at 400 μg?mL^-1 level. In addition, the 6-PP had significant inhibitory effect on conidial germination of F. oxysporum (EC₅₀ 151.81 μg?mL^-1) and sclerotial germination of R. solani (300 μg?mL^-1 for complete inhibitory concentration). 【Conclusion】 The 6-PP is an antagonistic compound isolated from T. viridescens, which showed strong inhibitory activities against the fruiting bodies of P. melonis, P. capsici, F. oxysporum and R. solani. It was concluded that the 6-PP is a type of broad-band antagonistic compound, which has great potential values of application in biocontrol.

Role of Extracelluar Enzymes of the Entomopathogenic Fungi During Degrading the Integument of Two Species of Scale Insects

DONG Jing, XIE Ying-ping, LIU Wei-min, NIU Xiu-ping, XUE Jiao-liang

Scientia Agricultura Sinica. 2015, 48(5): 889-899. doi:10.3864/j.issn.0578-1752.2015.05.07

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【Objective】The objective of this study is to explore the effect of extracellular enzymes and their virulence during entomopathogenic fungi degrading the integument of scale insects and to provide evidence for biological control by applying entomopathogenic fungi. 【Method】 The strains of the entomopathogenic fungi, Lecanicillium lecanii V3.4505, V3.4504, L. fungicola HEB02, and Fusarium incarnatum-equiseti HEB01 were used and the two species of scale insects, Ceroplastes japonicus Green and Rhodococcus sariuoni Borchsenius, as the target were studied. The cuticle of the two scale insects was used as the sole carbon source in the medium for fungal culturing. The activities of four extracellular enzymes, including lipase, protease (Pr1), chitinase, and N-acetyl-D-glucosaminidase (NAGase), produced by the four strains were determined, and based on this, the function of the extracelluar enzymes during the strains penetrating the integument of scale insects was analyzed. Meanwhile, the cumulative mortalities of the two scale insects infected with the four strains for eight days were assayed to evaluate the virulence of the strains and the extracellular enzymes. 【Result】 The activities of the four enzymes all changed significantly during the scale insect integument-degrading process, their changing trend all exhibited rising in the first few days and falling in the later. The activity peak of lipase appeared earliest that was at two days after inoculation, and the lipase activity of the strains cultured on the cuticular medium of C. japonicus was obviously higher than that on R. sariuoni. The activity peak of Pr1 came after 3-4 d while the activity peak of chitinase and NAGase appeared at 6 d and 4-5 d, respectively. Among the four strains, V3.4505 strain caused the highest mortality rate of 73% for C. japonicus and 81% for R. sariuoni, respectively, and that showed significant difference with other two strains, HEB02 and HEB01. The average values of Pr1 activity of four strains were significantly related to the cumulative mortality of the two scale insects, and the linear equations were y=0.082x+5.822 (R²=0.823) for C. japonicas and y=0.119x+14.75 (R²=0.764) for R. sariuoni. Similarly, the average chitinase activity of four strains were also significantly related to the cumulative mortality of the two scale insects, the linear equations were y=-0.148x+15.89 (R²=0.645) for C. japonicas and y=0.095x+10.46 (R²=0.762) for R. sariuoni. 【Conclusion】 The four extracellular enzymes worked together to degrade the wax and the integument of the scale insects in the infecting process. The lipase degraded the wax on the scale insects and its peak came earliest, and its activity of the four strains cultured on the cuticular medium of C. japonicus was obviously higher than that on the R. sariuoni because C. japonicus has a thicker wax layer. Among other three enzymes, Pr1 worked first to degrade the protein in the procuticle of scale insects then chitinase and NAGase worked together to degrade the chitin in the integument. The activity peak and level of each enzyme were corresponding to the distribution and content of its substrate in the integument of scale insects. The correlation analysis on the enzyme activities of the strains and mortality of the scale insects indicated that Pr1 and chitinase should be used as a virulence indication for the strains. Among the four strains, L. lecanii V3.4505 showed better pathogenic characteristics, which means this strain possesses higher virulence against scale insects and it can be considered as a better pathogenic fungus for biological control.

Key Research Priorities for Agricultural Land System Studies

TANG Hua-jun, WU Wen-bin, YU Qiang-yi, XIA Tian, YANG Peng, LI Zheng-guo

Scientia Agricultura Sinica. 2015, 48(5): 900-910. doi:10.3864/j.issn.0578-1752.2015.05.08

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Agricultural land use and its dynamics have attracted much attention from researchers due to their ecological and socio-economic implications for agricultural sustainability. Several international programs such as the Land-Use and Land-Cover Change (LUCC) and the Global Land Project (GLP) have promoted the emergence of Land System Sciences. Based on the latest progress in Land System Science, this review paper provides a definition of the Agricultural Land System (ALS) and conceptualizes a framework for the ALS studies relating to global change, food security, and sustainability studies. It is proposed that: 1) Multi-faceted patterns of ALS are the basis for subsequent analysis. It should consider not only the characteristics ALS at the land use and land cover level, e.g. the transitions between cropland and other land cover types, but also the characteristics of cropping system, crop allocation, intensification and productivity within cropland. Interdisciplinary approaches and data integration are necessary for understanding the complex characteristics of ALS. 2) Multi-model coupling through the interpretation and intercorrelation of ALS patterns and underlying drivers is an essential way to represent ALS dynamic changes, processes and its mechanisms, by which it is able to better understand the coupled human-environment interactions across different time, space and scales. 3) It is important to link the ALS with other parallel systems to understand their synergies and trade-offs, in order to build up a sustainable pathway for future agricultural land use. Those solutions for ALS studies would substantially promote the interdisciplinary integration and will contribute to the development of Land System Science and its relevant sciences.

Effect of Different Fertilization Regimes on the Main Groups of Soil Fauna in Cropland of Purple Soil

ZHU Xin-yu, ZHU Bo

Scientia Agricultura Sinica. 2015, 48(5): 911-920. doi:10.3864/j.issn.0578-1752.2015.05.09

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【Objective】Management of cropland, especially long-term fertilization, can change the species and quantity of plant residue and root exudates, which will affect the soil fauna community composition. To investigate the effect of different fertilization regimes on the main groups of soil fauna, the response of main groups of soil fauna to changes of soil fertility and the relationships between main groups of soil fauna and soil properties were studied in the cropland of purple soil.【Method】Taking the Yanting Agro-ecological Experimental Station of Purple Soil, Chinese Academy of Sciences as a research area, modified Tuggren, Baermann and hand-sorting methods were used to investigate the main groups of soil fauna (nematode, earthworm and oribatida) in different long-time fertilization croplands. Correlation analysis, multiple regression analysis and canonical correlation analysis were used to clarify the relationships between the main groups of soil fauna and soil fertility variables. The long-term fertilization experiment was conducted with a no fertilizer control (CK) and five fertilization regimes: NPK (synthetic fertilizer: nitrogen, phosphorus and potassium), OM (pig manure), OMNPK (pig manure plus nitrogen, phosphorus and potassium), RSD (crop residues returned) and RSDNPK (crop residues returned with nitrogen, phosphorus and potassium).【Result】The number of individuals of nematode, earthworm and oribatida in the OMNPK and RSDNPK treatments were significantly higher than in the NPK treatment (P＜0.05). The highest number of total individuals of the three main soil fauna was found in the RSDNPK, and was significantly higher than in the other fertilization regimes (P＜0.05). Statistical analysis showed that soil organic matter (SOM), soil microbial biomass carbon (MBC) and nitrogen (MBN) and available potassium (AK) were significantly correlated to the individuals of soil nematode, earthworm and oribatida (P＜0.05). The soil fertility properties were well explained by the individuals of soil nematode, earthworm and oribatida, with the explained percent of 78.03%，80.82% and 50.86%, respectively. Thus, the individuals of soil nematode, earthworm and oribatida can be applied to indicate certain characteristics of soil fertility.【Conclusion】The application of organic fertilizers promoted the number of individuals of nematode, earthworm and oribatida due to the abundant organic matter the fertilizers supplied for the survival and development of soil fauna. Organic-inorganic compound fertilizers were beneficial to the total individuals of the three main soil fauna, especially in the RSDNPK regime. The individuals of soil nematode, earthworm and oribatida can be used for indicating certain characteristics of soil fertility such as soil organic matter content. However, they cannot be used to quantify the integrated characteristics of soil fertility.

Tissue Location and Protein Expression Analysis of Auxin Binding Protein ABP1 in Peach Fruit (Prunus persica L.)

YU Jia, LI Yang, GONG Shuo, GUAN Wei, LIU Yue-ping

Scientia Agricultura Sinica. 2015, 48(5): 921-930. doi:10.3864/j.issn.0578-1752.2015.05.10

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【Objective】Auxin is almost involved in all aspects of plant development regulation, and ABP1 (Auxin binding protein 1) is established as a crucial component of auxin signaling．ABP1 as a kind of auxin fast response protein participates in a series of physiological processes such as regulation of fruit development. It is distributed mainly in the developing embryo and surrounding tissue of embryo. The purpose of this paper is to study whether ABP1 plays a role in peach fruit development and explore its expression characteristics and hence to provide a theoretical foundation for the further research．【Method】The peach ‘No.24’ was used as experimental material. Three periods of development were divided through the assay of growth curve. The fruits from different stages were separated into mesocarp, endocarp and seeds, small tissue was cut from seed and mesocarp, then put them into the EDAC solution, fixed by glutaraldehyde and paraformaldehyde after vacuumizing. The fixed samples continued dehydrated, paraffin embeded, and then used in paraffin section. The other samples which was used for extracting protein immediately froze in liquid nitrogen, then stored at -80℃. ABP1 rabbit polyclonal antibody was made successfully. The immunohistochemical localization technology was used to analyze the ABP1 distribution in seed and mesocarp at different development stages of peach fruit．The protein expression in mesocarp, endocarp and seed was analyzed by Western blot．【Result】According to peach fruit growth curve, it could be divided into three periods that are the first fast growing period, the hardcore period, and the second fast growing period. The results of immunohistochemical localization showed that ABP1 was distributed in both seed and mesocarp at all stages. ABP1 was evenly distributed in different parts of the seeds of all stages, the signal had no difference among different parts of seed at the same development stage. The stronger signals were detected in cells of outer and inner integument and around vascular tissue of seed coat. In the whole growth period, ABP1 signal did not significantly change. Between the outer and inner integuments, there were sporadic, zonal signals. The expression of ABP1 was around vascular tissue in mesocarp during the hardcore period of fruit development. The level of ABP1 expression was higher at the first rapid growth period（46DAFB）and the start of second rapid growth period (76DAFB) according to Western blot result．Its expression level was the lowest at 39 DAFB. In different tissues, the expression of ABP1 in the mesocarp and endocarp were lower than that in seed．【Conclusion】The distribution and expression level of ABP1 have a difference in tissue and at developmental stage in peach fruit, ABP1 plays an important role in peach fruit development.

Study on the Origin of Tree Peony Cultivars from Southwest China Based on ISSR Technology

LI Zong-yan1, QIN Yan-ling1, MENG Jin-fang2, TANG Dai1, WANG Jin1

Scientia Agricultura Sinica. 2015, 48(5): 931-940. doi:10.3864/j.issn.0578-1752.2015.05.11

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【Objective】 Forty-one tree peony samples consisting of 21 from Xinan group, 18 from Zhongyuan group, 1 from Jiangnan group and 1 wild species were used to detect their genetic diversity and verify their phylogenetic relationship and discuss their genetic backgrounds for the new cultivars breeding. 【Method】Total genomic DNA was extracted from the fresh leaves of tree peony by a modified CTAB method. A total of 27 primers were selected from 60 ISSR universal primers designed by UBC on the basis of the establishment of an optimal ISSR-PCR reaction system. They were used in the PCR amplification to compare the genetic difference among different cultivars. Comparative analysis on DNA fragments amplified by ISSR-PCR technology was made by the relative genetic software. Genetic parameter such as percentage of polymorphic loci (PPB) was calculated by using POPGENE 32. Cluster analysis (UPGMA) and dendrogram based on Nei’s genetic distance were made to construct the relationship between cultivars and cultivars groups by using the Numerical Taxonomy Multivariate Analysis System (NTSYS-pc) ver. 2.1 statistical package.【Result】A total of 317 bands were obtained by amplifying 41 cultivars from 27 primers, among which 304 bands were polymorphic and percentage of polymorphic bands (PPB) attained to 95.41%. In average, 11.74 bands were produced by each primer. The genetic similarity coefficients among all the tested 41 samples ranged from 0.483 to 0.811, which ‘Daguanfen 4’ and ‘Lijiangzi 5’ had the highest similarity coefficient with 0.811, otherwise ‘Daguanfen 4’and ‘Caihui’ had the lowest coefficient with 0.483. The 41 cultivars were divided into four branches based on UPGMA cluster at the coefficiency of 0.625. The first branch was composed of 19 samples, which included 5 Zhongyuan peony cultivars and 14 Xinan cultivars, the genetic similarity coefficient of ‘Lijiangzi 5’ and ‘Daguanfen 4’ had the closest phylogenetic relationship, ‘Lijiangzi 5’ and ‘Shouanhong’ had the farthest phylogenetic relationship with a genetic similarity coefficient of 0.609; The second branch included 10 samples in which there were 5 Zhongyuan cultivars and 9 Xinan cultivars. The closest phylogenetic relationship existed between ‘Zijinhe 2’ and ‘Sichuanfenzi’ with a genetic similarity coefficient of 0.770. However, the farthest phylogenetic relationship was between ‘Zijinhe 2’ and ‘Luowuxianrui’,with the minimum genetic similarity coefficient at 0.625; The third branch contained 11 samples: one Jiangnan cultivar, 7 Zhongyuan cultivars and 3 Xinan cultivars. The closest phylogenetic relationship was between ‘Linghuazhanlu’ and ‘Zhushalei’ with the genetic similarity coefficient of 0.779. The farthest genetic relationship was between ‘Zhushalei’ and ‘Fendangbai’. The fourth branch involved only one wild species of Paeonia lutea. The result showed that most of the Zhongyuan cultivars with similar flower color had the closer relationship. ‘Yanzhilou’ belonged to Tianpeng cultivars with red flower had the much more closer genetic relationship with the Zhongyuan cultivars bloomed in dark red flower, while other Tianpeng cultivars with dark red flower had the closer genetic relationship with Zhongyuan cultivars bloomed in purplish red, pink, purple and red. Yunnan cultivar with purple flower had a certain genetic relationship with Zhongyuan cultivars bloomed in purple and purplish red flower. But other pink flower cultivars had some certain genetic relationships with Zhongyuan cultivars in purplish red, light red, light purplish red and light purple flower color. Tianpeng cultivars firstly clustered with Zhongyuan cultivars and then with Yunnan cultivars. While Yunnan cultivars except ‘Shishanhuanguan’ and ‘Xianyuban’ gathered together firstly and then they clustered with Zhongyuan cultivars, which Yunnan cultivars from different places with similar and same flower-color had the closer genetic relationship. 【Conclusion】The origin of Xinan cultivar group is complicated. Tianpeng cultivars have a closer genetic relationship with Zhongyuan group than Yunnan cultivars. However, Yunnan cultivars could not share the common ancestors with Tianpeng cultivars. Yunnan cultivars might be differentiated by several ancestors under different conditions. More evidences were supposed to make certain their origins. Based on the cluster results, it could be inferred that P. lutea is almost involved in the origin of Yunnan cultivars.

Effect of Postharvest Treatment on the Storage Quality and Antioxidant Enzyme System of Pleurotus eryngii

TIAN Ping-ping, WANG Jie, QIN Xiao-yi, LI Dan-qing

Scientia Agricultura Sinica. 2015, 48(5): 941-951. doi:10.3864/j.issn.0578-1752.2015.05.12

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【Objective】This research was conducted to study the effect of postharvest treatment on the storage quality and antioxidant enzyme system of Pleurotus eryngii and to provide a theoretical guide for the application and development of postharvest preservation of Pleurotus eryngii. 【Method】Fresh mushrooms, treated by 1.5% chitosan coating, 0.03 mm and 0.05 mm PE film packaging, respectively, were stored at 4℃ and RH 85%-95%. Mushrooms packed with 0.05 mm PE without sealing were used as the control. Based on analysis of main quality indexes, including weight loss rate, color, firmness, total soluble solids (TSS) content, reducing sugar content and total soluble protein content, antioxidant enzyme activities of peroxidase (POD), superoxide dismutase (SOD) and catalase (CAT), conductivity, malondialdehyde content, superoxide anion and H₂O₂ content, effects of different postharvest treatment methods on the storage quality and antioxidant enzyme system were studied in stored P. eryngii.【Result】The results showed that compared with the control, 0.05 mm and 0.03 mm PE film packaging could significantly reduce the weight loss rate (P＜0.01). On the 18 d of storage, the weight loss rates of 0.05 mm and 0.03 mm PE treatments were 0.20% and 0.35%, respectively. The firmness of mushrooms in two PE treatments were significantly higher than those in the control (P＜0.05). Moreover, there were two peak values in the firmness curves of 0.05 mm and 0.03 mm PE treatments, appearing on 3rd day and 12th day, respectively. Browning in two PE treatments was not obvious. However, browning degree of 0.03 mm PE treatment significantly increased during the final storage (P＜0.05). Two PE treatments could significantly inhibit the increase of total soluble solids (TSS) and soluble proteins and slow down the decrease of reducing sugar content (P＜0.05). The 1.5% chitosan coating treatment could significantly increase the firmness during 0-9 d storage (P＜0.05), then the firmness decreased rapidly. But mushrooms in 1.5% chitosan coating treatment exhibited higher weight loss rate, which had no significance compared with the control (P＞0.05). Chitosan coating mushrooms showed low browning index, but at the end of storage, browning in chitosan coating treatment was significantly higher (P＜0.05). The changes of total soluble solids (TSS), reducing sugar and total soluble protein of chitosan coated mushrooms were significantly lower than those of the control in the mid and late storage. To a certain extent, the three treatments enhanced the antioxidant ability of stored P. eryngii compared with control. Three treatments could significantly decrease the cell membrane permeability and the content of membrane lipid peroxidation products MDA (P＜0.05). The conductivity and malondialdehyde content of chitosan coated mushrooms were significantly higher than those of two PE treatments in the mid and late storage. The 0.03 mm PE packaging and chitosan coating treatment could effectively reduce superoxide anion (O₂^-.) generation rate and inhibit hydrogen peroxide (H₂O₂) generation rate (P＜0.05). Meanwhile, 0.05 mm PE packaging treatment could significantly reduce hydrogen peroxide (H₂O₂) generation rate (P＜0.05). However, the superoxide anion (O₂^-.) generation rate of 0.05 mm PE treatment was significantly higher than the control during 3-12 d storage. And the higher peroxidase (POD) and catalase (CAT) activities were observed in these treatments in the early and late storage (P＜0.05). However, no significant difference in superoxide dismutase (SOD) activity was observed in these treatments throughout storage.【Conclusion】From the above mentioned, better storage qualities and higher antioxidant ability were observed in two PE packaging treatments during the whole storage, of which, 0.05 mm PE packaging was clearly superior in terms of maintaining overall quality of stored P. eryngii.

Influence of Constant High Ambient Temperature on Fat Metabolism of Different Parts in Finishing Pigs

WU Xin, FENG Jing-hai, ZHANG Min-hong, SU Hong-guang, JIA An-feng

Scientia Agricultura Sinica. 2015, 48(5): 952-958. doi:10.3864/j.issn.0578-1752.2015.05.13

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【Objective】 The objective of this study is to investigate the effects of constant high ambient temperature on fat metabolism in finishing pigs and to preliminarily explore the mechanism of the impact. 【Method】 Sixteen Duroc × Landrace × Large White castrated male pigs were randomly assigned into a high-temperature environment (HT group: 30℃, ad libtum ) and a normal thermal group (NT group: 22℃, ad libtum ) with eight pigs in each treatment. Pigs were housed in individual wire cages under a 14-h lighting schedule and had free access to water. The experiment lasted for 3 weeks, and the temperature kept unchanged during this time. The relative humidity in the room was controlled at (55±5)%. The pigs were electrically stunned and exsanguinated after a 12-h period of feed withdrawal with free access to water at the end of the experiment. 【Result】 The results of the experiment showed that the carcass weight and backfat depth at 30℃ were lower than that at 22℃, but the differences were not significant (P＞0.10). And high ambient temperature had a trend to increase the proportion of flare fat in carcass weight (+22.06%, P=0.07), to decrease the lipid content of longissimus dorsi (LM) (-22.39%, P=0.08). The activities of fatty acid synthase (FAS) (P＜0.05) and malic enzyme (ME) (P＜0.05) in backfat and flare fat were lower at 30℃ than at 22℃, the amounts of acetyl-CoA-carboxylase (ACC) (P＜0.01) and FAS (P＜0.05) were decreased in LM in HT group, and the activity of FAS in liver was also inhibited by high temperature (P＜0.01). These enzymes (ACC, FAS, ME) were key ones in de novo synthesis of the fatty acids, above results indicated that high ambient temperature inhibited de novo synthesis of fatty acids in adipose tissues. High ambient temperature had no significant effects on the content of hormone-sensitive lipase (HSL) in all adipose tissues (P＞0.10). High ambient temperature significantly increased the content of LPL in flare fat (P=0.05), and decreased the content of LPL in LM (P=0.05). The rule how high ambient temperature influenced fat deposition of the three parts was in accordance with the rule how high ambient temperature influenced the contents of LPL in the same part, which means that high ambient temperature may influence the fat deposition by regulating the content of LPL. The activities of β-hydroxyacyl coenzyme A dehydrogenase (HAD) at the front (P＜0.05) or back (P＜0.01) of LM were lower at 30℃ than at 22℃, and the enzyme of HAD is a key one in the β-oxidation of fatty acids, the findings indicated that high temperature inhibited fatty acid oxidation in skeletal muscle. High ambient temperature significantly increased the content of cAMP in LM (P＜0.01), but had no significant effects on the content of cAMP in backfat and flare fat (P＞0.10). The plasma concentration of nonesterified fatty acid (NEFA) was higher (P＜0.05), and very low density lipoprotein (VLDL) tended to be higher at 30℃ than at 22℃ (P=0.07). High ambient temperature had no significant effects on the plasma concentration of total cholesterol (CHOL), triglyceride (TG), high density lipoprotein-cholesterol (HDL-C) and low density lipoprotein-cholesterol (LDL-C) (P＞0.10). 【Conclusion】The results demonstrated that high ambient temperature had different effects on adipose tissues in different parts. High ambient temperature may influence fat deposition in different parts by regulating the content of LPL in different parts. High ambient temperature depressed de novo fatty acid synthesis in adipose tissues and in the liver. However, β-oxidation of fatty acid in skeletal muscles was also inhibited in the high-temperature environment, which may result in an increased concentration of NEFA in the plasma, and NEFAs were esterified to synthesize VLDLs in the liver, reabsorbed by adipose tissues ultimately. But the mechanism that plasma concentrations of NEFA and VLDL were higher in the high-temperature needs to be further studied.

Expression of IFNGR in the Celiac Superior Mesenteric Ganglion of Goats

LI Qiang, WANG Zhi-hao, JIN Xiu-fang, XU Yong-ping, GUO Xiao, DONG Wei, LIU Wen-gang

Scientia Agricultura Sinica. 2015, 48(5): 959-965. doi:10.3864/j.issn.0578-1752.2015.05.14

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【Objective】This experiment was conducted to detect the existence of IFNGR in the celiac superior mesenteric ganglion (CCMG) in goats.【Method】The CCMG were taken from male and female goats, respectively. The CCMG were fixed with 40 g·L^-1 paraformaldehyde in phosphate buffer for 4 hours. Fixation was followed by thorough rinsing in water for 10 h, then dehydrated in graded alcohols, embedded in paraffin and cut into thick sections to prepare for immunohistochemical and H.E. staining. The sections were divided into 4 groups. The first group stained with H.E. staining. The second group was deparaffinized with xylene and ethanol, and processed for immunohistochemical SP staining. The third group was stained with immunohistochemical SP staining and hematoxylin counterstain. The last group was used as the negative control group. After staining, the specimens were examined and photographed with Motic microscope with digital camera. The digital images were analytically processed and got the relative expression of IFNGR with image-analysis software. All data were processed by SPSS18.0 software, mono factor analysis of variance was used for Significance test. After CCMG thoroughly grinded, the total RNA was extracted with extract RNA kit, and primers were designed according to IFNGR1 cDNA sequence of bovine. Then, the IFNGR1 cDNA of goat was cloned and amplified by PCR. The amplification was detected whether there was the target band by agarose gel electrophoresis and DNA sequencing. The sequencing results were compared and affirmed in the NCBI, and compared with sequences of other species. 【Result】The results of immumohistochemical staining showed that: IFNGR immunopositivity was widely distributed in CCMG of goat, and different levels of staining existed in neurons, satellite cells and nerve fibers. All the neurons were IFNGR positive. Almost every cytoplasm of neurons were dyed tan, were IFNGR positive, only a few cytoplasmic stained weak were weakly positive. In the nucleus of neurons, the karyoplasms were strongly positive, and the nucleolus were weakly positive or negative. In the non-neuronal structure, the satellite cells and nerve fibers were moderately or weakly positive. The relative expression of IFNGR in neuron was very significantly higher than that in non-neuronal structure (P＜0.01). The PCR detection results showed that there was a clear white target band at 300-400 bp in the line of PCR production. The DNA sequenced analysis showed that the full length of IFNGR1 gene amplified by PCR was 376 bp. Homologous comparison with other species indicated that IFNGR1 gene of goat homology with sheep (98%, XM_004011371.1) was the highest, followed by cattle (97%, NM_001035063.1), Sus scrofa (84%, NM_001177907.1), Oryctolagus cuniculus (73%, XR_085137.1) and Rattus norvegicus (66%, NM_053783.1), in the NCBI.【Conclusion】The results suggested that the IFNGR in the CCMG of goats mainly expressed and located in sympathetic postganglionic neurons which were provided with the conditions for the role of IFN-γ, which implied that the CCMG may act as critical point to coordinate the immune regulation of IFN-γ with neuroregulation of autonomic nerve on gastrointestinal tract.

Effect of Dietary Fat Sources and Dosages on Growth, Health, Serum Lipid and Liver Cholesterol Metabolism of Mice

HUANG Yang, LI Ping-hua, HE Li-chun, WANG Han, NIU Qing, SHI Lei,ZHOU Bo, HUANG Rui-hua

Scientia Agricultura Sinica. 2015, 48(5): 966-975. doi:10.3864/j.issn.0578-1752.2015.05.15

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【Objective】This experiment was conducted to investigate the effect of dietary fat source and dosages on growth performance, health status, serum lipid indicators and liver cholesterol metabolism-related gene mRNA expression level and to explore the mechanism of dietary fats source and dosage on hepatic cholesterol metabolism of mice. Results will contribute to select a suitable amount and type of oil for mammal.【Method】 Forty eight 3-week-old healthy KM mice whose body weights were 16-19 g were randomly assigned into four groups with 4 replicates per group and 3 mice each. Mice were fed: normal diet (control group); 4% bean oil diet (group B); 4% emulsified coconut powder diet (group L); 8% emulsified coconut powder diet (group H)for fourteen days, respectively. During the whole experiment, daily feeding times, feed quantity and remaining amount of feed of each time were recorded. All animals were fed and watered ad libitum. According to the recorded data, body weight and average daily feed intake (ADFI), the average daily gain (ADG), feed gain ratio (F/G) were calculated. Blood distribution, health index and liver weight of mice were measured. The concentrations of triglyceride (TG), total cholesterol (TC), high density lipoprotein cholesterol (HDL-C), and low density lipoprotein cholesterol (LDL-C) in serum were determined. The expression of mRNA of 3-Hydroxy-3-methylglutaryl coenzyme A reductase (HMGCR), cholesterol 7 alpha-hydroxylase (CYP7A1) and low density lipoprotein-receptor (LDLR) in liver were determined by real time PCR.【Result】Supplementation of 4% bean oil significantly increased body weight, ADFI and liver index of mice compared with the control group (P＜0.05), but had no significant effects on ADG, F/G or liver weight (P＞0.05). Supplementation of 4% emulsified coconut powder failed to significantly change growth performance of mice compared with the control group (P＞0.05). Supplementation of 8% emulsified coconut powder significantly increased ADFI of mice compared with the control group (P＜0.05), but had no significant effects on other growth performance (P＞0.05). Healthy status: Fat had no significant effects on blood distribution and health index (P＞0.05). Compared with the control group, TC and HDL-C levels in the serum significantly decreased in 4% bean oil group (P＜0.05), TG and LDL-C levels have no significant difference. Supplementation of 4% emulsified coconut powder had no significant effects on serum lipid index of mice (P＞0.05). Supplementation of 8% emulsified coconut powder significantly decreased serum TC levels (P＜0.05), but had no significant effects on TG, HDL-C and LDL-C levels (P＞0.05). Supplementation of 8% emulsified coconut powder significantly increased the expression of mRNA of CYP7A1 of mice compared with the control group (P＜0.05). Four percent bean oil diet and 4% emulsified coconut powder diet had no significant effects on the expression of mRNA of CYP7A1 (P＞0.05). There were no significant differences in the expression of mRNA of HMGCR and LDLR among four groups (P＞0.05). 【Conclusion】 Supplementation of bean oil increased the growth performance on ADFI and body weight of mice, decreased serum total cholesterol and high-density lipoprotein cholesterol. Supplementation of high doses emulsified coconut powder improved ADFI and decreased serum total cholesterol, and increased CYP7A1 mRNA expression in liver of mice. Compared to the high dosage of emulsified coconut powder, low dosage had no significant effects on improving lipid metabolism of mice. Fat in the diet probably affected liver cholesterol metabolism by regulating the gene expression levels of cholesterol metabolism-related enzymes in the liver to maintain cholesterol metabolism homeostasis in mice.

PCV2 Virus Like Particles Vaccine Produced with Recombinant Cap Protein Expressed in E.coli

ZHAO Xiao-yun, QIAO Xu-wen, CHEN Jin, LI Peng-cheng, YU Xiao-ming, ZHU Guo-qiang, ZHENG Qi-sheng, HOU Ji-bo

Scientia Agricultura Sinica. 2015, 48(5): 976-986. doi:10.3864/j.issn.0578-1752.2015.05.16

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【Objective】The objective of this study is to develop recombinant virus like particles vaccine for Porcine circovirus type 2 through soluble prokaryotic expression of ORF2 gene. 【Method】 The optimized ORF2 gene of PCV2 NJ strain was synthesized according to codon usage of E.coli, and then the optimized ORF2 gene was cloned into pQZ1 to get the recombinant prokaryotic expression vector named pQZ-Cap. The target gene was expressed with 1.0 mmol·L^-1 IPTG induction for 24 h under 15℃ on the prokaryotic expression platform in authors’ laboratory following the recombinant plasmid pQZ-Cap transformed into host E.coli BL21. Firstly, SDS-PAGE and Western blotting were used to identify the expression and solubility of the recombinant protein. Secondly, assemble for recombinant Cap VLP was evaluated through electron microscope analysis technology. Furthermore, swines were inoculated with recombinant Cap protein emulisified in SPPEIC 206 adjuvant, and the immunogenicity of recombinant Cap protein vaccine was evaluated by PCV2 specific antibody detection and virus attack.【Result】SDS-PAGE and Western blotting results indicated that the optimized ORF2 gene of PCV2 NJ strain could be efficiently expressed in E.coli BL21 in form of completely soluble. Abundant PCV2 VLP with diameter about 17 nm could be observed through electron microscope, exist in supernatant of the induced E.coli after ultrasonication. VLP vaccine composed of recombinant Cap protein possessed satisfactory immunity. Swine immunized with this VLP vaccine generated perfect antibody response with 100% qualified at 21 d post one sole vaccination and could be completely protected from pathogenic virus attack.【Conclusion】To summarize, optimized ORF2 gene of PCV2 NJ strain was successfully expressed in E.coli completely soluble and recombinant Cap protein can auto-assemble into VLP particles after ultrasonication. The VLP particles pose perfect immunity and could be used as subunit vaccines to prevent PCV2 infection.

Gene Cloning and Expression Analysis of 5-HT Receptors in Silkworm (Bombyx mori)

LI Hai-yin, LI Yan, CHEN Xi, CHEN Peng, CHEN Ping

Scientia Agricultura Sinica. 2015, 48(5): 987-1001. doi:10.3864/j.issn.0578-1752.2015.05.17

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【Objective】The objective of this study is to clone four kinds of 5-HT receptor genes and investigate their expression in different tissues of Bombyx mori, and to provide basic knowledge for further functional studies of these 5-HT receptor genes.【Method】Four 5-HT receptors were cloned based on genome database and using RT-PCR techniques. The bioinformatic method was used to analyze 5-HT receptor genes of B. mori and homology between species. The expression profiles of these genes in different tissues of larvae and adults were investigated by using semi-quantitative real-time PCR.【Result】Based on the predicted gene sequences, special primers were designed to clone the 5-HT receptor genes. Four 5-HT receptor genes 5-HT1ABm, 5-HT1BBm, 5-HT2Bm and 5-HT7Bm (GenBank accession number: KM236100-KM236103) were cloned. The open reading frame (ORF) of 5-HT1ABm, 5-HT1BBm, 5-HT2Bm and 5-HT7Bm was 1 395, 1 341, 1 881 and 1 497 bp, which encoded a polypeptide of 464, 446, 626 and 498 amino acids, respectively. They were typical of G protein-coupled receptors with seven transmembrane protein domains. The sequences were aligned and the phylogenetic tree of four 5-HT receptors was analyzed with other insects and vertebrates. Similarity of the amino acid sequence of 5-HT1ABm, 5-HT1BBm, 5-HT2Bm and 5-HT7Bm was only 30.4%. While, the similarity of 5-HT1A, 5-HT1B, 5-HT2 and 5-HT7 were 45.4%, 61.4%, 48.4%, and 54.1%, respectively in insects. In addition, 5-HT receptors had high homology and more conservative transmembrane region than non-transmembrane region in insects and vertebrates. The phylogenetic tree indicated that the same type receptors from different species got together, then the same receptor formed branch as species genetic relationships. The evolutionary relationships of 5-HT receptors in silkworm were close to Manduca sexta. The results of semi-quantitative real-time PCR showed that 5-HT1Abm, 5-HT1BBm expressed in all tissues in larvae, 5-HT2Bm only had expression in head, ventral chain and testis in larvae, 5-HT7Bm had expression in head, ventral chain, midgut, fat body, testis and ovary in larvae. 5-HT1ABm, 5-HT1BBm,and 5-HT7Bm expressedin other tissues except head in male of adults, whereas 5-HT7Bm in the reproductive system expression of male adults was significantly higher than other tissues. 5-HT2Bm did not express in adult.【Conclusion】Four 5-HT receptor genes were cloned in silkworm. They have more conservative in insects and vertebrates, and homologous relationships are close to M. sexta. The results of semi-quantitative real-time PCR showed that the tissue expression patterns were diverse.

Genetic Diversity Analysis of 98 Collections of Sugarcane Germplasm with AFLP Markers

ZAN Feng-gang, YING Xiong-mei, WU Cai-wen, ZHAO Pei-fang, CHEN Xue-kuan, MA Li, SU Huo-sheng, LIU Jia-yong

Scientia Agricultura Sinica. 2015, 48(5): 1002-1010. doi:10.3864/j.issn.0578-1752.2015.05.18

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【Objective】Cane sugar accounts for 92% of sugar production in China, and hybridization is the most widely used and the most effective way for developing new sugarcane cultivars. Sugarcane germplasm is essential for sugarcane breeding. Selecting parental clones and the cross combinations for hybridization contributes directly to the breeding efficiency. Aimed at providing reference for selecting parental clones and cross combinations, the genetic diversity and similarity among 98 sugarcane germplasm were studied.【Method】Good genomic DNA was extracted from young leaves of 98 sugarcane germplasm collected from 10 countries following the CTAB method, and then were amplified by sequence-related amplified polymorphism molecular markers to analyze genetic diversity and genetic similarity. Separation of the amplified fragments was performed on 5% denaturing polyacrylamide gels, the gels were stained with AgNO3, then “0,1” matrix was obtained according to the electrophoresis result. The number of polymorphic loci, percentage of polymorphic loci, quantity of polymorphic information, effective number of alleles and the indexes of genetic diversity were estimated by POPGENE version 32. The genetic similarity that estimated by NTSYS pc-V. 2.1 was used for UPGMA (unweighted pair group method analysis) and PCA (principal component analysis) to group the sugarcane germplasm. 【Result】Among 1 392 bands detected by 10 selective primer pairs proved by Yunnan Key Laboratory of Sugarcane Genetic Improvement, 1 344 (96.55%) were polymorphic. On average, each primer combination amplified 139.2 loci and 134.4 polymorphic loci. The genetic similarity of 98 sugarcane germplasm ranged from 0.484 to 0.929 with an average of 0.734, the number of polymorphic information was 0.2495, the number of effective alleles for each loci was 1.4092, the average index of genetic diversity was 0.3890. The highest genetic similarity (0.929) was found between KN90-418 and KN90-455, and the lowest (0.484) was found between Yunzhe94-375 and IS76-126. According to the genetic similarity of 0.64, 98 sugarcane germplasm were divided into 4 groups, 5 sugarcane germplasm IK76-48, IS76-126, IK76-22, SES309 and E.SARPET collected from Australia was classified as group I. 1 sugarcane germplasm IS76-199 collected from Australia was classified as group II. KN93-06, 90-110-9 and BURMA were classified as group III; other 89 sugarcane germplasm were classified as group IV which was divided into 9 subgroups (A, B, C, D, E, F, G, H and I ) at the genetic similarity of 0.79. The coefficient of Jaccard was used in PCA and indicated a similar result with cluster analysis that the germplasm with the same region shares high similarity, the similarity within Australian sugarcane germplasm was much lower, and the lowest was found within the germplasm belongs to Erianthus fulvus or Saccharum spontaneum. 【Conclusion】It was concluded that 98 sugarcane germplasm share high genetic similarity and low genetic diversity, the Australian sugarcane germplasm is relatively high in genetic diversity. 90-110-9, KN93-06 and Yuetang00-236 are 3 unique germplasm and are worth utilizing in hybridization.

Characterization and Transcriptional Expression Analysis of ABA Biosynthesis Related Genes from Mulberry (Morus alba L.)

ZHU Pan-pan, LIU Chang-ying, ZHAO Ai-chun, PEI Rui-chao, LI Jun, WANG Xiao-hong, LI Zhen-gang, WANG Xi-ling, LU Cheng, YU Mao-de

Scientia Agricultura Sinica. 2015, 48(5): 1011-1022. doi:10.3864/j.issn.0578-1752.2015.05.19

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【Objective】 9-cisepoxycarotenoid dioxygenase (NCED), ABA-aldehyde oxidase(AAO), Zeaxanthin epoxidase (ZEP) are the key enzymes involved in biosynthesis of abscisic acid (ABA) via indirect pathway. The aim of this study was to analyze the transcriptional expression of ABA biosynthesis related genes and detect the content of ABA during Jialing 40 (Morus atropurpurea Roxb.) fruit development. The effects of ABA and its synthesis inhibitors on fruit development were also explored. This work will lay a foundation for further studying the role of ABA on mulberry (Morus alba L.) fruit maturity and senescence. 【Method】In this study, the sequences of six putative ABA biosynthesis related genes were obtained from Morus notabilis genome database and its deduced amino acid sequences were analyzed by using bioinformatics tools. Total RNA was extracted from mulberry fruit using RNAiso Plus (TaKaRa) and reverse transcribed to synthesize cDNA. The relative transcriptional expression of ABA biosynthesis related genes in different developmental stages and treated mulberry fruits was investigated by using qRT-PCR. The content of ABA was determined by using HPLC.【Result】Six ABA biosynthesis related genes were isolated from mulberry, including one AAO gene, two ZEP genes and three NCED genes. Multiple-sequence alignment results showed that MnNCEDs amino acids are relatively conserved in higher plants. Cluster analysis revealed that MnNCED1-3 are closely related with dicotyledons plants and farther with monocotyledons plants. Transcription analysis indicated that the expression of ABA biosynthesis related genes was higher in leaves and the lowest expression was detected in roots. Particularly, the expression level of MnNCED2 and MnNCED3 was higher in different plant tissues compared to other genes. The concentration of ABA was strongly increased after the conversion stage of fruit. Exogenous ABA significantly promoted fruit maturity, whereas fluridone remarkably inhibited fruit ripening compared with the negative control treated with distilled water. The results of real-time PCR showed that the expression abundance of MnNCEDs was higher in the middle and latter periods than the early stage. Again, ABA biosynthesis related genes have shown different expression patterns under the same treatment. The expression level of ABA biosynthesis related genes was up-regulated in the fourth day after ABA treatment while down-regulated in the first day after fluridone treatment, unlikely the expression level of MnNCED2 was down-regulated after both ABA and fluridone treatments. 【Conclusion】 In this study six putative ABA biosynthetic genes in M. notabilis genome were obtained. The expression levels of MnNCEDs were higher in middle and later stages than early fruit developmental stage, which is in coincidence with the trend of ABA content during fruit development. Thus, MnNCEDs may play important roles in ABA biosynthesis. Exogenous ABA significantly promoted fruit maturity, while fluridone markedly inhibited fruit ripening.

The Structure and Formation Characteristics of Super-High Yield Population with Late Yongyou Series of Indica-Japonica Hybrid Rice in Double-Cropping Rice Area

HUA Jin, ZHOU Nian-bing, ZHANG Jun, ZHANG Hong-cheng, HUO Zhong-yang, ZHOU Pei-jian, CHENG Fei-hu, LI Guo-ye, HUANG Da-shan, CHEN Zhong-ping, CHEN Guo-liang, DAI Qi-gen, XU Ke, WEI Hai-yan, GAO Hui, G

Scientia Agricultura Sinica. 2015, 48(5): 1023-1034. doi:10.3864/j.issn.0578-1752.2015.05.20

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【Objective】Under the conditions of late rice of double cropping systems, Yongyou series of indica-japonica hybrid rice were used to investigate the structure and formation characteristics of super-high yield population. And it will provide a theoretical basis for cultivation of late Yongyou series of indica-japonica hybrid rice in double cropping systems. 【Method】A field experiment was conducted using the representative indica-japonica hybrid rice varieties Yongyou 538, Yongyou 2640, Yongyou 1540, and Yongyou 1538 to compare systematically yield and its components, development of stem and tiller number, development of leaf area and its composition, photosynthetic potential, dry matter accumulation and population growth rate between super-high yield and high yield populations formed by the regulation of cultivation measures. 【Result】Super-high yield population had more panicle and spikelets per panicle compared with high yield population. There was no significant difference in filled-grain percentage and 1000-grain weight between populations of super-high yield and high yield. Super-high yield population exhibited fewer tillers at the early growth stage and achieved expected number of stems and tillers at critical leaf-age for productive tillers, whose max number of stems and tillers was at jointing stage. Then, the number of population stems and tillers began to decrease stably, which achieved the expected number again. At last, the ratio of productive tillers to total tillers of super-high yield population was more than 75%, which was higher than that of high yield population. The leaf area index of super-high yield population was lower than that of high yield population, and the max leaf area index was about 8.1 at booting. Then the leaf area index of super-high yield population decreased stably, whose leaf area index, the ratio of effective leaf area, the ratio of effective leaf area and the ratio of grain to leaf were significantly higher than those of high yield population at heading and was above 3.5 at maturity. The photosynthetic potential of super-high yield population was small at early stage and larger at middle and late stages, as compared with high yield population. The total photosynthetic potential was above 580×10⁴m²·d·hm^-2, of which more than 50.0% was from heading to maturity. The dry matter accumulation was smaller compared with high yield population before jointing, increased faster after jointing, and reached about 10.0 t·hm^-2 at heading and 19.0 t·hm^-2 at maturity, which was significantly higher than that of high yield population. Before the critical leaf-age for productive tillers, the population growth rate of super-high yield population was larger than that of high yield population, which was lower than that of high yield population from the critical leaf-age for productive tillers to jointing. After jointing, the population growth rate of super-high yield population was significantly larger than that of high yield population.【Conclusion】 With higher population started quality and earlier tiller, the population of super-high yield had obvious advantages, especially, higher photosynthetic matter production and greater dry matter accumulation in the middle and late growth stages. That building of enough total spikelets through adequate panicles and the large spikelets per panicle, and keeping a high seed-setting rate and 1000-grain weight are the suitable yield formation characteristics of rice under super-high yield cultivation.

Effects of Biochar on Physico-Chemical Properties of Acid Red Soil Under Simulated Rainfall Condition

ZHU Pan, YING Jie-guan, PENG Shu-ang, JIANG Cun-cang

Scientia Agricultura Sinica. 2015, 48(5): 1035-1040. doi:10.3864/j.issn.0578-1752.2015.05.21

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【Objective】 Purposes of this study are to determine if biochar has effects on red soil under precipitation conditions, and the difference between no-fertilization soil and fertilization soil.【Method】A rainfall simulation experiment was carried out with different biochar levels (biochar/soil: 0%, 1%, 2% and 3%) on red soil, and some characters such as soil available phosphorus (SAP), available potassium (SAK), nitrate nitrogen (SNN), ammonium nitrogen (SAN), organic carbon (SOC) and active aluminum (SAA) were determined.【Result】Under precipitation conditions, the variation of SAN, SOC, SAA and pH in red soil was observably. When 3% biochar was added, the change of soil characters was most significantly. Compared with the no-fertilization control (CK), soil pH raised by 0.60, SAA decreased by 91.1%, the descent range of SAK decreased by 9.5%, the descent ranges of SAP and SNN, respectively, increased by 33.2%, 40.5% in no-fertilization soil with 3% biochar application. Compared with the fertilization control (F), soil pH raised by 1.09, SAA decreased by 94.8%, the descent range of SAK decreased by10.3%, the descent ranges of SAP and SNN, respectively, increased by 23.4% and 21.9%, SOC and SAN, respectively, increased by 23.6% and 5.4% in fertilization soil with 3% biochar application rate.【Conclusion】Under precipitation conditions, biochar was propitious to maintain SAP, improved soil pH and SOC, and decreased SAA, especially in red soil with fertilizer application.

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