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    16 November 2016, Volume 49 Issue 22
    CROP GENETICS & BREEDING·GERMPLASM RESOURCES·MOLECULAR GENETICS
    SPECIAL FOCUS Guidance: QUALITY - THE FUTURE OF CROP PRODUCTION
    ZHANG Ai-min, LI Xin, LIU Dong-cheng, SUN Jia-zhu, YANG Wen-long
    Scientia Agricultura Sinica. 2016, 49(22):  4265-4266.  doi:10.3864/j.issn.0578-1752.2016.22.001
    Abstract ( 522 )   HTML ( 9 )   PDF (220KB) ( 767 )   Save
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    Progresses in Research on Cloning and Functional Analysis of Key Genes Involving in Rice Grain Quality
    ZHANG Chang-quan, ZHAO Dong-sheng, LI Qian-feng, GU Ming-hong, LIU Qiao-quan
    Scientia Agricultura Sinica. 2016, 49(22):  4267-4283.  doi:10.3864/j.issn.0578-1752.2016.22.002
    Abstract ( 740 )   HTML ( 9 )   PDF (509KB) ( 1156 )   Save
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    Rice (Oryza sativa L.) is one of the most important cereal crops in worldwide and also a major stable food in China, thus it is very important to breed novel rice cultivars with high yield as well as good grain quality. Rice grain quality is a complex trait, and usually means rice or rice products meeting the demand of end-users. Therefore, the concept of rice grain quality covers multiple features revealed by the physical and chemical characteristics, including milled rice ratio, grain shape, appearance, cooking time, aroma and its retention after cooking, eating palatability, and nutrition. In general, rice grain quality includes as milling quality, apparent quality, eating and cooking quality (ECQ), and nutritional value. The grain shape is not only the factors associated with yield but also crucial aspects of grain quality. In the past decade, there were rapid and great achievements in the cloning and functional analyses of the genes involving in rice grain qualities. For grain size and shape, numerous QTLs and genes have been cloned and characterized. These cloned genes could be divided into three groups based on the phenotypes of the mutants. The first group is associated with not only grain shape but also plant phenotype, such as D1, D2, D11, D61 and SMG1. The second group appears to specifically affect grain trait, including GS3, GL3.1, GW7, GW2, GW5, GS5, GS6, TGW6, GW8, BG2, GW6a and GS2, which are well valuable for improvement of grain yield and quality. The third group is called small and round seed, such as the SRS gene. Chalkiness is associated with both grain appearance and milling property, and only few such QTLs have been finely mapped and cloned, including Chalk5, cyPPDK, G1F1, OsRab5a, FLOURYENDOSPERM2, PDIL1-1 and SSG4. The starch comprises about 90% of the dry matter of rice endosperm, and thus the grain quality is greatly affected by starch composition and structure. Therefore, the starch biosynthesis plays a crucial role in the formation of rice quality, especially the eating and cooking quality. Recent studies had made deep understanding of the regulation network of starch biosynthesis related enzymes, and several transcriptional regulators had also been proven for involving in starch biosynthesis, such as Dull, OsEBP89, OsEBP5, OsRSR1 and OsbZIP58. For seed protein content, most of the genes for seed storage proteins have been well characterized, and some other genes, such as OsSar, OsRab5a, OsAPP6, RISBZ1, RPBF, OsVPS9A, OsGPA3 and GEF2 have also been identified associating with protein sorting and transporting. The aroma of cooked rice contributes to consumer sensory acceptance, and recent studies have confirmed that the BADH2 and OsP5CS genes are responsible for the synthesis of fragrance material 2-AP. As for the other nutritional factors, such as the contents of essential amino acid lysine, vitamins, anthocyanin and minerals, also many functional genes have been cloned or elucidated. Taken together, all of the above traits are known to be genetically controlled by multiple genes, and also interact with each other. In present review, the genetic networks involving in regulation of rice grain quality in the last decade were summarized and updated. It will give a better understanding of the genes that contribute to the overall grain quality as well as lay a foundation for development of new strategies for grain quality improvement with high yield in rice.
    Progress in Research on Genetic Improvement of Nutrition and Health Qualities in Wheat
    ZHANG Yong, HAO Yuan-feng, ZHANG Yan, HE Xin-yao, XIA Xian-chun, HE Zhong-hu
    Scientia Agricultura Sinica. 2016, 49(22):  4284-4298.  doi:10.3864/j.issn.0578-1752.2016.22.003
    Abstract ( 790 )   HTML ( 26 )   PDF (481KB) ( 1252 )   Save
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    The role of nutrition and health has become one of the main targets of research and breeding for major crops in the world. The research progress on micronutrients involving iron and zinc, resistant starch, dietary fibre as arabinoxylans, and a range of phytochemicals involving phenolic acids and sterols related to wheat quality, as well as wheat sensitivity and fusarium head blight related deoxynivalenol that have an impact on human health was reviewed from breeding point of view. Laboratory evaluation methods, germplasm screening, and QTL mapping on nutrition quality parameters as well as breeding, were summarized. China’s major strategies on wheat breeding program for nutrition and health improvement were proposed, with the following four areas being advanced: (1) analysis of the contents of micronutrients involving iron, zinc, as well as the bioavailability related factors phytate content and phytase activity, dietary fibres such as arabinoxylans and resistant starch, and phytochemicals involving phenolic acid and sterols should be strengthened, to screen materials with high quality of micronutrients, dietary fibre, and phytochemicals; (2) more efforts should be made in study on fusarium head blight and the results of study should be used in wheat breeding as early as possible; (3) development and utilization of molecular markers, especially functional markers in conventional breeding programs for speeding up wheat breeding, on the basis of gene mapping and cloning; (4) establishment of initiative project through strengthening international cooperation and domestic collaboration in research on addressing wheat quality, to find and extend the utilization of high quality methods on nutrients analyzation. The review addressed some crucial information on wheat related research and breeding programs for nutrition and health quality improvement.
    Recent Advances in Identification and Functional Analysis of Genes Responsible for Soybean Nutritional Quality
    ZHANG Yu-qin, LU Xiang, LI Qing-tian, CHEN Shou-yi, ZHANG Jin-song
    Scientia Agricultura Sinica. 2016, 49(22):  4299-4309.  doi:10.3864/j.issn.0578-1752.2016.22.004
    Abstract ( 609 )   HTML ( 7 )   PDF (411KB) ( 1055 )   Save
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    Soybean is one of the most important cash crops and provides edible oil and vegetable proteins for human beings. The study of soybean is recently focused by researchers, breeders and public people, because its value is mainly determined by the content of oil, protein and isoflavones and the quality of soybean is directly related to the health of the human body. The profile of fatty acids in soybean oil has a great influence on the nutritional value, storage and processing technology. And the profile and accumulation of soybean oil was determined by activity of oil-biosynthesis-related genes, which regulated by many genes at pre-transcriptional, transcriptional and post-transcriptional levels. Recent study reveals that GmDof4 and GmDof11 were found to increase the content of total fatty acids and lipids in GmDof4 and GmDof11 transgenic Arabidopsis seeds, which activated the acetyl CoA carboxylase gene and long-chain-acyl CoA synthetase gene. GmMYB73 overexpression enhanced lipid contents in both seeds and leaves of transgenic Arabidopsis plants by promoting PLDα1 expression whose promoter can be bound and inhibited by GL2. The GmbZIP123 transgene promoted expression of two sucrose transporter genes (SUC1 and SUC5) and three cell-wall invertase genes (cwINV1, cwINV3, and cwINV6) by binding directly to the promoters of these genes, and increased seed oil-content. And GmNFYA promoted master regulator WRI and oil-biosynthesis-related genes to increase seed oil-content. Soybean protein contains 8 kinds of essential amino acids, and is a kind of excellent quality of vegetable protein which can replace some animal protein in the diet. The accumulation of plant oil and protein is often negatively related. GmDof4 and DmDof11 down-regulated the storage protein gene, CRA1, through direct binding promoter although GmDof4 and GmDof11 enhanced seed oil-content. Soybean isoflavones are secondary metabolites formed during the growth of soybean, which have a wide range of biological activities and physiological functions in animals and plants. In recent years, soybean isoflavones have become one of the most attractive functional components, and are also one of the hot spots in food and nutrition research. Flavonoids may regulate the development, growth, propagation and nitrogen fixation of plants by regulating the production of nodules. Beneficial effects of soybean isoflavones are shown in the treatment of breast cancer, prostate cancer, cardiovascular disease and osteoporosis. GmMYB176 can regulate the expression of CHS8, and the interference of GmMYB176 expression decreased the soybean isoflavones levels in hair, indicating that GmMYB176 is essential for isoflavones biosynthesis. This review summarized the recent progresses in the gene cloning and regulation of soybean oil, storage protein and isoflavones accumulation. Other relevant advances and prospects were also compared and discussed. This review may give the current status of the studies on the regulatory mechanisms of soybean nutritional quality.
    Progress in Studies on Genes Related to Fiber Quality Improvement of Cotton
    YANG Jun, MA Zhi-ying, WANG Xing-fen
    Scientia Agricultura Sinica. 2016, 49(22):  4310-4322.  doi:10.3864/j.issn.0578-1752.2016.22.005
    Abstract ( 428 )   HTML ( 19 )   PDF (837KB) ( 1037 )   Save
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    Cotton is an excellent and the most widely used natural fiber. With the improvement of living standards of people, the demand for more and better natural cotton fabrics is increasing continuously. Therefore, improving fiber yield and quality has become an important objective of cotton genetic breeding. To achieve this goal, cloning and functionally identifying cotton fiber development-related genes is the main foundation. Cotton fiber development consists of four distinct but overlapping stages, including fiber initiation, elongation (primary cell wall synthesis), secondary cell wall biosynthesis, and drying and maturation. The number of fibre cells per ovule is established at the initiation stage, and the length and strength of fibres are determined mainly at the stages of elongation and secondary cell wall synthesis. Cotton fiber development is a complicated and ordered process regulated by a large number of genes. To date, it has been reported that some genes play important roles in cotton fibre development, including various transcription factors, genes controlling the metabolism of plant hormones, cell wall and cytoskeleton-associated proteins, gene involving in the release or consumption of ROS, and lipid- and sugar- metabolism genes, etc. In order to provide reference for the future study of cotton fiber development and quality improvement, advances in the cloning and functional analysis of genes related to cotton fiber development were systematically summarized in this paper.
    TILLAGE & CULTIVATION·PHYSIOLOGY & BIOCHEMISTRY·AGRICULTURE INFORMATION TECHNOLOGY
    Immobilization Effect and Its Physiology and Biochemical Mechanism of the Cadmium in Crop Roots
    WANG Xue-hua, DAI Li
    Scientia Agricultura Sinica. 2016, 49(22):  4323-4341.  doi:10.3864/j.issn.0578-1752.2016.22.006
    Abstract ( 559 )   HTML ( 4 )   PDF (2604KB) ( 1036 )   Save
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    A certain degree of cadmium stress has seriously influenced crop growth, development, yield and quality of farm produce. In this review, the authors comprehensively summarized the damages of Cd to crops and a human being, and the characteristics of the Cd absorption, transport and accumulation as well as their dependent main regulatory genes and their functions. The resistant and tolerant mechanisms of crops to the Cd toxicity were simply summarized, while the physiology and biochemical mechanism of the root cadmium immobilization effect which was one of them were emphatically introduced. The Cd got into the plant via the root absorption mostly, and in the root, the Cd2+ entered the “free space” firstly, which is constituted by the cell space, the cell wall micropore and the space between cell wall and plasma membrane, afterwards, passed through the plasma membrane into the cytoplasm by the way of active or passive absorption, and then were transported into the xylem vessel through symplast or apoplast pathway. Rice and others crops mainly use the following ways to adapt the Cd stress: the retention effect of cell wall, the chelation effect in symplast, the compartmentation effect of vacuole, the accumulation of stress protein and proline, the enhancement of antioxidase system activity and the immobilization effect of the root. As an important way to resist and tolerate the cadmium toxicity, the immobilization effect of cadmium in crop root plays a crucial role in regulating the Cd absorption, transport, distribution and accumulation in crop, preventing Cd entering into bioplasts and shoots of plant, and reducing the harm of Cd to the growth and development of the crop and the yield and quality of the farm produce. It mainly contains the retention caused by the low root-to-shoot Cd transport volume, the cell wall and the vacuolar. (i) The retention caused by the low root-to-shoot Cd transport volume. This retention effect is mostly influenced by the xylem Cd loading capacity of the root and the content of the cadmium long distance transport carrier – phytochelatins (PCs); and they were controlled by the ion transporter HMA2 and HMA4 on plasma membrane and the PCs synthetase in cell and their relevant regulatory genes, such as HMA2, HMA4 and PCs1. And they have a negative regulation to the Cd retention in the xylem. (ii) The cell wall immobilization effect. This effect happens in the apoplast (including the cell wall and intercellular layer) of the root cell, and is mainly related to the component and structure of the apoplast, and in which the pectin has played a key role, moreover, the hemicellulose also has some effect in it. On the basis of the different action way in the pectin and hemicellulose Cd retention, the cell wall immobilization effect can be divided into physical immobilization (PIB) and chemical immobilization (CIB). The PIB is mostly related to the pectin content and the pectin methyl esterase (PME) activity. While, the CIB is the result of electrostatic binding effect between the Cd2+ and carboxyl group or other groups with negative charge on the pectin, hemicellulose and other components of the cell wall. And they were controlled by the genes like PME14 and XCD1. (iii) The vacuole immobilization effect. This effect is closely related to the PCs in the cytoplasm and the vacuole, as well as the transport protein on the tonoplast; and its retention ability to Cd is restricted by the vacuolar sequestration capacity (VSC). In the vacuole Cd immobilization, the PCs with different molecular weight play an important part, they participated in the chelation of Cd in the cytoplasm, the Cd transformation between cytoplasm and vacuole, and the Cd sedimentation in the vacuole finally. At the same time, the transport proteins on the tonoplast are in charge of transferring the low molecular weigh PC-Cd compound from cytoplasm solution into the vacuole through active transport, making the Cd insulated. In crop roots, the successively combined action of the three immobilization effects has reduced the cadmium transfer amount from the root to shoot, then eased the pernicious effects of the Cd to the shoot, and decreased the content in the crop harvesting organs like grain. However, due to the finiteness of the percentage of PC-Cd in the general Cd number, the total charge number of the cell wall and the VSC of vacuole, making the intensity and effectiveness of the root-Cd-retention effects are limited to a certain degree.
    TILLAGE & CULTIVATION·PHYSIOLOGY & ECOLOGY
    Effects of Seed Maturity on Maize Hybrid Seed Vigor and F1 Yield
    LIU Guo-liang, ZHAO Ya-li, WANG Xiu-ling, LI Hong-ping, LI Chao-hai
    Scientia Agricultura Sinica. 2016, 49(22):  4342-4351.  doi:10.3864/j.issn.0578-1752.2016.22.007
    Abstract ( 463 )   HTML ( 6 )   PDF (3199KB) ( 540 )   Save
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    【Objective】 Investigations on the effects of seed maturity on maize (Zea mays L.) hybrid seed vigor and F1 production in corn seed production base along Hexi corridor in China are the bases of determining optimum harvest time of maize seed, protecting seed from the early frost and ensuring seed quality safety. 【Method】A field experiment combined with biochemical test were carried out with two maize hybrids, i.e. Yudan603 (partial dent maize variety, YD603) and Yudan606 (rigid grain maize variety, YD606). Ear samples were taken at an interval of 5 days after 40 days after pollination to test milk line development, grain water content, hundred-grain weight, standard germination, electrical conductivity, F1 field performance and production, and study the effects of maize seed maturity on seed vigor and F1 production.【Result】 There was a significant difference in the occurrence time and development process of milk line between the two hybrids YD603 and YD606. Compared with YD606, milk line of YD603 appeared earlier while disappeared later. On 40 days after pollination (DAP), the milk line position of YD606 was at 0.16, while that of YD603 had not yet appeared. On 65 DAP, both hybrids had a similar milk line position about 0.50, and the grain water content of YD603 was 31.66% and that of YD606 was 35.09%. On 40 DAP, grain water content of YD603 was higher than that of YD606. After 45 DAP, opposite changes in grain water content were found between YD603 and YD606. At physiological maturity, grain water contents of YD603 (75 DAP) and YD606 (80 DAP) were 26.71% and 27%, respectively. The germinating ability of F1 increased with the seed maturity and reached the maximum at physiological maturity. On 40 DAP, the seeds of two hybrids had partly germinating ability. YD603 reached physiological maturity on 75 DAP with a standard germination rate of 96.67%, while YD606 reached physiological maturity on 80 DAP with a standard germination rate of 98%. Seed simple vigor index of YD603 and YD606 also increased with the seed maturity, and reached the maximum values 5.89 and 8.70, respectively, on 65 DAP. Afterwards, seed simple vigor index of both hybrids decreased slightly. In accordance with standard germination rate, artificial accelerated aging germination rate of the two hybrids increased with the seed maturity. On 40 DAP, aging germination rates of YD603 and YD606 were only 58.33% and 58.33%, respectively, for the low seed maturity, while that were 90.00% (75 DAP), and 92.33% (80 DAP), respectively at physiological maturity. The field germination percentage, uniformity, dry matter accumulation and yield of F1 generation of two hybrids increased with the seed maturity improvement, and reached maximum value at physiological maturity without significant difference compared with those of 65 DAP, which suggested that good seedling quality and yield of F1 could be obtained when F1 seed was harvested on 65 DAP . 【Conclusion】The results suggested that the optimum harvest time of hybrid seed is on 65 DAP, when the seed milk line position was at 0.5 and seed moisture is between 30% and 35%. The harvest time is 15 days earlier than the conventional harvest time (milk line almost disappeared) in corn seed production base along Hexi corridor of Zhangye, which not only can provide more time for seeds air-drying to avoid frost damage, but also can ensure high-quality hybrid seeds.
    Analysis of Temporal and Spatial Variation of Vegetation Phenology in the Loess Plateau
    LI Qiang, ZHANG Chong, REN Zhi-yuan
    Scientia Agricultura Sinica. 2016, 49(22):  4352-4365.  doi:10.3864/j.issn.0578-1752.2016.22.008
    Abstract ( 426 )   HTML ( 6 )   PDF (12067KB) ( 465 )   Save
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    【Objective】The Loess plateau is in the transitional region from wetness to dryness, from forest to grassland, from farming to animal husbandry, being the sensitive zone in climate change and agricultural development in China, the research on phenological feature of surface vegetation coverage in the region is of directive significance to agricultural production, environmental protection and ecological construction. Analysis of difference in phenological trends of vegetation in different time series and altitudes and hydrothermal conditions shall offer theoretical support and decision basis for current agricultural ecological environment improvement and sustainable development on loess plateau.【Method】Phenological feature values of vegetation on Loess Plateau every year were determined and phenological change trend was analyzed based on NDVI of ten-day values of SPOT VEGETATION from 1998 to 2012 and combined with harmonic analysis method and linear trend method.【Result】(1) From 1998 to 2012, the start of growing season advanced by 0.9 d on average every year and the end of growing season delayed by about 0.8 d on average every year, the length of growing season every year extended by 1.7 d on average under the joint action of advance at the start of growing season and delay at the end of growing season. (2) Hydrothermal condition on Loess Plateau has an immediate impact on phenological difference, the restrictive temperature for vegetation growth is 9,with the restrictive precipitation of 475 mm and 540 mm, respectively and restrictive altitude of 1 750 m. (3) The spatial partial correlation coefficients between the length trend of growing season of vegetation and altitude and air temperature are 0.0591and 0.0139 respectively, the spatial partial correlation coefficients between the length trend of growing season of vegetation and precipitation is -0.0174, therefore, the degree of correlation between three factors and the trend at the start of growing season is higher than that at the end of growing season.【Conclusion】The zones showing a significant and stable trend of phenological feature of vegetation on loess plateau are primarily distributed on plateau in the north of Shaanxi and Beishan in the middle of Shanxi. The phenological change in arid area and desert and grassland area in northwest is mainly subject to control by air temperature. The phenological change in semi-arid area and farming and grassland areas is mainly subject to control by precipitation. The phenological change in agricultural area in Fenwei Basin is subject to hydrothermal condition. Difference in hydrothermal condition has an insignificant impact on vegetation phenology in broad leaved forest zone. Altitude has an insignificant impact on change trend of vegetation phenology on loess plateau. The extension trend of growing season is on the increase with the increase of altitude and air temperature under the joint action of the start and end of growing reason, the shortening trend of growing season is on the increase with the increase of precipitation, the change characteristics of the same type of vegetations in terms of phenological trend based on the change of altitude, precipitation and air temperature are consistent, the change characteristics at the start of growing season have a greater influence on length change of growing season compared with that at the end of growing season.
    PLANT PROTECTION
    Development of IC-RT-nested PCR for the Detection of Blueberry shock virus
    XIE Li-xue, ZHENG Shan, ZHANG Li-jie, ZHANG Xiao-yan, LI Tao
    Scientia Agricultura Sinica. 2016, 49(22):  4366-4374.  doi:10.3864/j.issn.0578-1752.2016.22.009
    Abstract ( 425 )   HTML ( 1 )   PDF (1463KB) ( 494 )   Save
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    【Objective】 Blueberry shock virus (BlShV) is one of the major viruses infecting blueberry, which can cause serious impacts on the yield of blueberry. In order to provide a reliable technique for rapid detection and identification of BlShV, IC-RT-nested PCR assay was developed.【Method】 A primer set containing outer primers (BlShV-F/BlShV-R) and inner primers (BlShV-1/BlShV-2) was designed according to the reported BlShV gene sequences, and IC-RT-nested PCR assay was established by combining immunocapture reverse transcriptase polymerase chain reaction and nested PCR with BlShV infected leaf as material. The specificity of the IC-RT-nested PCR was determined by testing extracts of leaf tissues from BlShV, Blueberry scorch virus (BlScV), Blueberry shoestring virus (BSSV), Blueberry leaf mottle virus (BLMoV), Tobacco ringspot virus (TRSV), Tomato ringspot virus (ToRSV)and healthy blueberry leaf. PCR products were purified, and then ligated to pMD18-T vector. After transformed into Escherichia coli DH5α, positive clones were screened and sequenced to further confirm the validation of the IC-RT-nested PCR assay. To test the sensitivity of the IC-RT-nested PCR, serial ten-fold dilution of the extracts prepared from BlShV-infected blueberry leaves was made by addition of corresponding amount of healthy plant extracts. The solutions were subsequently subjected to the IC-RT-nested PCR assay and DAS-ELISA, respectively. A total of 68 samples of blueberry from different regions (53 samples from Fujian, Jilin and Liaoning regions in China and 15 samples from America) were collected and detected for the presence of BlShV with the established IC-RT-nested PCR, and the result was validated by conventional RT-PCR. 【Result】 The established IC-RT-nested PCR amplified fragment of about 746 and 486 bp only from BlShV infected leaf extract by the first and second round PCR, respectively. No target fragment was observed from healthy blueberry leaves extract and blank control. The IC-RT-nested PCR assay showed a good specificity to BlShV with the expected 746 and 486 bp fragments for the first and second round PCR, respectively. No cross-reaction was observed from BlScV, BSSV, BLMoV, TRSV, ToRSV and healthy blueberry leaves. Sequence analysis showed that the sequences of the first and second round PCR product (746 and 486 bp, respectively) was identical with the expected size, and shared an extremely high homology (99%) with the previously published BlShV gene sequence. The results of sequence analysis confirmed that the two PCR products were BlShV specific products, and further verified the accuracy of amplification results. Sensitivity assay showed that the first round of IC-RT-nested PCR could successfully detect BlShV from leave extracts diluted up to 102, which was as sensitive as DAS-ELISA. After the second round PCR, the sensitivity of IC-RT-nested PCR was increased by 100 times, with a limit of 104 dilution of BlShV infected leaf extract. Field sample test revealed that the target fragment (about 486 bp) was amplified from two blueberry leaf samples from America by IC-RT-nested PCR, and the detection rate was 13.3%. However, no corresponding target fragment was amplified from samples from Fujian, Jilin, Liaoning regions of China. The result of field sample test was in accord with the results of conventional RT-PCR. 【Conclusion】 The established IC-RT-nested PCR is a rapid, specific, accurate and sensitive method for the detection and identification of BlShV on blueberry samples from field and port of entry-exit.
    Molecular Characteristics and Functional analysis of Trehalase Genes in Locusta migratoria
     
    LIU Xiao-jian, SUN Ya-wen, CUI Miao, MA En-bo, ZHANG Jian-zhen
    Scientia Agricultura Sinica. 2016, 49(22):  4375-4386.  doi:10.3864/j.issn.0578-1752.2016.22.010
    Abstract ( 366 )   HTML ( 2 )   PDF (2555KB) ( 430 )   Save
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    【Objective】 Trehalase, the only enzyme that hydrolyzes one trehalose molecule into two glucose molecules, plays key roles in insect energy metabolism and chitin synthesis. So trehalase could be served as a potential target for insect pest control. In this paper, the molecular characteristics and functions of trehalase genes were explored in an important agricultural pest Locusta migratoria. The results will provide a reasonable basis for the effective management of locusts. 【Method】By searching the transcriptome and genome of L. migratoria, four full-length cDNA of trehalase genes were obtained. Sequence characteristics of these four trehalases were analyzed by using blast, TMHMM and SignalP softwares. Multiple amino acid sequence alignment of trehalases was made using ClustalW. The phylogenetic tree was constructed by MEGA 7. The expression patterns of trehalase genes in different tissues and developmental days were studied in the 5th-instar nymphs by RT-qPCR. The dsRNAs of four trehalase genes were synthesized in vitro, and then injected into the 5th-instar nymphs on day 2, respectively. Control nymphs were injected with dsGFP alone. The whole body after the dsRNA injection for 48 h was used for total RNA extraction and cDNA synthesis. RT-qPCR were performed to determine the transcript levels of four trehalase genesand genes involved in chitin synthesis such as UDP-N-acetylglucosamine pyrophosphorylase gene LmUAP1 andchitin synthase 1 gene LmCHS1. The abnormal nymphs displayed phenotypes were carefully observed. 【Result】 Blast analysis showed that all these four trehalases contained two conservative regions and a glycine enrichment region. A membrane-bound trehalase with a typical transmembrane domain was named LmTreM (GenBank accession number KX371563). A transmembrane like domain was predicted in another trehalase, named LmTreM-like (GenBank accession number KX371565). The two remains were soluble trehalases, named LmTreS1 and LmTreS2, respectively (GenBank accession number KX371564 and FJ795020). Phylogenetic analysis showed that LmTreM and LmTreM-like, LmTreS1 and LmTreS2 were clustered with membrane-bound and soluble trehalases, respectively. RT-qPCR was carried out to analyze the expression patterns of four trehalase genes in different tissues and developmental days in the 5th-instar nymphs of L. migratoria. The results indicated LmTreM, LmTreS1 and LmTreS2 were mainly expressed in specific tissues, and LmTreM-like was consistently expressed in all selected tissues. mRNA transcripts of four trehalase genes were different during the development of 5th-instar nymphs. RNAi results suggested that expressions of four trehalase genes were significantly reduced compared to the control, and the injection of LmTreM, LmTreS1, LmTreS2 and LmTreM-like dsRNA only repressed the expression of the targeted LmTre without reducing that of other LmTres, the expressions of LmUAP1 and LmCHS1 involved in chitin synthesis were not affected. However, nymphs could successfully molt to adults after injected with four trehalase dsRNAs, respectively.【Conclusion】One membrane-bound, one membrane-bound like and two soluble trehalase were found in L. migratoria. The four trehalase genes showed different tissue and developmental expression characteristics, silencing of each of these four trehalase genes in the 5th-instar nymphs could not affect the normal molting to adults.
    Spatio-Temporal Expression of Sphingosine Kinase and Its Response to Insecticide Stress in Laodelphax striatellus Fallén
    JIAO Wen-juan, LI Fei-qiang, ZHANG Min-jing, SHI Xiao-xiao, ZHU Mu-fei, MAO Cun-gui, ZHU Zeng-rong
    Scientia Agricultura Sinica. 2016, 49(22):  4387-4397.  doi:10.3864/j.issn.0578-1752.2016.22.011
    Abstract ( 461 )   HTML ( 1 )   PDF (2289KB) ( 251 )   Save
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    【Objective】The objective of this study is to understand the characteristics of sphingosine kinase (SK) and research on the spatio-temporal expression of SK and its response to insecticide stress in Rice stripe virus (RSV) -infected Laodelphax striatellus Fallén (small brown planthopper, SBPH) and RSV-free L. striatellus.【Method】A SK sequence (LsSK) from L. striatellus was cloned using PCR. qRT-PCR was employed to analyze the expression pattern of SK gene at different stages (1st-5th instar nymphs, female and male adult) and various organs (head, salivary, midgut, malpighian tubules, ovary and spermary) of the two above mentioned L. striatellus colonies. In order to calculate the sublethal concentration (LC50) of three insecticides (deltamethrin, buprofezin and imidacloprid), those insecticides solutions were applied topically to the mesonotum of L. striatellus 4th-instar nymphs with a hand microapplicator. The SK mRNA level of L. striatellus was detected after exposure to LC50 of three insecticides. After knockdown SK of two L. striatellus colonies by using dsRNA injection, L. striatellus was exposed to LC50 of three insecticides and the mortality was recorded.【Result】a LsSK sequence from L. striatellus was cloned and its length is 1 282 bp(GenBank accession number KT989975). According to the phylogenetic analysis, SK of L. striatellus was clustered together with those from hemipteran insects. The amino-acid sequences of LsSK contained four conserved domains (C1-C4), and the catalytic domain–Diacylglycerol active site formed within C1-C3. qRT-PCR results showed that LsSK was most highly expressed in the 4th-instar nymphs of RSV-infected L. striatellus, followed by 3rd-instar nymphs. In RSV-free L. striatellus, LsSK was most highly expressed in 5th-instar nymphs, followed by 1st- and 4th-instar nymphs. Furthermore, LsSK levels were significantly higher in RSV-infected 3rd- and 4th-instar nymphs than that in RSV-free 3rd- and 4th-instar nymphs(P<0.05). At the adult stage, LsSK levels were significantly higher in RSV-infected adults than in RSV-free adults (P<0.05). Interestingly, LsSK showed extremely higher expression in the tissues of viruliferous males than that of viruliferous females and nonviruliferous adults. The highest expression of LsSK was found in salivary and malpighian tubules of viruliferous males, followed by heads. After exposure to sublethal concentrations (LC50) of three insecticides (imidacloprid 6.5 ng·μL-1, buprofezin 500 ng·μL-1 and deltamethrin 37.5 ng·μL-1), the mRNA levels of LsSK and response speed of two L. striatellus colonies were different. Exposure to buprofezin led to significantly increased expression of LsSK while no significant changes was observed after exposure to deltamethrin(P>0.05). The response of LsSK expression to buprofezin was relatively faster than to the other insecticides. Injection of L. striatellus with dsRNA against SK reduced the mRNA level of LsSK greatly, and silencing of SK led to increased susceptibility in RSV-infected and RSV-free L. striatellus to these three insecticides.【Conclusion】The LsSK was identified and cloned, and it was found significantly high expression in the 3rd and 4th instar of RSV-infected L. striatellus. Silencing the SK resulted in increased susceptibility in L. striatellus to three insecticides, indicated that SK plays a role in facilitating L. striatellus to response to insecticidal stress.
    SOIL & FERTILIZER·WATER-SAVING IRRIGATION·AGROECOLOGY & ENVIRONMENT
    Soil Organic Carbon Lability of Purple Soil as Affected by Long-Term Fertilization in a Rice-Wheat Cropping System
    ZHAO Ya-nan, CHAI Guan-qun, ZHANG Zhen-zhen, XIE Jun, LI Dan-ping, ZHANG Yue-qiang, SHI Xiao-jun
    Scientia Agricultura Sinica. 2016, 49(22):  4398-4407.  doi:10.3864/j.issn.0578-1752.2016.22.012
    Abstract ( 412 )   HTML ( 3 )   PDF (402KB) ( 474 )   Save
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    Objective Based on a 22-year fertilization experiment, soil organic carbon (SOC) and its lability under different long-term fertilization were studied to investigate the SOC quantity and quality of purple soil and their responses to long-term fertilization in a rice-wheat cropping system.MethodThere were six fertilization treatments including no fertilizer (CK), chemical N fertilizer alone (N), chemical NPK fertilizers (NPK), chemical NPK fertilizers plus straw (NPKS), high amount of chemical NPK fertilizers plus equal amount of straw (1.5NPKS) and chemical NPK fertilizer plus manure (NPKM). In soil samples at 0-20, 20-40 and 40-60 cm depths, the labile organic carbon (LOC) and its three fractions with different labilities, i.e., high LOC (HLOC), middle LOC (MLOC) and low LOC (LLOC), were determined according to the oxidation by 33, 167 and 333 mmol·L-1 potassium permanganate (KMnO4) solution, and carbon management index (CMI) was determined by total SOC (TOC) and LOC, and CK was used as reference.ResultThe TOC and LOC were 9.2-16.5 g·kg-1 and 1.58-3.67 g·kg-1 across all treatments and soil depths, respectively. Long-term fertilization could maintain or improve the TOC, LOC content and CMI, with greater improvement on the 0-20 cm soil layer than other layers. Compared with no fertilization, the increases in NPKS treatment were 32.5%, 25.7% and 5.3% for TOC, 37.0%, 44.7% and 9.3% for LOC, 38%, 49% and 9% for CMI on 0-20, 20-40 and 40-60 cm soil layers, respectively, which were relatively greater than other fertilization treatments. Long-term fertilization significantly improved the content of HLOC, MLOC and LLOC on three soil layers with greater increase in treatments with combined application of mineral and organic fertilizers (NPKS, 1.5NPKS and NPKM) than mineral fertilizers alone (NPK and N), while the effect of long-term fertilization on proportions of three labile fractions to LOC was relatively small, indicating that long-term fertilization did not alter the distribution pattern of different LOC fractions. However, the content and proportions of HLOC, MLOC and LLOC were significantly affected by soil depth. On the average, HLOC, MLOC and LLOC accounted for 23.6%, 35.6% and 40.7% of LOC on 0-20 cm soil layer while 30.5%, 44.8% and 24.7% in 20-40 cm soil due to great decline of LLOC content. The LOC, HLOC, MLOC and LLOC were linearly and positively correlated with TOC content, indicating that LOC and its fractions could be used as indicators of TOC change caused by management practices.ConclusionThese results suggested that long-term fertilization could maintain or improve the quantity and lability of SOC and thus CMI, and combined application NPK fertilizers with straw return is the recommended practice to promote both the TOC and LOC accumulation of purple soil in the rice-wheat cropping system.
    Responses of Micropopulation in Black Soil of Northeast China to Long-Term Fertilization and Crops
    DING Jian-li, JIANG Xin, GUAN Da-wei, MA Ming-chao, ZHAO Bai-suo, ZHOU Bao-ku, CAO Feng-ming, LI Li, LI Jun
    Scientia Agricultura Sinica. 2016, 49(22):  4408-4418.  doi:10.3864/j.issn.0578-1752.2016.22.013
    Abstract ( 489 )   HTML ( 13 )   PDF (1170KB) ( 651 )   Save
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    【Objective】This experiment was carried out to investigate the characteristics of micropopulation in northeast China black soil under more 35 years of fertilization of two crop seasons. The effects of inorganic fertilizer and manure on the abundance and structure of micropopulation were identified. Black soil micropopulation responses to the interaction of long-term fertilization and crops would be revealed. This study will provide evidences for further enhancing fertilization and tillage method.【Method】Based on a long-term fertilization experiment carried out in Heilongjiang Academy of Agricultural Sciences, four different fertilization treatment samples in soybean and maize growing seasons were selected including samples without fertilizer (CK); manure (M); inorganic fertilizer (NPK); and inorganic fertilizers with manure (MNPK). Letter “m” before treatment represented maize season sample, and letter “s” represented soybean season sample. The Illumina Miseq sequencing and quantitative PCR of 16S rRNA gene were applied to analyze the effects of fertilization and crops on micropopulation in black soil. Correlation analysis was carried out between micropopulation and the soil properties.【Result】The 16S rRNA gene copy numbers in maize growing season (6.32×108-8.83×108/ng DNA) was lower than that in soybean growing season (0.96×109-2.30×109/ng DNA). Alpha diversity in maize growing season (ACE index was between 3 674.58-4 034.84) was lower than that of soybean season (ACE index was between 4 167.47-4 887.36), too. The top phylum was Acidobacteria (24.47%-27.90%) in maize growing season, but it was Proteobacteria (27.78%-34.40%) in soybean growing season. The relative abundances of Bacteroidetes and Actinobacteria were significantly different between two crop growing seasons. The 16S rRNA gene copy numbers in treatment of inorganic fertilizers with manure was greater than that of inorganic fertilizers. Alpha diversity in treatment of inorganic fertilizers with manure was higher than that of inorganic fertilizers (Chao1 index of sMNPK compared with that of sNPK was more 11.89%). The composition of micropopulation in different fertilization treatments of one crop growing season was different. The relative abundances of Alphaproteobacteria in sMNPK and sNPK compared with that of sCK were more 3.31% and 5.24%, Gammaproteobacteria in sMNPK and sNPK were higher 1.72% and 1.20% than that in sCK, and were sensitive to fertilizers. Correlation analysis showed that 16S rRNA gene copy number was positively correlated with soil NO3--N and available K, the diversity index and soil total N, NO3--N, NH4+-N, available P and available K were closely related. 【Conclusion】Results of the research demonstrated that of long-term different fertilizations and different crop growing seasons have effects on microbial richness, α diversity and community structure. Inorganic and organic fertilizers improved the soil pH, slowed down soil acidification, changed microbial structures, increased microbial richness and diversity as well as the metabolic activity of micropopulation.
    HORTICULTURE
    Effect of Five Different Dwarfing Interstocks of SH on Growth, Yield and Quality in ‘Fuji’ Apple Trees
    LI Min-Ji, ZHANG Qiang, LI Xing-Liang, ZHOU Bei-Bei, SUN Jian, ZHANG Jun-Ke, WEI Qin-Ping
    Scientia Agricultura Sinica. 2016, 49(22):  4419-4428.  doi:10.3864/j.issn.0578-1752.2016.22.014
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    【Objective】Effect of different dwarfing interstocks of SH on growth, yield and fruit quality in ‘Fuji’ apple trees was in-depth studied to provide a theoretical basis and mentoring programs for the selection of suitable dwarfing interstock.【Method】 The ‘Fuji’ apple tree seedlings with different SH dwarf interstock (SH1, SH3, SH6, SH9 or SH40) planted in spring 2009 were used to investigate the dynamic changes of tree growth, yields and fruit qualities from 2010 to 2015. The spacing was 1.5 m×5 m, fine-spun spindle pruning.【Result】There were big differences in the tree growth, fruit yield and quality between ‘Fuji’ apple trees with different SH dwarf interstocks. The tree trunk and total amount of branch were increased along with growth of tree age during the second to seventh year. SH6 dwarfing interstock tree trunk circumference was significantly smaller than others, and the trunk circumferences of SH3 and SH40 dwarfing interstock trees were significantly larger than the other lines. In the first four years, with the growth of age, the proportion of long branch was decreased, the increasing proportion of short branches of all SH dwarf interstock was increased. From the fifth year, the number and proportion of branches of different lengths began to be stable. The ratio of short shoots of SH6 dwarfing interstock apple tree was the highest (63.81%), and the proportion of long branches was the smallest (7%). Variation of branch growth was firstly decreased year by year and then became stable. Annual growth of shoots of SH6 dwarfing interstock trees in the seven year maintained minimum, and the annual growth of shoots of SH3 dwarfing interstock tree showed that the previous two years was high and then quickly came down. All SH dwarfing interstock apple trees began to have production in the fourth year, the cumulative average yield per plant of SH3, SH6, SH9 and SH40 dwarfing interstock apple trees was more than 75 kg, and there was no significant difference among them. The production of SH1 tree was significantly lower than the other four rootstocks. The yield stability of SH6 dwarf interstock was the best, and the yield stability of SH9 dwarf interstock was the worst. The distribution of fruits of SH6 dwarf interstock in different parts of the canopy was the most uniform. The proportion of big fruits (the proportion of fruits weighted more than 200 g) from high to low in order was SH40>SH6>SH3>SH9>SH1. There was no significant difference in average weight of fruit weight, fruit shape and fruit hardness among all SH dwarfing interstock apple trees. The coefficient of variation of fruit shape index of SH6 dwarfing interstock apple trees was the least, and the consistency of its fruit shape was good. The content of soluble solid and solid-acid ratio of its fruit was significantly better than that of the other five stocks.【Conclusion】According to the survey data of seven years, the ‘Fuji’ apple with SH6 dwarf interstock showed small tree, large total branch amount, moderate growth, early bearing, high and stable yield, and good fruit quality compared with the others.
    STORAGE·FRESH-KEEPING·PROCESSING
    Effects of Controlled Freezing Point Storage on the Protein Phosphorylation Level
    ZHANG Yan, LI Xin, LI Zheng, LI Meng, LIU Yong-feng, ZHANG De-quan
    Scientia Agricultura Sinica. 2016, 49(22):  4429-4440.  doi:10.3864/j.issn.0578-1752.2016.22.015
    Abstract ( 376 )   HTML ( 1 )   PDF (2001KB) ( 563 )   Save
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    【Objective】 The aim of this study was to investigate the effect of controlled freezing point on protein phosphorylation level, so as to provide a new theoretical basis for controlling meat quality by controlled freezing point. 【Method】 The longissimus doris (LD) of large tail×small tail crossed sheep were obtained and divided into two groups: refrigeration group and controlled freezing point group. Each LD was assigned to 6 storage times (0.5 h, 12 h, 24 h, 3 d, 5 d and 9 d). Kit for protein kinase activity measurement and enzyme linked immunosorbent assay were applied to detect protein kinase activity. A gel-based phosphoproteomic study was performed. And SDS-PAGE electrophoresis and fluorescence staining were applied to gain the protein bands of sarcoplasmic proteins and myofibrillar proteins stained by SYPRO Ruby and Pro-Q Diamond. A software named Quantity One was used to analyze the protein phosphorylation level by measuring the fluorescence intensity.【Result】 At 12 h, the protein kinase activity of controlled freezing point group significantly increased (P<0.05) and that of refrigeration group significantly decreased. The protein kinase activity of the two groups were reduced from 12h to 9 d, which was significantly higher in controlled freezing group compared with refrigeration group (P<0.05). Seventeen protein bands distributed from 15-250 kDa in each sarcoplasmic protein sample were analyzed and the results suggested that storage temperature influenced (P<0.001) the protein phosphorylation level of all sarcoplasmic proteins significantly. The total phosphorylation level of sarcoplasmic proteins in refrigeration group increased at first and then decreased while the sarcoplasmic proteins in controlled freezing point group showed an opposite variation trend. The phosphorylation level of 3 d was the highest, while the lowest was 24 h. Globally, the sarcoplasmic proteins of refrigeration group had the higher total phosphorylation level than controlled freezing point group during 12 h to 3 d. However, the total phosphorylation level of the sarcoplasmic proteins of controlled freezing point was higher than refrigeration group on 9 d. Twenty protein bands distributed from 15-250 kDa in each myofibrillar protein sample were analyzed and the results suggested storage temperature influenced the protein phosphorylation level of all myofibrillar proteins very significantly (P<0.001). The total phosphorylation level of myofibrillar proteins increased firstly and then decreased in two groups. And the highest phosphorylation level of 24 h in refrigeration group and 12 h in controlled freezing point group was the highest. Total phosphorylation level of myofibrillar protein had no significant difference between two groups from 0.5 h to 12 h. However, total phosphorylation level of myofibrillar protein was higher in refrigeration group compared with controlled freezing group from 24 h to the end of storage.【Conclusion】 The controlled freezing point storage regulated the phosphorylation level of protein by affecting the activity of protein kinase, which can indirectly regulate the meat quality by influencing the glycometabolism, muscle contraction and degradation of skeleton protein.
    ANIMAL SCIENCE·VETERINARY SCIENCERE·SOURCE INSECT
    Effects of Rearing Environment on Physiological and Behavioural Responses of Pigs to Pre-Slaughter Handing, Carcass Straits, and Meat Quality
    ZHU Hong-long, YANG Jie, XU Xiao-bo, PAN Xiao-qing,QIN Feng, LI Jian, XU Ye-fei, ZHOU Xiao-yun, GU Hong-ru
    Scientia Agricultura Sinica. 2016, 49(22):  4441-4450.  doi:10.3864/j.issn.0578-1752.2016.22.016
    Abstract ( 386 )   HTML ( 10 )   PDF (431KB) ( 482 )   Save
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    【Objective】This study aimed to make a comparative analysis of salivary cortisol level before and after transport, behavior during lairage, blood welfare indicators at slaughter of pigs housed in two rearing environments. Carcass traits and meat quality also were evaluated. 【Method】A total of 144 DLY pigs (half of castrated male and female) with body weight (BW) of 27.08±1.06 kg were randomly assigned into either the concrete floor house (CFH) treatment or the deep litter house (DLH) treatment. Each treatment had 6 pens (6 replicates), and according to the similar rearing density (0.85m2/animal), each replicate for CFH and DLH had 10 and 14 pigs, respectively. Animals had always access to feed and fresh water. Feed intake was recorded during the experimental period, and pigs were weighed individually on day 64 and 106 to get the growth performance. At the end of experiment, 10 pigs (half of castrated male and female) with approximately BW of 105 kg from each treatment were selected for slaughter. Salivary samples were collected at home pens (-60 min), 0 and 120 min during lairage for the cortisol analysis, and behavior video during lairage was recorded for observation of pigs’ behavior. Blood samples at slaughter were collected for the analysis of the plasma level of glucose, lactate, cortisol and creatine kinase (CK). Carcass traits (carcass percentage, carcass drip loss, backfat depth and muscle depth) and meat quality (pH value, meat color, intramuscular fat and drip loss) in Longissimus dorsi (LD) were evaluated. 【Result】 Final body weight, average daily feed intake, average daily gain, and feed/gain ratio of pigs from CFH did not show difference from those of pigs from CFH (P>0.10). DLH pigs had higher salivary cortisol level in the home pen, and 0 and 120 min at lairage after transport than CFH pigs, but salivary cortisol rise at 0 and 120min lairage in DLH pigs as compared with the home pen measurement was lower than in CFH pigs (0 min, + 2.85±0.66 vs. + 5.08±1.33, P<0.01; 120 min, + 1.03±0.63 vs. + 2.66±1.54, P=0.04). There were no differences in pigs spending time in resting, standing, exploring, walking, fighting and drinking between DLH and CFH during 0-60 min at lairage (P>0.10). During 60-90 min, however, CFH pigs spent more time in exploring, walking and fighting compared with DLH pigs (P<0.05). Compared with CFH pigs, DLH pigs at slaughter had similar plasma glucose level (P>0.10), but lower levels of lactate and CK, and a higher tendency for cortisol level (P=0.07). Carcass percentage, carcass drip loss, backfat depth and muscle depth from DLH pigs did not show difference from those of from CFH pigs (P>0.10). Concerning the meat quality, DLH tended to have more intramuscular fat content and less drip loss in LD (P=0.10), whereas pH45min, pH24h and meat color (L*, a* and b*) were unaffected (P>0.10).【Conclusion】DLH could decrease time spent in fighting behavior during lairage and physiological response to pre-slaughter handling in pigs. These data showed DLH could improve the ability to copy with pre-slaughter stress in pigs. However, DLH had no effects on growth performance, carcass traits and meat quality.
    Construction of cDNA Library Derived from Human Lung Epithelial Cell Lines and Screening for Host Cellular Proteins Interacting with Influenza Virus Nucleoprotein
    LUO Wei-yu, ZHU Peng-yang, ZHANG Jie, HU Yong-hao, KONG Hui-hui, LIANG Li-bin, ZHOU Yuan, LI Cheng-jun, JIANG Li, CHEN Hua-lan
    Scientia Agricultura Sinica. 2016, 49(22):  4451-4459.  doi:10.3864/j.issn.0578-1752.2016.22.017
    Abstract ( 344 )   HTML ( 1 )   PDF (2066KB) ( 656 )   Save
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    【Objective】In order to study the biological function of influenza virus nucleoprotein and provide a basis for the understanding of mechanism of influenza virus replication and pathogenesis, a yeast two-hybrid library derived from human lung epithelial cell lines Calu-3and A549 was constructed, and the host factors that interact with nucleoprotein (NP) of influenza virus were screened. 【Method】Total RNA was extracted from equal numbers of Calu-3 and A549 cells and their cDNA was synthesized by reverse transcription. Double strand cDNA (dscDNA) was amplified by long-distance PCR (LD-PCR) and purified through CHROMA SPINTM+TE-400 column according to the user manual of Make Your Own “Mate & Plate” Library System (Clontech). The purified dsDNA containing homologous arms was transformed into component Y187 yeast cells together with linearized pGADT7-Rec plasmid, and then the samples were incubated on SD/-Leu plates at 30℃ for about 4 days. All colonies were harvested and aliquoted, resulting in the construction of yeast two-hybrid library of Calu-3 and A549 cells. The library quality was evaluated by capacity, titer, recombination efficiency and diversity. Meanwhile, the EcoRI and BamHI digested NP gene from influenza virus A/Anhui/2/2005 (H5N1) was inserted into pGBKT7 vector to generate the bait plasmid, which was further demonstrated without self-activating activity. The bait plasmid pGBKT7-NP was transformed into Y2H-Gold yeast strain, and then used to screen for interacting proteins from the yeast two-hybrid library. The selected pray plasmids with correct encoding insert and bait plasmids were co-transformed into the yeast cells, with BD-P53/AD-T7 and BD-Lam/AD-T7 plasmids as positive and negative controls, respectively. The blue colonies that grew well in medium containing SD/-Trp/-Leu/-Ade/-His/X-α-gal/AroA(SD/-4/X/A) were selected as candidate containing potential interacting partners of NP. After plasmid extraction and sequencing analysis, these candidates were annotated and classified by Blast and Gene Ontology (GO) analyses. 【Result】Analysis of RNA extracted from the two types of cells showed that both 28S and 18S RNA bands were clear whereas 5S band was faint, indicating that high quality RNA was obtained with almost no degradation. The dscDNA reverse transcribed from RNA was evenly dispersed on the gel with sizes ranged mostly between 500 and 2000 bp, demonstrating that RNA with different abundance and sizes were successfully reverse transcribed. The capacity and titer of the established yeast two-hybrid library were 1.5×107 and 2.2×108 cfu/mL, respectively, with 88% recombination efficiency and sufficient diversity. The screening of the library with the NP bait revealed 11 candidate interacting proteins. Through gene ontology analysis, it was found that these proteins are involved in several biological processes including regulation of cell apoptosis, embryonic development, alternative splicing, transcription regulation translation, metabolic process, regulation of cell proliferation etc. In addition, the molecular functions of these proteins included GTP binding, metal ion binding, DNA binding and transcription factor activity.【Conclusion】 In conclusion, the yeast two-hybrid library containing Calu-3 and A549 cells was successfully constructed, which laid a foundation for the screening of host factors interacting with other influenza virus proteins, and the identification of 11 potential interacting host factors provided preliminary data for further study of the biological function of influenza virus NP protein.
    RESEARCH NOTES
    The Grape Anthocyanin Biosynthesis Regulation by Different Color Fruit Bags
    JI Xiao-hao, WANG Hai-bo, ZHANG Ke-kun, WANG Xiao-di, SHI Xiang-bin, WANG Bao-liang, ZHENG Xiao-cui, WANG Zhi-qiang, LIU Feng-zhi
    Scientia Agricultura Sinica. 2016, 49(22):  4460-4468.  doi:10.3864/j.issn.0578-1752.2016.22.018
    Abstract ( 484 )   HTML ( 1 )   PDF (419KB) ( 701 )   Save
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    【Objective】The aim of this study was to ascertain the effect of different fruit color bags on grape anthocyanin biosynthesis and preliminarily clarify the mechanism, providing theoretical guidance for the development of grape fruit bags. 【Method】The experimental materials were five-year-old vines of ‘Kyoho’ grape using ‘Beta’ as rootstock, bagged with red, green, blue and white fruit paper bags 30 days after fruit setting, respectively, and no bagging as control. The berries were sampled every eight days from bagging to full mature stage. The fruit bag transmission spectrum analysis was tested with StellarNet® Black-Comet Spectrometers. Anthocyanin content in fruit peel was measured by HPLC. The expression of anthocyanin biosynthesis pathway structural genes VvCHS, VvLDOX, VvUFGT, and regulatory gene VvmybA1 and light signal transcription factor VvHY5 were analyzed by real-time PCR. 【Result】Red bag, green bag and blue bag had selective permeability in red, green and blue band, while white bag had no selective permeability for light quality. Different color fruit bags significantly affected grape anthocyanin biosynthesis. Red bag, green bag and blue bag treatments significantly delayed grape variation, and delayed anthocyanin accumulation, but anthocyanin contents in blue bag and white bag treatments were higher than the control at last. RT-PCR analysis showed that the expression of VvmybA1, VvCHS, VvLDOX and VvUFGT increased first and then decreased, which was consistent with anthocyanin accumulation. Different color fruit bag treatments delayed the expression increase of VvmybA1, VvCHS, VvLDOX and VvUFGT at the early stage of fruit ripening and also delayed their expression decrease at the late stage of fruit ripening. Their expression contents were higher in control and white bag treatment, followed by blue bag, green bag and red bag treatments before the expression peaks, while after the expression peaks, their expressions in control were lower than blue bag, white bag, green bag and red bag treatments. In anthocyanin rapid accumulation period and the late stage of fruit ripening, VvHY5 had two expression peaks, which was consistent with the variation patterns of anthocyanin accumulation and the expression of VvmybA1 during fruit ripening. The induction ability of different color fruit bags on VvHY5 expression was different, like that the blue paper bag was the strongest and the red paper bag was the worst.【Conclusion】Blue paper bag is of benefit to grape anthocyanin biosynthesis, while red paper bag is poor. The regulation of anthocyanin accumulation in grape by different color fruit bags is probably through the light signal transcription factor VvHY5, and then regulated the expression of anthocyanin biosynthesis pathway regulatory gene VvmybA1 and structural genes VvCHS, VvLDOX and VvUFGT.