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    01 January 2015, Volume 48 Issue 1
    CROP GENETICS & BREEDING·GERMPLASM RESOURCES
    Identification and Transition Analysis of Photo- /Thermo-Sensitive Genic Male Sterile Genes in Two-Line Hybrid Rice in China
    ZHANG Hua-li, CHEN Xiao-yang, HUANG Jian-zhong, E Zhi-guo, GONG Jun-yi, SHU Qing-yao
    Scientia Agricultura Sinica. 2015, 48(1):  1-9.  doi:10.3864/j.issn.0578-1752.2015.01.001
    Abstract ( 801 )   HTML ( 7 )   PDF (479KB) ( 1316 )   Save
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    【Objective】The objective of this study is to identify and reveal the transition of the male sterile gene(s) in photoperiod- and temperature-sensitive genic male sterile (P/TGMS) lines utilized in the two-line hybrid rice system in China.【Method】A total of 90 environment-conditioned genic male sterile (EGMS) lines including descendents of Nonken 58S, Annong S-1 and Zhu 1S, were used in the present study. Genomic DNAs were extracted from rice leaves by modified CTAB. One functional CAPS marker based on the C to G mutation in the long non-coding RNA (lncR) gene was designed for PGMS genotyping; Namely, a pair of primers NK-F (5′-ATCCCACAAATCCTTTAGCA-3′) and NK-R (5′-CCGTTATAGATAGACCCGAGA-3′) were used to amplify segments harboring the mutation site, followed by digestion overnight at 37℃ with restriction endonuclease RsaⅠ and separation on 1% agarose gel electrophoresis. Homozygous PGMS allele (lncRm) (329 bp) can be readily distinguished from homozygous wild type (lncRwt) (414 bp) and heterozygous type (lncRm/lncRwt) (414 and 329 bp) based on the sizes of digestion products. For TGMS genotyping, functional dCAPS markers were deployed with the following steps: Two pairs of primers RNZ1F (5′-ACCGCGCCGCCACCGGGTCGGCCGGAG-3′)/RNZR (5′-TGAAGAGGAACTCCTGCGAGACGG-3′), RNZ2F (5′-ACCGC GCCGCCACCGGGTCGGCCCAAG-3′)/RNZR were used to amplify segments harboring the mutation site (SNP-+70TA/TC/GC); Amplified products were digested overnight at 37℃ with restriction endonucleases HinfⅠand StyⅠ, respectively, and separated on 8% polyacrylamide gels. Homozygous lines with the TGMS allele (RNZm) cannot be digested by these two restriction enzymes; on the other hand, homozygous wild type lines with the alleles of RNZtc or RNZgc, can be digested completely by HinfⅠ and StyⅠ, respectively; heterozygous genotypes, RNZm/RNZtc and RNZm/RNZgc, can be digested incompletely by HinfⅠand StyⅠ, respectively. By using these functional molecular markers, the PGMS (lncRm) and TGMS (RNZm) genes were identified in commercial EGMS lines that had been utilized in the two-line hybrid rice system. Meanwhile, the transition of P/TGMS genes utilized in two-line hybrid rice production in China (1993-2012) was analyzed according to the information of pedigree and growing area. 【Result】Out of the 47 EGMS lines derived from the PGMS line Nongken 58S, 12 lines carry the PGMS gene lncRm, 29 lines have the TGMS gene RNZm, two lines carry both genes, while the remaining four lines contain none of them. All 18 lines derived from Annong S-1 and Zhu 1S carry RNZm gene. All P/TGMS descendants from crosses between a Nongken 58S derivative (Pei’ai 64S) and Annong S-1 carry RNZm gene. In the two lines derived from Pei’ai 64S and Zhu 1S, one carries both genes, the other has RNZm gene. In addition, in 16 EGMS lines with EGMS progenitors independent from Nongken 58S, Annong S-1 and Zhu 1S, six of them have lncRm, nine of them have RNZm gene, while one of them contains neither lncRm nor RNZm gene. A pedigree map with P/TGMS genes was drawn for 92 EGMS lines, including derivatives from Nongken 58S, Annong S-1, Zhu 1S and others. Furthermore, the transition from lncRm-based to RNZm-based two-line hybrid rice production was shown after examination of statistics data of the two-line hybrid rice planting area during 1993-2012, with the RNZm-based hybrids occupying >95% planting area in the two-line hybrid rice production in 2012. 【Conclusion】The study systematically revealed the P/TGMS genes in commercial EGMS rice and the presence of discrepancy between pedigree of EGMS lines and their P/TGMS gene. The spontaneous emergence of RNZm can be the reason for the transition from PGMS to TGMS in some EGMS lines derived from Nongken 58S. EGMS lines with RNZm currently dominate the two-line hybrid rice production in China.
    CROP GENETICS & BREEDING·GERMPLASM RESOURCES·MOLECULAR GENETICS
    Identification of QTL/Segments Related to Agronomic Traits Using CSSL Population Under Multiple Environments
    XIANG Shi-hua, WANG Wu-bin, HE Qing-yuan, YANG Hong-yan, LIU Cheng, XING Guang-nan, ZHAO Tuan-jie, GAI Jun-yi
    Scientia Agricultura Sinica. 2015, 48(1):  10-22.  doi:10.3864/j.issn.0578-1752.2015.01.02
    Abstract ( 497 )   HTML ( 8 )   PDF (3045KB) ( 775 )   Save
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    【Objective】 The present study was aimed to take a first step of the improvement of the previously reported chromosome segment substitution line (CSSL) population SojaCSSLP1, and to explore superior QTL/gene-alleles related to some agronomic traits from the wild parent (Glycine soja Sieb. et Zucc.) for broadening the genetic basis of cultivated soybean (Glycine max (L.) Merr.). 【Method】 The SojaCSSLP1, with the wild soybean N24852 as donor parent and the cultivated soybean NN1138-2 as recurrent parent, was treated with adding markers and removing a number of lines with segment of single marker, the new population was designated as SojaCSSLP2. By using the new population, the QTL/segments for flowering time (FT), plant height (PH), node number (NN), pod number per plant (PN), 100-seed weight (100SW) and seed weight per plant (PSW) were detected through joint comparisons among CSSLs significantly different from the recurrent parent based on QTL mapping with the methods of single marker analysis (SMA), interval mapping (IM), inclusive composite interval mapping (ICIM) and mixed linear composite interval mapping (MCIM), for experiments in three years each with two locations. 【Result】SojaCSSLP2 was composed of 150 CSSLs, of which 130 ones were the same as SojaCSSLP1, added 40 new SSR markers into the previous molecular map, resulted in the average genetic distance and the number of lines with genetic distance more than 30 cM between adjacent markers reduced from 16.15 cM and 32 to 12.91 cM and 17, respectively. The total length of the genetic map increased by 103.52 cM in comparison to the original map (2 063.04 cM). The genome component of NN1138-2 in CSSLs ranged from 79.45% to 99.70% with an average of 94.62% in SojaCSSLP2. Tested in three years and two locations, 4, 5, 5, 7, 14 and 3 working QTL/segments were identified for FT, PH, NN, PN, 100SW and PSW, respectively, with the improved population. Of those, 15 QTL/segments were joint working QTL which could be detected in more than one environment. The directions of additive effects for wild alleles were consistent with that expected from the parental phenotypes except segment of Sct_190-Sat_293 for NN. Among the QTL, the individual QTL could explain 5% to 64% of the phenotypic variation. There were 3, 2 and 2 QTL/fragments that interacted with locations for PH, NN and NP, respectively, and the interactions associated with Fengyang could increase the value of phenotype, which may be due to its higher latitude than that of Nanjing. These detected loci distributed on 26 substituted segments, among them seven related to more than one trait, which might be the genetic basis of correlation among the traits. Compared with the results in the literature, 3, 3, 2, 2, 8 and 2 QTL could also be detected in other cultivated soybeans for FT, PH, NN, 100SW and PSW, respectively, indicating allele differentiation happened not only between wild and cultivated but also among cultivated soybeans. The other 18 loci/segments were newly discovered in the wild soybean.【Conclusion】 The genetic base of FT, PH and NN are much simpler than that of 100SW. There was a large effect QTL (PV<10%) in the former, while the later is controlled by many small effect loci (PV<10%) with complicated genetic bases. Using the wild soybean, the novel alleles with the capability of broadening the genetic base of cultivated soybean can be explored.
    Differentially Proteomics Analysis of Pre-Harvest Seed Deterioration and Deterioration Resistance in Spring Soybean
    SONG Li-ru, WANG Shuang, NIU Juan, MA Hong-yu, SHU Ying-jie, YANG Yan, GU Wei-hong, MA Hao
    Scientia Agricultura Sinica. 2015, 48(1):  23-32.  doi:10.3864/j.issn.0578-1752.2015.01.03
    Abstract ( 457 )   HTML ( 4 )   PDF (2420KB) ( 690 )   Save
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    【Objective】 Soybean seed is susceptible to high temperature and humidity (HTH) stress resulting in pre-harvest deterioration during its development and maturation (R6 or R7), which limits the production and utilization of soybean in south China. The aim of the present study is to reveal the pre-harvest seed deterioration and deterioration resistance mechanism of spring soybean under HTH stress at proteomic level, thus laying a foundation for breeding new cultivars.【Method】When the deterioration-sensitive cultivar Ningzhen No.1 and deterioration-tolerant cultivar Xiangdou No.3 developed to physiological period (R7), their plants were treated under HTH stress and control for 1, 5, 10, 16 and 24 h, respectively. The extracted proteins from the developing seed of treatment and control were analyzed by two-dimensional electrophoresis (2-DE), and the differentially expressed proteins were identified by MALDI-TOF/TOF. 【Result】Approximately 700 protein spots were detected on each 2-DE gel. Of them, 50 were found to be significantly changed in abundance, with 33 being successfully identified by MALDI-TOF/TOF. The identified differentially expressed proteins were involved in cell rescue and defense (9%), redox homeostasis (12%), protein synthesis (3%), energy metabolism (15%), transport pathway (15%), protein destination and storage (31%). Moreover, the function of 5 identified proteins is unknown. 【Conclusion】Pre-harvest deterioration-resistant cultivar Xiangdou No.3 possessed the greater ability of ROS scavenging and cell rescue and defense than deterioration-sensitive cultivar Ningzhen No.1 under HTH tress, which may be the major reasons why it is more deterioration-resistant than the latter.
    TILLAGE & CULTIVATION·PHYSIOLOGY & BIOCHEMISTRY·AGRICULTURE INFORMATION TECHNOLOGY
    Vigor and Physiological Changes of Different Genotypes of Maize Seed (Zea mays L.) Under Critical Stress Storage Conditions
    CHENG Guang-lei, ZHANG Hai-jiao, ZHAO Jiu-ran, LIU Chun-ge, WANG Yuan-dong, WANG Xiao-guang, WANG Rong-huan, CHEN Chuan-yong, XU Tian-jun
    Scientia Agricultura Sinica. 2015, 48(1):  33-42.  doi:10.3864/j.issn.0578-1752.2015.01.04
    Abstract ( 627 )   HTML ( 9 )   PDF (478KB) ( 990 )   Save
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    【Objective】This experiment was carried out to understand the viability and physiological variation rule of different genotypes maize seeds under critical stress storage conditions, evaluate its of storability objectivity, provide a theoretical basis for clearing the physiological and biochemical mechanisms of maize seed storability. 【Method】 Major maize hybrids(ZD958, ND108, XY335, ZD619, JK968) were used as experimental materials. Seed initial moisture content, vigor and physiological indicators was measured. The moisture content of experimental materials wet back to 14% and stored at temperature of 35, for one year, then the germination energy, germination rate, germination index, vigor index, electrical conductivity, MDA concentration, soluble protein and soluble sugar content and dehydrogenase activity of materials were measured every month. The germination experiment was conducted by using sand culture method, DDS-ⅡA conductivity meter was used to measure the seed electrical conductivity, the TBA was used to measure the MDA content, coomassie brilliant blue G-250 method was used to analyze the soluble sugar content, and dehydrogenase activity was determined by TTC method. The vigor and physiological indicators change of different genotypes maize seeds were compared under the critical stress storage conditions.【Result】Seed genotype was the determinative factor under the same storage condition. The initial moisture content of experimental materials was at a low level, between 7.39% and 8.71%. The germination potential and germination rate of different genotypes maize seed were over 90%, showing a stronger germination ability. After storage for one year, the germination potential and germination rate of JK968 was 50%-60%, germination index was 25%, and vigor index was 0.3. The germination potential and germination rate of ND108, XY335, ZD619 was 15%-25%, germination index was 8%-25%, and vigor index was 0-0.08. The germination potential, germination rate, germination index and vigor index of ZD958 decreased to 0 after storage for one year. Different degrees in decline of germination potential, seed soluble protein content and soluble sugar content changes showed a close relationship with seed vigor. Germination rate, germination index, vigor index were almost the same, however, there was a number of differences in amplitude of variation. Different germination speeds lead to different germination indexes of each genotype of maize seed, seed vigor index declined was always ahead of seed germination ability, which reflected the degree of real seed aging and deterioration. With the storage time extension, the membrane permeability variation of different genotypes of maize seeds, electrical conductivity and MDA content were increased and the change trend was negatively related with seed vigor(R2=0.752). A membrane permeability difference existed among different genotypes of maize seeds. The vigor of different genotypes of maize seed had no significant correlation with the MDA(R2=-0.171-0.094), it significantly positively correlated with soluble protein, soluble sugar and dehydrogenase (R2=0.284- 0.517), but the mechanism of physiological changes of different genotypes of maize seed was complex, the gap was bigger.【Conclusion】 Different genotypes of maize seed were more sensitive to critical stress storage condition, ZD958 was sensitive to critical stress, the vigor and physiological indicator changes of JK968 were stable, showed a higher storability.
    Relationship Between Light Interception and Light Utilization of Soybean Canopy in Relay Strip Intercropping System
    CUI Liang, SU Ben-ying, YANG Feng, YANG Wen-yu
    Scientia Agricultura Sinica. 2015, 48(1):  43-54.  doi:10.3864/j.issn.0578-1752.2015.01.05
    Abstract ( 452 )   HTML ( 1 )   PDF (1383KB) ( 1101 )   Save
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    【Objective】 Intercropping, as one of the most universal multiple cropping systems based on biodiversity, plays an important role in enhancing crop productivity for more efficient use of resources, such as land, light, temperature and water as well as reducing incidence of weeds, insects, pests and diseases to meet the future human demand. Cereal-legume relay intercropping system is commonly used in China, because of the different distances between maize and soybean which influence the PAR distribution above the soybean canopy within relay strip intercropping system, and it is not only bad to the development of canopy structure but also decreases the light interception of intercropped soybean, thus resulting in limited photosynthetic active radiation use efficiency of intercropped soybean which restricts the productivity of maize-soybean relay strip intercropping systems. Therefore, there is a need for exploring an optimum spatial-temporal configuration management of intercropping system based on competition so that light resources requirement of soybean can be realized in this system. 【Method】 An experiment with maize (Zea mays L.) cultivar Chuandan 418 and soybean (Glycine max L. Merr.) cultivar Gongxuan1 were used in this study. The relay strip intercropping systems were designed as the distances of maize intercropped with soybean were 40 cm (A),50 cm (B) and 60 cm (C), and both of maize monoculture (SM) and soybean monoculture (SSB) were used as control. The soybean population canopy structure, light interception, dry matter weight in the relay strip intercropping combinations were measured and analyzed under maize/soybean relay strip intercropping systems in order to optimize the reasonable group configuration.【Result】 The PAR density was different above the soybean canopy under different maize-soybean relay strip intercropping systems, and lower than SSB significantly (P<0.05). During the co-growth stage of maize and soybean, the PAR density occurred in treatment A was lower than that in treatments B and C by 44.1% and 60.4%, respectively. This means that the PAR density decreased as the degree of low light stress increasing due to the distance between maize and soybean decreased. The LAI, LA and plant height of intercropped soybean varied considerably in different maize-soybean relay strip intercropping systems. During the stages of V5, V7 and R1, the LAI of treatment B was higher than treatment A and treatment C significantly. Treatment B was higher than that in treatments C and A by 16.4%, 13.1%, 12% and 30.3%, 32.2%, 29.3%. LA of treatment B was higher than that in treatments C and A by 15%, 16%, 14% and 34%,31%,26%. Plant height of treatment B was lower than that in treatments C and A by 7%, 8.8%, 7.9% and 13.5%, 16.7%, 14.8%, which means that suitable soybean canopy structure can improve RUE. The LI of intercropped soybean was decreased significantly under different maize-soybean relay strip intercropping. During the stages of V5, V7 and R1, the LI of treatments A and C was lower than that in treatment B by 43%, 22%, 33% and 21%, 10%, 17%. A positive correlation of LAI and LI was significant (0.977**), which means that LAI might be the main factor due to the LI increasing. The photosynthetic active radiation use efficiency of intercropped soybean varied considerably in different maize-soybean relay strip intercropping systems. During the stages of V5, V7 and R1, the RUE of treatment B was higher than that in treatments C and A by 8.6%, 7%, 5.8% and 40%, 23%, 13%. The dry matter weight of soybean in all intercropping systems was lower than monoculture significantly (P<0.05), and varied considerably in different maize and soybean relay strip intercropping systems. During the stages of V5, V7 and R1, the dry matter weight of treatments A and C was lower than that in treatment B by 59%, 36%, 41% and 27%, 16% and 22%. A positive correlation of DMW and LI was significant (0.989**), which means that the dry matter increased with the light interception increasing. The yield of intercropped soybean and total yield varied considerably in different maize and soybean relay strip intercropping systems(P<0.05). The yield of treatment B was higher than that in treatments C and A by 10% and 27%, the total yield of treatment B was higher than that in treatments C and A by 1% and 3%. This means that the yield of intercropped soybean increased/decreased as influenced by the intensified low light stress due to the difference of distance between maize and soybean. 【Conclusion】In this case, suitable distance of maize and soybean configuration could optimize intercropped soybean canopy structure, improve the light use efficiency and enhance yield.
    PLANT PROTECTION
    Establishment of SYBR Green I Real-Time PCR for Quantitatively Detecting Rhizoctonia cerealis in Winter Wheat
    SUN Bing-jian, CHEN Qing-qing, YUAN Hong-xia, SHI Yan, LI Hong-lian
    Scientia Agricultura Sinica. 2015, 48(1):  55-62.  doi:10.3864/j.issn.0578-1752.2015.01.06
    Abstract ( 563 )   HTML ( 6 )   PDF (1216KB) ( 1200 )   Save
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    【Objective】Wheat sharp eyespot (WSE) caused by Rhizoctonia cerealis is one of the most important soil-born diseases on wheat in China. Early accurate quantitative detection is a foundation of forecast and control. Traditional method of organization isolation and identification of pathogen is time consuming, complicated and can’t be accurately quantified. In order to implement the early and quick quantitative determination of wheat sharp eyespot, a SYBR Green I real-time PCR method of R. cerealis was established based on the pathogen sequence information.【Method】 Based on the β-tubilin of R. cerealis, a pair of specific primers was designed. The SYBR Green I real-time PCR reaction system was established and optimized. The sensitivity, specificity and repeatability of the system were also evaluated, and RBipolaris sorokiniana, Fusarium graminearum, F. pseudograminearum were used for control fungi. The indoor potted plants of wheat which infected by R. cerealis were detected with optimized reaction system after inoculated for 5, 10 and 60 days, respectively.【Result】The primers were of great specificity, the specific PCR fragment was amplified from the DNA of R. cerealis isolates, but not from the DNA of other fungal isolates by conventional PCR. The real-time PCR assays also did not amplify DNA from control fungi. The sensitivity of conventional PCR was 6.5×103 copies/μL plasmid, while the sensitivity of real-time PCR was 6.5×102 copies/μL. The standard curve established by recombinant plasmid showed a fine linear relationship between threshold cycle and template concentration. The melt curve was specific with the correlation coefficient of 0.997 and with high amplification efficiency (0.91). For the indoor potted experiments, the detection results of real-time PCR of infected wheat samples, were showed a significant positive correlation with disease index and inoculum, respectively. 【Conclusion】 The developed real-time PCR assay for R. cerealis is fast, highly specific, sensitive, and reproducible. This method can be used to detect R. cerealis in wheat, and guidance prediction and control of wheat sharp eyespot.. cerealis, R. solani, AG-A, AG-F, Gaeumannomyces graminis var. tritici,
    Effect of Candidatus Liberibacter asiaticus Infection on Carbohydrate Metabolism in Citrus sinensis
    WU Yue, SU Hua-nan, HUANG Ai-jun, ZHOU Yan, LI Zhong-an, LIU Jin-xiang, ZHOU Chang-yong
    Scientia Agricultura Sinica. 2015, 48(1):  63-72.  doi:10.3864/j.issn.0578-1752.2015.01.07
    Abstract ( 431 )   HTML ( 1 )   PDF (433KB) ( 986 )   Save
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    【Objective】The objective of this experiment is to study the effect of Huanglongbing (HLB) infection on carbohydrate metabolism in Citrus sinensis, elucidate the relationship between HLB infection and starch accumulation, and provide a theoretical basis for HLB pathogenesis. 【Method】C. sinensis were used as the experimental materials, plants were inoculated by grafting three bark pieces onto the rootstock portion of each plant. For infected plants, bark pieces were derived from trees infected with Candidatus Liberibacter asiaticus, which were confirmed positive for Ca. L. asiaticus by PCR. For non-infected plants, bark pieces were obtained from healthy trees. Three plants in good growth state were chosen for successive test, mature leaves were collected every month until October. DNA and RNA were extracted, the content of soluble sugar and starch were measured immediately, the other leaf tissue was snap-frozen with liquid nitrogen and stored at -80 for testing carbohydrate metabolism related key enzyme activities. At the same time, the expression levels were compared using real-time quantitative PCR analysis. 【Result】The content of soluble sugar and starch tended to increase with time extended, and their peak value were respectively reached 1.59 and 3.73 times of control in June, then slightly decreased later, the ratio of soluble sugar to starch dropped down. Various key enzymes imposed different hands on carbohydrate metabolism at different stages of infection. Sucrose phosphate synthase activity rapidly rose to the top 58.44 μg?g-1?min-1 and had become lower than control in late infection; acid invertase had been consistently high and the difference was significant (P<0.05), the highest activity was 2.91 times of control at middle stage; the activity of neutral invertase was kept at a lower level and had the same change trend in experimental group and control group; soluble starch synthase activity was closely related to granule-bond starch synthase, coordinating the synthesis of acting on the starch, while amylase activity had dropped to some extend at each stage, even as low as 0.38 U?g-1 at middle stage. Real-time quantitative PCR indicated that there was a significant relationship between the expression level of sucrose phosphate synthase and its enzyme activity (P<0.05). Among the sucrose decomposition related genes, sucrose synthase gene was not changed obviously and had lower change range while cell wall-bound invertase gene CSCWI was up-regulated with the highest value of about 7.4 times and expressed abundantly during the whole process. Starch decomposition-related genes BAM3, MEX1 and DPE2 had a relatively reduced expression and the difference was significant (P<0.05) in different periods.【Conclusion】HLB infection has influence on the formation and transportation of photosynthate in C. sinensis and results in the disruption of carbohydrate metabolism balance, it is in deep connection with starch accumulation and the late stage symptoms.
    Molecular Characterization and RNAi-Based Functional Analysis of Obstructor Family Genes in Locusta migratoria
    WANG Yan, LI Da-qi, LIU Xiao-jian, LI Tao, MA En-bo, FAN Ren-jun, ZHANG Jian-zhen
    Scientia Agricultura Sinica. 2015, 48(1):  73-82.  doi:10.3864/j.issn.0578-1752.2015.01.08
    Abstract ( 361 )   HTML ( 1 )   PDF (582KB) ( 722 )   Save
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    【Objective】 The objective of this study is to obtain cDNA sequences of Obstructor (Obst) family genes of cuticular proteins from Locusta migratoria, clarify their molecular characterization and biological function, and to get new molecular target for pest management. 【Method】 The cDNA fragments of Obst family genes were searched from locust transcriptome database and the sequences were further analyzed by BLAST at NCBI, the candidated cDNA fragments belonging to the Obst family genes were confirmed by sequence alignment with other known insect Obst family genes. The primers were designed for non full-length cDNA sequences and RACE-PCR was performed to amplify 3′ cDNA sequences, the full-length cDNA sequences of Obst family genes were assembled by overlap region. All the full-length cDNA sequences were translated into amino acid sequences and signal peptides were analyzed by SignalP tool, functional domains were predicted by SMART website. Then phylogenetic tree was constructed by using Mega 5.10 software with homologous amino acid sequences encoded by the Obsts from Drosophila melanogaster and the CPAP3 family genes (the Obst homologous genes) from Tribolium castaneum. The real-time quantitative PCR (qPCR) was applied to analyze the gene expression patterns of LmObst genes in different tissues and developmental stages of the 5th instar nymphs in the integument. RNA interference (RNAi) technology was used to explore biological function of these Obst genes. 【Result】 EightcDNA fragments assumed to be Obst family genes were got from locust transcriptome database, which showed high similarity with CPAP3 family genes of T. castaneum and Obst family genes of D. melanogaster by BLAST analysis. Five of them were the full-length cDNA sequences, and three of them missed 3′ end sequences. Then three full-length cDNA sequences were further amplified by using RACE-PCR technique. The functional domain analysis of eight LmObstsshowed that they all had a signal peptide and three chitin binding domain ChtBD2, which are in accordance with the characteristics of the representative insect Obst cuticular protein. According to the result of phylogenetic analysis, eight LmObst genes were named as LmObst-A1, LmObst-A2, LmObst-B, LmObst-C, LmObst-D1, LmObst-D2, LmObst-E1 and LmObst-E2, respectively. The qPCR results showed the tissue distribution of eight LmObst genes. LmObst-E1 and LmObst-E2 werespecifically expressed in foregut and hindgut, LmObst-D1 was mainly expressed in integument and foregut, the others were highly expressed in integument, foregut and hindgut, and lowly expressed in gastric caeca, midgut, Malpighian tube and fat body. Developmental expression patterns showed that they had a similar trend, eight LmObst genes were highly expressed at the early stage of the 5th instar nymphs, gradually reduced to a minimum at the middle stage, then got raised before molting. RNAi was applied to explore their biological function, eight dsLmObsts were injected into the 5th day of 5th instar nymphs, respectively, and the control group was injected with equal amount dsGFP. The silencing efficiency was detected at 48 h after dsRNA injection, and the mRNA expression was significantly reduced. Further phenotypic observation showed that 80% nymphs injected with dsLmObst-E1 appeared no molting and died, the rest 20% nymphs could molt to the next stage, but the molting time delayed about 1-2 d, after molting to adults, the development was retarded and died within 16 h. However, the control nymphs with dsGFP injection successfully molted to the adults and developed well. The nymphs injected with the other dsObsts displayed slow development, the molting time delayed about 1-3 d, but no visible abnormal phenotypes were found. 【Conclusion】 The eight full-length cDNA sequences of Obst family genes were obtained from L. migratoria. LmObsts had typical domain structure of Obst protein with a signal peptide and three chitin binding domain (ChtBD2). LmObsts were mainly responsible for the formation of integument, foregut and hindgut, which developed from ectoderm. LmObst-E1 is essential for locust development, silencing LmObst-E1 was lethal to L. migratoria, the mRNA suppression of the other seven LmObsts displayed developmental delay, but no lethal effect was observed.
    SOIL & FERTILIZER·WATER-SAVING IRRIGATION·AGROECOLOGY & ENVIRONMENT
    Carbon Footprint Analysis of Crop Production in North China Plain
    WANG Zhan-biao, WANG Meng, CHEN Fu
    Scientia Agricultura Sinica. 2015, 48(1):  83-92.  doi:10.3864/j.issn.0578-1752.2015.01.09
    Abstract ( 659 )   HTML ( 10 )   PDF (2533KB) ( 1001 )   Save
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    【Objective】Based on the statistic data of cultivated area, sown area, crop yield, production value and farmland investment of the crop production in the period of 1993 to 2012, the dynamics, distributions and compositions of carbon footprint (CF) of the crop production were estimated in North China Plain (NCP), which will provide a theoretical support and scientific basis for the development of low-carbon agriculture. 【Method】Based on the historical statistic data of cultivated area, sown area, crop yield, production value and farmland investment of the crop production of 47 prefecture-level cities in 5 provinces and cities (Hebei, Beijing, Tianjin, Shandong and Henan) in North China Plain, and used the theory of carbon footprint and life cycle assessment method in the agricultural sector, CF per unit sown area (CFs), CF per unit cultivated area (CFc), CF per unit yield (CFy) and CF per unit production value (CFv) of the crop production were estimated in North China Plain. The dynamic and distributions of carbon footprint were analyzed briefly. In addition, the proportions of different agricultural inputs in carbon footprint of crop production were explored.【Result】 CFs and CFc of crop production showed an increasing trend in the last twenty years. The CFs of crop production was 4.08 tCO2eq·ha-1 in period I (1993-2002) and 4.72 tCO2eq·ha-1in period II (2003-2012), respectively; The CFc of crop production was 6.81 tCO2eq·ha-1 in period I (1993-2002) and 8.12 tCO2eq·ha-1in period II (2003-2012), respectively. CFy and CFv of crop production showed a decreasing trend in the last twenty years. The CFy of crop production was 0.54 tCO2eq·t-1 in period I and 0.42 tCO2eq·t-1in period II, respectively. The CFv of crop production was 3.91 tCO2eq·¥10-4 in period I and 2.51 tCO2eq·¥10-4 in period II, respectively. The carbon footprints were different in different regions of the NCP. Most coastal regions (Tangshan, Tianjin and Yantai, etc.) have bigger CFs and CFc and smaller CFy and CFv. On the contrary, most cities of South Henan (Zhumadian, Xinyang, and Pingdingshan, etc.) have smaller CFs and CFc and bigger CFy and CFv. The agricultural inputs per unit sown area and the vegetable crops sown area percentage in high yield region were significantly higher than that in low yield region, respectively. The results presented here indicated that the main components of carbon footprint were electricity for irrigation (30.25%), N fertilizer (23.07%), and direct emissions of N2O (19.83%).【Conclusion】In the last twenty years, the CFs and CFc of crop production increased, but the CFy and CFvdecreased in North China Plain. Most coastal regions have bigger CFs and CFc and smaller CFy and CFv, but the regions of southern Henan province were the opposite. In addition, the electricity for irrigation and N fertilizer were the major resources of emissions in North China Plain.

    Research Progress and Analysis of Carbon Footprint of Livestock Products
    HUANG Wen-qiang, DONG Hong-min, ZHU Zhi-ping, LIU Chong, TAO Xiu-ping, WANG Yue
    Scientia Agricultura Sinica. 2015, 48(1):  93-111.  doi:10.3864/j.issn.0578-1752.2015.01.10
    Abstract ( 646 )   HTML ( 11 )   PDF (2250KB) ( 993 )   Save
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    Livestock production is one of the important emission sources of greenhouse gases (GHGs), evaluation of the carbon footprint of livestock products is vital for selection of mitigation technology and promotion of low-carbon agriculture. Based on current evaluation methods of carbon footprint, this study summarized the domestic and overseas researches on assessment of the carbon footprint of animal products (eggs, pork, beef and milk), and made a comprehensive analysis based on the present research achievements. Carbon footprint of livestock products varies with unit of animal products. The carbon footprint in producing 1 kg of beef is the greatest and reaches (20.51±8.39) kg CO2-eq, followed by 1 kg of pork and eggs production with (4.24±1.07) kg CO2-eq and (2.24±0.83) kg CO2-eq, respectively, while that in producing 1 kg milk is the minimum of (1.19±0.40) kg CO2-eq. The carbon footprint in producing 1 kg protein from animal products is in a descending order as beef>milk>pork and egg, with values of (103.05±42.14), (39.72±13.20), (32.09±8.14) and (19.37±7.15) kg CO2-eq, respectively. The carbon footprint in producing 1 kg fat from animal products is in a descending order as beef>milk>egg and pork, with values of (488.25±199.65), (37.23±12.37), (29.28±10.80) and (11.45±2.91) kg CO2-eq, respectively. The carbon footprint of 1 000 kcal energy from animal products are beef, milk, egg and pork in descending order, with values of (16.41±6.71), (2.21±0.73), (1.56±0.57) and (1.07±0.27) kg CO2-eq, respectively. Analyses on different links of animal products revealed that the share of greenhouse gas emissions reaches maximum in feed crop planting and producing & processing during egg and pork production, accounting for (74.0±16.5)% and (61.3±7.6)%, respectively. The methane emission from enteric fermentation delivers the greatest contribution to carbon footprint during beef and milk production, accounting for (53.7±8.2)% and (52.7±6.1)%, respectively. Analyses on GHGs emissions from animal products showed that CO2 contributes the maximum to the carbon footprint during egg production, whose emission covers (55.42±2.7)% of the entire system. N2O contributes the maximum during pork production, with (56.8±10.4)% of the entire system. CH4 contributes the maximum during beef and milk production, with (50.2±8.3)% and (58.6±8.3)%, respectively. Although researches on carbon footprint of livestock products based on different methodologies are mostly in abroad, unified assessment guidance needs to be developed to evaluate the carbon footprint of livestock products. There is little research on carbon footprint in China, so it’s suggested that an assessment method suitable for actual production in China should be established. The result of this study could provide some preliminary data to the assessment of carbon footprint of livestock product and identification of mitigation options.
    HORTICULTURE
    QTL Mapping for Parthenocarpy in Cucumber
    WU Zhe, LI Lei, ZHANG Ting, ZHANG Ting-lin, LI Ji, LOU Qun-feng, CHEN Jin-feng
    Scientia Agricultura Sinica. 2015, 48(1):  112-119.  doi:10.3864/j.issn.0578-1752.2015.01.11
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    【Objective】Cucumber is one of the ten vegetables in the world and parhenocarpy is an important trait closely related to production and quality of cucumber. To explore the inheritance and QTL mapping for parthenocarpy in cucumber could provide a preliminary basis for further study on mechanism of parthenocarpy and molecular assistant selection breeding, and lay a theoretical foundation for breeding of parthenocarpy.【Method】In this study, The authers clipped eight female flowers on main stem and branches, respectively, for every individual plant and investigated parthenocarpic fruit once when all plants treament finished 8-10 days later to calculate the parthenocarpy percentage (numbers of parthenocarpis fruit/numbers of clipped female flower) in order to evaluate parthenocarpy ability. Two F2 progenies derived from two crosses between EC1, a gynoecious parthenocarpic line, and two monoecious non-parthenocarpic lines 8419 and 14519 were constructed to determine the inheritance of parthenocarpy in cucumber. A linkage map from part of F2 plants from the cross of EC1×8419 was constructed with JoinMap4.0 software by screening 1 335 SSR from 9930 and gy14 cucumber genome sequencing and 143 Indel primers from two parents resequencing and QTL detection for parthenocarpy was conducted with WinQTLcart2.5 software using F2:3 families from the same cross. The candidate genes in major QTL region were predicted using bioinformatic analysis method.【Result】Parthenocarpy in EC1 was inherited quantitatively but segregated towards different parents in two F2 progenies. A linkage map containing 7 chromosomes, 116 SSR and 9 Indel markers was constructed, which total length was 802.9 cM and average distance between two markers was 6.3 cM. QTL analysis identified 7 QTLs, Parth1, Parth2-1, Parth2-2, Parth3-1, Parth3-2, Parth5, and Parth7,distributing on chromosomes 1, 2, 3, 5, and 7. The major QTL Parth2-1 locating between SSR00684-SSR22083 was the only locus detected in two seasons, having two LOD scores of 9.0, 6.2 and R2 of 17.4%, 10.2%, respectively, and its genetic and physical distance was 17.1 cM and 2.9 Mb. There were 307 genes in this region and two gene among them, Csa2M035330.1 and Csa2M070880.1, involved in plant hormone signal transduction would be candidate genes closely related to parthenocarpy, the rest were minor QTLs.【Conclusion】The inheritance of pathenocarpy was quantitative. The QTL Parth2-1 locating on chromosome 2 was the major QTL controlling parthenocarpy in cucumber and two genes in plant hormone pathway would be candidate genes. The results in this study would lay a foundation for fine mapping and gene cloning of major QTL of parthenocarpy in cucumber and for use in MAS breeding.
    Effect of Exogenous Spermidine on Levels of Endogenous Hormones and Chloroplast Ultrastructure of Ginger Leaves Under Heat Stress
    LI Xiu, GONG Biao, XU Kun
    Scientia Agricultura Sinica. 2015, 48(1):  120-129.  doi:10.3864/j.issn.0578-1752.2015.01.12
    Abstract ( 523 )   HTML ( 7 )   PDF (1242KB) ( 861 )   Save
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    【Objective】Ginger is a thermophilic vegetable crop, but not tolerate high temperature. It is easily damaged under high temperature. To study the effect of exogenous spermidine on endogenous hormones and chloroplasts, the relationship among exogenous spermidine, endogenous hormones, and the chloroplast ultrastructure of ginger under heat stress was investigated.【Method】Laiwu Big Ginger was sandy cultured in a climate chamber with 12 h/12 h photoperiod under 28℃/18℃ (normal) and 38℃/28℃ (heat stress) conditions. Ginger root was treated with 0.5 mmol·L-1spermidine. Relative water content, chlorophyll concentration, malondialdehyde content, electrolyte leakage, chlorophyll fluorescence parameters, reactive oxygen content and endogenous hormones metabolism of ginger leaves were investigated on 0, 5, 10, 15 and 20 d after treatment, moreover, the ultrastructures of chloroplasts and thylakoid were observed on 20 d after treatment. 【Result】Under heat stress, chloroplasts and thylakoid of ginger leaves were seriously damaged, and chlorophyll concentration significantly decreased with continuing stress. Chlorophyll fluorescence parameters including Fv/Fm, ФPSII, Fv′/Fm′, qP and P decreased, and NPQ, β/α-1 and D increased, which mainly showed that its photochemical activity of PSII was decreased, so the  and H2O2 accumulated extensively resulted by excess energy, and then the MDA content and electrolyte leakage increased. Under heat stress, superoxide dismutase activity, abscisic acid and proline contents were significantly accumulated and then decreased in different treatment times. Ascorbate peroxidase activity, cytokinin and kinetin content kept a decreasing trend with continuing stress. However, relative water content, chlorophyll concentration, malondialdehyde content and electrolyte leakage of ginger leaves were recovered by exogenous application of spermidine. Besides, it also maintained the integrity of chloroplasts and thylakoid, regulated chlorophyll fluorescence parameters to normale, increased antioxidant enzymes activity and endogenous hormones concentration, and reduced the reactive oxygen content. 【Conclusion】Heat stress can damage ginger leaves, leading to function of chloroplasts and PSII disorder, and reactive oxygen and endogenous hormones abnormity. Exogenous application of spermidine can effectively reduce the damages of ginger caused by heat stress and improve its tolerance to heat stress, involving maintain endogenous hormones regular metabolism and chloroplasts normal physiological function.
    Effects of Orchard Mulching Grass on the Microstructure and Function of Photosystem in Apple Leaves
    Lü San-san, DU Guo-dong, LIU Zhi-kun, Lü De-guo, LI Shuang
    Scientia Agricultura Sinica. 2015, 48(1):  130-139.  doi:10.3864/j.issn.0578-1752.2015.01.13
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    【Objective】 The influencing mechanisms of orchard mulching grass on the photosynthetic apparatus as well as the physiological function of apple leaves in the northern of the Bohai Gulf were studied in order to provide a theoretical basis for organic cover measures in orchard. 【Method】 ‘Hanfu’ apple (Malus domestica Borkh./GM256/M.baccata Borkh.) and Digitaria sanguinalis (L.) Scop. were used as materials, a potted experiment was conducted to determine the effects of orchard mulching grass together with using N on microstructure, photosynthetic pigment contents, gas exchange parameters and overall function of photosynthetic apparatus of apple leaves. The experiment were divided into four parts which were mulching grass treatment (C1N0, 1 kg grass per pot), nitrogen fertilizer (C0N1, 3.4 g N per pot) and mulching grass + nitrogen fertilizer treatment (C1N1, 1 kg grass and 3.4 g N per pot) and control (CK). 【Result】 The results showed that C1N0 and C0N1 treatments increased the palisade tissue thickness as well as the ratio of PT/ST, however, compared with the control, there was no obvious difference in spongy tissue and the leaf thickness. Leaf microstructure showed that C1N1 treatment increased the thickness of the palisade tissue, spongy tissue and leaf by 8.45%, 12.91%, and 19.34%, respectively. It also significantly improved the ratio of PT/ST. Straw mulching treatment could change photosynthetic pigment contents, the ratio of chlorophyll and photosynthetic gas exchange parameters, among them, chlorophyll a content, total chlorophyll content, the ratio of chlorophyll a/b and net photosynthetic rate (Pn) increased by 22.7%, 12.71%, 23.42% and 22.83%, respectively. The contents of chlorophyll a, chlorophyll b and total chlorophyll in C1N1 treatmentwere 1.42, 1.04 and 1.37 times than that of control, it also increased the net photosynthetic rate (Pn) and water use efficiency (WUE) by 41.71% and 21.99%, respectively. Photosynthetic pigment content and photosynthetic physiological parameters were higher in C0N1 and C1N1 treatments than that of C1N0 treatment, and control was the lowest. The chlorophyll a fluorescence transient and 820 nm reflection kinetics of apple leaves changed obviously. The JIP-test showed that different treatments increased the chlorophyll fluorescence parameter including maximal photochemistry efficiency of PS Ⅱ (Fv/Fm), efficiency/probability that an electron moves further than QA- (ψo), quantum yield for electron transport (φEo), which showed C1N1>C0N1>C1N0>CK. Photosynthetic performance index (PIABS), biggest redox ability (ΔI/Io) of PSⅠ were significantly higher than that of control. Different kinds of treatments promoted the development of apple plant. There was no significant difference between C1N0 treatment and control in leaf length. However, C0N1 treatment increased leaf length by 10.63% compared with the control. C1N0 and C0N1 treatments not only increased leaf width and leaf area, but also promoted the development of the trunk circumference by 8.82% and 12.35%. In addition, apple leaf length, leaf width, leaf area and trunk circumference of C1N1 were 1.14, 1.19, 1.44 and 1.21 times higher than that of control, respectively. 【Conclusion】 Under potted experiment, orchard mulching grass and nitrogen fertilizer treatment could improve the overall function of photosynthetic apparatus, and the development level of apple plant significantly.
    ANIMAL SCIENCE·VETERINARY SCIENCERE
    Association of GHSR and GHRL Gene Genetic Variation with Growth Traits in Two Guizhou Goat Breeds
    SONG Tao-wei, CAI Hui-fen, LUO Wei-xing, LIU Ruo-yu, ZHANG Yi-yu, SUN Yan-yan, LIU Bin
    Scientia Agricultura Sinica. 2015, 48(1):  140-153.  doi:10.3864/j.issn.0578-1752.2015.01.14
    Abstract ( 578 )   HTML ( 3 )   PDF (3896KB) ( 632 )   Save
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    【Objective】 The aim of this study was to explore the relationships between GHSR and GHRL gene and body weight & size traits, and search the molecular genetic markers related to goat growth traits.【Method】Guizhou white goat and Guizhou black goat were chosen as subjects, the DNA pooling was constructed and the technology of direct sequencing of PCR products and PCR-SSCP were used to detect the single nucleotide polymorphism of GHSR and GHRL genes, and its association of polymorphisms and different genotypes and combined genotypes with growth traits was analyzed by SPSS(18.0). Meanwhile, the bioinformatics analysis of GHSR gene was conducted by online software.【Result】The result showed that two SNPs (G200A and T628C) were detected in exon 2 and 3′UTR but no SNPs in exon1 and 5′UTR of GHSR gene, respectively. G200A was a sense mutation site and devided into three genotypes GG, GA and AA by PCR-SSCP. Genotype GG was the dominant genotype and allele G was dominant allele with the frequencies of 0.6731 and 0.5243 in Guizhou white goat and Guizhou black goat (♀) groups instead of allele A in Guizhou black goat (♂). All populations were moderate polymorphic in G200A site (0.25<PIC<0.50). G141A was found in intron 2 and two sense mutation sites (T78C and C14T) were identified in exon2 and exon4 of GHRL gene, respectively. There were two genotypes (CT and CC) in C14T site, genotype CC was the dominant genotype, which the frequency were 0.7692, 0.9417 and 0.9390, allele C was dominant allele with the frequency of 0.8846, 0.9709 and 0.9695, respectively. These populations were low polymorphic (PIC<0.25). χ2 test indicated that SNPs(G200A and C14T) fit with Hardy-Weinberg equilibrium in the population (P>0.05). Correlation analysis revealed that G200A site was significantly associated with body weight, body height, body length and hucklebone width (P<0.05), GG genotype was superior genotype. Additionally, body weight, body height and chest girth of individuals of Guizhou black goat (♂)with genotype CC was significantly higher than those with genotype CT(P<0.05) in C14T site. Meanwhile, the combined genotype was significantly associated with body height and chest girth, the individuals with CCGG genotype had significantly better body height than that of genotype CCAA (P<0.05). Bioinformatics analysis indicated that no core promoter regions and CpG island were found but a CAAT-box and several important transcription factors in 5′UTR region of GHSR gene. G200A had led to the change of secondary structure of mRNA. The secondary structure of protein was mainly α-helix (48.90%). Tertiary structure and transmembrane helix prediction showed that GHSR protein was a membrane protein.【Conclusion】The results revealed that SNPs and combined genotype of GHSR and GHRL gene had effect on growth traits in goat, G200A and C14T sites could be used as effective genetic markers for goat molecular breeding.
    Effect of Different Nano-Zinc Levels in Dietary on Semen Quality, Activities of Antioxidant Enzyme and Expression of Copper Zinc Superoxide in Epididymis of Ram Lambs
    ZHANG Chun-xiang, QIN Xiao-wei, GUO Li-na, ZHANG Guo-lin, ZHANG Jian-xin, REN You-she
    Scientia Agricultura Sinica. 2015, 48(1):  154-164.  doi:10.3864/j.issn.0578-1752.2015.01.15
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    【Objective】 The objectives of this study were to investigate the effects of nano-zinc supplementation in dietary on the semen quality parameters, the activities of antioxidant enzyme in seminal plasma and the expression of copper zinc superoxide dismutase in epididymis of ram lambs, and to analyze the correlation between semen quality parameters and the activities of antioxidant enzyme.【Method】Sixteen 9-month-old Jinzhong ram lambs with good health and approximate weight were randomly divided into 4 groups, fed with a basal diet with supplementation of 0, 50, 100 and 150 mg·kg-1 DM nano-zinc, respectively. The experimental period was 90 d. Semen was collected on 78 d and 79 d in consecutive two days, samples of 100 µL fresh semen was used to analyze semen quality parameters, the rest of fresh semen sample was centrifuged at 2 000 r/min for 10 min, the supernatant (seminal plasma) was collected for measurement of the activities of copper zinc superoxide dismutase, glutathione peroxidase and total anti-oxidation competence. All rams were castrated for collection of epididymis caput, corpus and cauda at the end of experiment. Expression of Cu-ZnSOD protein in epididymis was detected and located by immunohistochemistry. The mean optical density was analyzed with Image Pro Plus 7.0 software.【Result】The results showed that there was no significant difference in ejaculate volume between the treatment groups and control group (P>0.05). Compared with the control group, the groups with supplementation of 50 mg·kg-1 or 100 mg·kg-1 nano-zinc had higher sperm concentration, sperm motility and membrane integrity, and less percentage of abnormal sperm. The group with supplementation of 150 mg·kg-1 had higher membrane integrity than control group, while it had no significant difference in sperm concentration, sperm motility with control group. The rams in groups with supplementation of 50 mg·kg-1 or 100 mg·kg-1 nano-zinc also had higher activities of Cu-ZnSOD, GPxs and total anti-oxidation competence, and less MDA concentration in seminal plasma. The rams in group with supplementation of 150 mg·kg-1 had higher total anti-oxidation competence than the control group, while they had no significant difference in activities of Cu-ZnSOD, GPxs and MDA content with rams in the control group. The result of immunohistochemistry showed the positive signals were detected in pseudostratified columnar epithelium in epididymis caput and corpus, and simple columnar epithelium in epididymis cauda. The order of expression of Cu-ZnSOD protein in epididymis from rams in the treatment group was corpus>caput>cauda, while those in the control group was caput>corpus>cauda. The mean optical density of Cu-ZnSOD was higher in the group with supplementation of 50 mg·kg-1 or 100 mg·kg-1 nano-zinc in epididymis caput and corpus. The activity of Cu-ZnSOD in seminal plasma had a positive correlation with sperm concentration, sperm motility and membrane integrity, and a negative correlation with percentage of abnormal sperm.【Conclusion】The optimal supplementation of 50 mg·kg-1 or 100 mg·kg-1 nano-zinc in diet could improve semen quality and seminal plasma anti-oxidase activities, and the expression of Cu-ZnSOD in epididymis of rams. The activity of Cu-ZnSOD in seminal plasma could be regarded as index of evaluation of semen quality in sheep production. The further researchs need to be done in molecular mechanism of trace element zinc regulation on semen quality.

    Melanin Synthesis of Alpaca Melanocytes Regulated by miR-663 Through Targeting TGF-β1
    JIA Xiao-yun, JIN Lei-hao, MIAO Lian-juan, DING Na, FAN Rui-wen, DONG Chang-sheng
    Scientia Agricultura Sinica. 2015, 48(1):  165-173.  doi:10.3864/j.issn.0578-1752.2015.01.16
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    【Objective】 The objective of the present study is to identify the target genes of miR-663 and investigate the role of miR-663 in melanin synthesis in alpaca melanocytes.【Method】 The potential targets and binding sites of TGF-β1 were predicted and analyzed by Targetscan, RNAhybrid and RNA22. The similarity of 3′UTR of TGF-β1 sequences from various species was analyzed by DNAMAN. The dual-luciferase construct of pmirGLO-TGF-β1-3′UTR was created by inserting partial TGF-β1 3′UTR into the pmirGLO vector by Sacand XbaⅠ restriction sites. The regulation of TGF-β1 by miR-663 was validated by co-transfecting pmirGLO-TGF-β1-3′UTR construct with miR-663 mimic into 293T cells. The over-expression of miR-663 was achieved by transfecting melanocytes with miR-663 mimic. The mRNA and protein levels of TGF-β1, Smad3, Smad4, Smad7 and β-catenin in melanocytes transfected with miR-663 mimic were analyzed by qRT-PCR or Western blotting, respectively. The effects of miR-663 on melanin synthesis were evaluated by measuring the melanin content of the cells.【Result】There are 68 potential targets for miR-663 predicted by bioinformatics, including 74 conserved binding sites and 44 less conserved binding sites. DNAMAN analysis showed that all 3′UTR sequences of TGF-β1 from analyzed species are highly conserved and enriched potential target sites. One of the potential targets of miR-663 is TGF-β1, which is involved in the development of hair follicle as well as melanin pigmentation. The alpaca 3′UTR sequence of TGF-β1 contains three miR-663 potential binding sites. To confirm the regulation of TGF-β1 by miR-663 through its 3′UTR, a dual-luciferase reporter vector pmirGLO-TGF-β1-3′UTR was successfully constructed and co-transfected into 293T cells with miR-663 mimic. The luciferase assay experiments showed that the luciferase activity was 31.01% lower in cells co-transfected with pmirGLO-TGF-β1-3′UTR and miR-663 mimic than that in control cells, suggesting TGF-β1 is a direct target of miR-663. When miR-663 mimic was transfected into melanocytes, the mRNA level of miR-663 increased to 345% but those of TGF-β1, β-catenin and Smad4 were reduced by 89%, 41%, and 34%, respectively. The protein level of TGF-β1 was reduced by 21%. The contents of melanin were significantly reduced by 42%.【Conclusion】TGF-β1 is a direct target gene of miR-663. Overexpression of miR-663 led to the decreased expression of TGF-β1 both at protein and mRNA levels. The miR-663 may influence/affect synthesis through regulation of TGF-β1 directly as well as TGF-β/Smad and Wnt signal pathways indirectly in skin melanocytes of Alpaca.
    RESEARCH NOTES
    Cd Uptake and Distribution Characteristics of Cd Pollution-Safe Rice Materials
    ZHANG Lu, ZHANG Xi-zhou, LI Ting-xuan, JI Lin, ZHENG Tao
    Scientia Agricultura Sinica. 2015, 48(1):  174-184.  doi:10.3864/j.issn.0578-1752.2015.01.17
    Abstract ( 463 )   HTML ( 1 )   PDF (394KB) ( 765 )   Save
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    【Objective】 It is important to minimize the influx of cadmium (Cd) to the human food chain through consumption of agricultural products. The characteristics of the uptake and distribution of Cd in pollution-safe materials were studied to provide Cd safety rice germplasm resources. 【Method】 A field experiment was conducted to investigate the differences of 56 rice parent materials in Cd uptake and distribution in polluted paddy field. According to cluster analysis, the pollution-safe materials were chosen by the Cd content of the brown rice, and Cd uptake and distribution characteristics of rice parent materials were analyzed.【Result】There were significant differences in the Cd contents and Cd accumulations at tillering stage(CV=44.05% and CV=50.21%), booting stage(CV=23.57% and CV=28.62%) and mature stage(CV=44.98% and CV=44.69%) among the 56 rice parent materials when the field soil Cd content was 13.89 mg·kg-1. Meanwhile, the Cd contents in brown rice ranged from 0.15 to 1.77 mg·kg-1 among the parent materials, the ratio of maximum and minimum value reached 11.80, and the minimum value of Cd content was lower than the National Food Safety Standard. The 56 rice parent materials were divided into pollution-safe materials, general materials and high accumulation materials depending on the Cd content of brown rice. The Cd content of brown rice of pollution-safe materials was 0.2 mg·kg-1 which was significantly lower than that of the general materials (0.65 mg·kg-1) and the high accumulation materials (1.57 mg·kg-1). Moreover, the lowest Cd contents of chaff and grain partition coefficient were also observed in pollution-safe materials. Shoot Cd contents in the three kinds of materials were significantly decreased with the growth stage prolonged. Furthermore, shoot Cd contents in the pollution-safe materials were significantly lower than that of the general materials and high accumulation materials at tillering, booting and mature stages. Especially, the Cd contents in shoot of the general materials and high accumulation materials were 1.35 and 3.39 times higher than the pollution-safe materials at mature stage. The pollution-safe materials exhibited significantly lower Cd accumulations in shoots compared to the general materials and high accumulation materials at the three growth stages. The maximum differences among the three kinds of materials were observed at maturity stage. The Cd accumulations in shoots of the general materials and high accumulation of materials were 2.23 and 3.86 times higher than that of the pollution-safe materials at mature stage. The maximum differences among the three kinds of materials were also observed at maturity stage. The greatest Cd accumulation in shoots of pollution-safe materials was observed at sowing-tillering stage. However, there were no difference among the three growth stages in the general materials and high accumulation materials. Due to the lower metastatic ability of Cd to grain, the pollution-safe materials have lower Cd content in grain. Meanwhile, the distribution ratio of Cd accumulations in grain was 8.11% of the total Cd accumulations in aboveground of the pollution-safe materials, which was lower than that of the general materials (11.60%) and high accumulation materials (17.59%).【Conclusion】Among the pollution-safe materials, the Cd contents in the brown rice of D62B, IRBN95-90 and GRlu 17/ai TTP//lu 17_2 were lower than the National Food Safety Standard (0.2 mg·kg-1). Thus, D62B, IRBN95-90 and GRlu 17/ai TTP//lu 17_2 can be considered as Cd safety rice germplasm resources for Cd-polluted farmlands.
    Cloning and Expression Analysis of Sugarcane NADP+-Dependent lsocitrate Dehydrogenase (SoNADP-IDH) Gene
    XIE Xiao-na, YANG Li-tao, WANG Sheng, ZHANG Xiao-qiu, LI Yang-rui
    Scientia Agricultura Sinica. 2015, 48(1):  185-196.  doi:10.3864/j.issn.0578-1752.2015.01.18
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    【Objective】The aim of this study was to clone the full-length cDNA of sugarcane NADP+- dependent lsocitrate dehydrogenase, a key enzyme gene relating to tricarboxylic acid cycle (TCA) in sugarcane, to provide a candidate gene for breeding of sugarcane disease resistance and other stress tolerance.【Method】Two-dimensional electrophoresis (2-DE) was used to study the variation expression of proteins, and the differential proteins were identified by MALDI TOF/TOF. The SoNADP-IDH gene cDNA sequence was cloned from sugarcane variety GT11 using RT-PCR techniques. Online software was used to analyze the putative amino acid sequence, and qRT-PCR method was used to study the expression of SoNADP-IDH gene in different tissues and under different stresses. 【Result】 The full-length cDNA of SoNADP-IDH (GenBank accession number KF808326) in sugarcane was cloned. The full-length cDNA was 1 497 bp with an intact open reading frame of 1 239 bp, encoding a polypeptide of 412 amino acids. The bioinformatics analysis showed that the protein was a hydrophilic protein, did not contain a signal peptide, but contained four transmembrane regions. This protein was a soluble and stable protein. Secondary structure analysis showed that the protein contains alpha helix, bend region, extended strand, random coil, and alpha helix and random curl occupied most of the protein secondary structure. Online software analysis showed that the gene contains 19 phosphorylation sites, 4 N-glycosylation sites, 4 casein kinase phosphorylation sites Ⅱ, 4 N-myristoylation site of action, 6 protein kinase C phosphorylation sites and 1 tyrosine kinase phosphorylation site. Homology analysis showed that the deduced SoNADP-IDH protein was highly homologous to other IDH proteins from different species. The qRT-PCR analyses showed that the SoNADP-IDH expressed in root, stalk and leaf. Furthermore, SoNADP-IDHtranscription level was impacted under the treatment of the pathogen of ratoon stunting disease, low temperature, PEG, NaCl and ABA stresses, but the expression patterns were different. In PEG simulated drought treatment, the transcript of SoNADP-IDH showed first increase and then decrease, reached the peak at 6 h, then continued to decline, and reached the minimum at 48 h. In the 4 cold stress treatment, the expression of SoNADP-IDH reached the minimum at 3 h, with the extension after treatment, the expression of SoNADP-IDH increased, reached the peak at 24 h, then slightly decline at 48 h. After ABA application, the transcript of SoNADP-IDH showed a “suppression-increase-suppression-increase” pattern, the transcript of SoNADP-IDH levels decreased at 3 h, reached the peak at 6 h, to a minimum at 24 h, and rose again at 48 h. In salt-treatment, the expression of SoNADP-IDH showed an “increase-suppression-increase” pattern, reached the peak at 6 h, then began to decline sharply, down to the lowest at 24 h, then began to rise, and basically flatted with the control at 48 h 【Conclusion】 The gene SoNADP-IDH was firstly isolated and characterized from sugarcane, and the pathogen of ratoon stuning disease, low temperature, PEG, NaCl and ABA stresses impacted the expression of the gene, indicating that the SoNADP-IDH gene may play an important role in resistance to oxidative stress of sugarcane.
    Cloning, Subcellular Localization and Expression Analysis of Gibberellin 2-Oxidase Gene in Diospyros kaki Linn. cv. Nantongxiaofangshi
    TU Xu-tong, ZHANG Shi-jie, CHEN Xiao-yun, LI Ning-ning, XIN Lu, XUE Xiao-hui, ZHANG Zhen, QU Shen-chun
    Scientia Agricultura Sinica. 2015, 48(1):  197-206.  doi:10.3864/j.issn.0578-1752.2015.01.19
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    【Objective】 This paper aims to isolate the cDNA of GA2ox from Diospyros kaki Linn. cv. Nantongxiaofangshi, and do some preliminary study on their functions and expression level, in order to lay a foundation for further exploration of the dwarf mechanism and breeding of new dwarf cultivars. 【Method】 Total RNA was extracted from leaves of ‘Nantongxiaofangshi’ persimmon by improved CTAB method. Twofragments were identified from cDNA of ‘Nantongxiaofangshi’ by degenerate primers, and their full length cDNA were acquired by RACE amplification and named as DkGA2ox1 and DkGA2ox2, respectively. Gene structure characteristics were analyzed using the bioinformatics software. Quantitative real-time PCR (qRT-PCR) was performed to determine the expression pattern during pre-budding period, budding period, leaf expanding period, tip buds dying period, flowering period, physiological fruit-falling period, fruit coloring period, fructescence and abscission period. 【Result】 The full length cDNA of DkGA2ox1 and DkGA2ox2 were 1 318 bp and 1 267 bp, respectively, containing 5′ untranslated region (UTR) with lengths of 198 bp and 61 bp, 3′ UTR with lengths of 97 bp and 172 bp, and coding region with lengths of 999 bp and 1 005 bp which encoded 332 and 334 amino acids. The two amino acid sequences shared 73%-77% in homology compared with Populus tomentosa (JX102472.1), oleander (AY594292.1), tobacco (AB125232.1), Petunia (GU059939.1), apple (FJ571521.1), pears (JF441168.1) and grapes (JQ608472.1). The conserved structural domain analysis revealed that DkGA2ox1 and DkGA2ox2 had the typical functional domains of GA2ox protein, containing Fe2+ binding sites (DkGA2ox1: His-205, Asp-207, His-262; DkGA2ox2: His-204, Asp-206, His-261) and 2-oxoglutarate binding sites (DkGA2ox1: Arg-272, Ser-274; DkGA2ox2: Arg-271, Ser-273), as well as the 2OG-Fe(II)-Oxy protein domains. The protein molecular weights were 36 596.1 Da and 37 544.2 Da, respectively. Both of them are stable proteins, have no signal peptide, transmembrane domains, and significant hydrophobic region, as well as belong to C19-GAoxs. After construction of transient expression vector and onion epidermal cell transformation, subcellular localization assays showed that the GA2ox1 protein was located in the nucleus and cytoplasm. The quantitative RT-PCR results showed that the highest expression levels of DkGA2ox1 and DkGA2ox2 were detected in florescence, and all higher than those in vigorous cultivar ‘Dafangshi’ during all the 7 phenological periods. 【Conclusion】 The expression of gibberellin 2-oxidase genes in ‘Nantongxiaofangshi’ is related with the dwarf trait.