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Table of Content

    01 April 2017, Volume 50 Issue 7
    CROP GENETICS & BREEDING·GERMPLASM RESOURCES·MOLECULAR GENETICS
    Identification of Heterotic Loci for Plant Traits Using Chromosomal Segment Substitution Lines Test Population in Maize
    LIU XiaoYang, WEI XiaoYi, CHEN Hao, LIU Kun, XIE HuiLing, GUO ZhanYong, FU ZhiYuan, LI WeiHua
    Scientia Agricultura Sinica. 2017, 50(7):  1179-1188.  doi:10.3864/j.issn.0578-1752.2017.07.001
    Abstract ( 502 )   HTML ( 6 )   PDF (371KB) ( 848 )   Save
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    【Objective】 Plant characters including plant height, ear height, etc., are important traits that can decide planting density and grain yield in field. They also have high heterosis, and used as a model to dissect the genetic basis of heterosis in studies, so identifying the heterotic loci for plant characters can provide useful information for selecting elite commercial maize hybrids.【Method】 A set of single segment substitution lines (SSSLs) population, which was constructed using the inbred line Xv178 as the receptor parent and the inbred line Zong 3 as the donor parent. The test populations were crossed with the receptor parent Xv178, and were used as the material in this study. The populations were evaluated in the field by following three replicates in randomized complete blocks carried out at Xunxian, Xinxiang and Xuchang locations in Henan province in 2014. Ten plants of each material were selected for measuring plant height, ear height, leaf number after the pollen shedding in the field. The heterotic loci for plant characters were identified through significant analysis comparing to each test hybrid and its mid-parent value by mean of t test. 【Result】 The three traits of plant characters of SSSL test population all had some heterosis, the mid-parent heterosis data of plant height were 4.74%, 3.61% and 1.09% in the Xunxian, Xinxiang and Xuchang locations, respectively, the mid-parent heterosis values of ear height were 6.06%, 7.77% and 7.51%, and leaf number had a little heterosis. A total of 9 QTL for plant height, 10 QTL for ear height, 5 QTL for leaf number were identified in the SSSL population in three environments. In the test population, 6 heterotic loci for plant height were identified, and 3 heterotic loci were detected in three environments simultaneously. For ear height, 8 heterotic loci were detected, including 1 heterotic locus identified in three locations simultaneously. There were 5 heterotic loci detected for leaf number, and 1 heterotic locus was identified in three environments simultaneously. A total of 24 QTL and 19 heterotic loci were identified for the 3 measured traits of plant character in the SSSL and its test population, 5 QTL and heterotic loci were identified in the same SSSLs. 【Conclusion】 The results revealed that the heterosis in plant height and ear height was greater than that in plant leaves number. Of the detected QTL and HL, a certain number of them were conserved across different environments. The chromosomal regions of these QTL/HL probably contain the important genes for corresponding trait. And, a few of chromosomal segments were proved to be associated with multiple traits, which indicated that these traits interact with each other during the plant development. Most of QTL/HL for plant height and ear height sowed over-dominant effects, but less over-dominant QTL/HL for plant leaf number. It implies that high heterosis is associated with high proportion of QTL/HL. Here the three tested traits are associated with each other, achieving balance among them is an important lodging-resistant breeding target. The above mentioned QTL and HL can be used by MAS (molecular assistance selection) to realize the target of elite plant architecture breeding.
    Genome-Wide Association Analysis of Salt Tolerance Related Traits in Brassica napus and Candidate Gene Prediction
    HE YaJun, WU DaoMing, YOU JingCan, QIAN Wei
    Scientia Agricultura Sinica. 2017, 50(7):  1189-1201.  doi:10.3864/j.issn.0578-1752.2017.07.002
    Abstract ( 716 )   HTML ( 30 )   PDF (1163KB) ( 1132 )   Save
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    【Objective】To find the SNP loci and candidate genes associated with salt tolerance in rapeseed, genome-wide association analysis of salt tolerance related traits in B. napus was performed. 【Method】 The seeds of 307 inbred lines of the association panel were germinated in Petri dishes under 1.2% NaCl. The control was under the sterile dH2O. Three salt tolerances related traits, root length, fresh weight and germination rate of each line were measured seven days after planting. The relative value of root length, fresh weight and germination rate under the salt tolerance condition were calculated. The software TASSEL5.0 were employed to examine salt tolerance related traits in B. napus using a genome-wide association study with a 60K Brassica Illumina® Infinium SNP array. The software SPAGeDi v1.4 was used to calculate the relative kinship matrix comparing all pairs of the 307 accessions. STRUCTURE v2.3.4 was employed to analyze the population structure of the association panel. In this study, to avoid the false negative associations, three mixed models controlling relative kinship, K model, Q+K model and PCA +K model were chosen to determine the statistical associations between phenotypes and genotypes to evaluate the effects of population structure and relative kinship on salt tolerance related traits. The optimum model was determined according to observed P values and expected P values of all SNPs. TASSEL 5.0 was used to perform genome-wide analysis under the optimum model. Based on the physical positions of the associated SNP loci, the corresponding genomic sequences of the regions and flanking sequences (200 kb upstream and downstream of the SNPs) were extracted. The genes in the target intervals were extracted subsequently. According to the salt tolerance related genes in Arabidopsis, the homologous genes of rapeseed were screened out in the target genome interval.【Result】The association analysis identified 164 SNP loci significantly associated with the root length, 23 SNP loci with fresh weight, and 38 SNP loci with germination rate. The SNP loci which were most significantly associated with root length, fresh weight and germination rate were located on chromosomes A08, A02 and A06, and explained 23.84%, 18.59% and 31.81% of the phenotypic variance, respectively. Fifty candidate genes were found to be related to salt tolerance in the target interval of B. napus. These candidate genes include transcription factors Myb, WRKY, ABI1, bZIP, ERF1, CZF1, XERICO and some different functional genes which are regulated by the transcription factors, such as NHX1, PTR3, CAT1, HKT, CAX1, ACER, STH, STO, etc. The common salt tolerance gene BnaA03g14410D on chromosome A03 was screened out according to two different traits, root length and germination rate. In addition, the salt tolerance candidate genes contain two pairs of tandem repeats genes which are BnaA03g18900D and BnaA03g18910D located on chromosome A03 and BnaC09g19080D, BnaC09g19090D and BnaC09g19100D located on chromosome C09. Moreover, the salt tolerant candidate genes also contain two duplicated genes BnaC02g39600D and BnaC02g39630D with very close distances.【Conclusion】Totally, 225 SNP loci were detected significantly associated with salt tolerance related traits and fifty candidate genes were found.
    Lignin and Lignin Monomer Synthetic Characteristics of Culm in Common Buckwheat with Different Lodging Resistance Capabilities
    SHE HengZhi, NIE Jiao, LI YingShuang, LIU XingBei, HU Dan, MA Shan, Ciren zhuoga, WANG Can, WU DongQian, RUAN RenWu, YI ZeLin
    Scientia Agricultura Sinica. 2017, 50(7):  1202-1209.  doi:10.3864/j.issn.0578-1752.2017.07.003
    Abstract ( 349 )   HTML ( 8 )   PDF (957KB) ( 898 )   Save
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    Objective】 This research aims to study the content change rule of lignin, total lignin monomer, G, S and H type lignin monomer of culm with different lodging resistance capabilities of buckwheat cultivars at different stages, so as to determine the lignin and lignin monomer synthetic characteristics.【Method】The varieties of Youqiao2 (YQ2, high lodging resistance), Xinnong1 (XN1, medium lodging resistance) and Ukraine Daliqiao (UD, sensitive to lodging) were used as test materials. The field experiment arranged in a factorial randomized complete design, with three replicates. Acetyl bromide method was used to determine the lignin content of different lodging resistance varieties at different growth stages. GC-MS method was conducted to determine the content of total lignin monomer which includes G, S and H type lignin monomer. Stems in common buckwheat were used to determine stem bending force and lodging index. 【Result】 At same growth stages, YQ2 was superior in culm breaking resistance and lodging index compared with XN1 and UD. The culm breaking resistance increased firstly and then decreased from the stage of branching to milk ripe, the maximum values arrived at full bloom stage. The lodging index among different varieties changed differently, the lodging index of YQ2 and UD increased and then decreased from branching to milk ripe stage, and the maximum values arrived at full bloom stage. The lodging index of XN1 increased gradually from branching to milk ripe stage, and the maximum values arrived at milk ripe stage. The content of culm lignin and lignin monomer increased from branching to milk ripe stage. The YQ2 contained higher lignin and lignin monomer than XN1 and UD. At the same growth stage, the content of S type monomer was higher than G type monomer, and the H type monomer was the lowest. From branching to milk ripe stage, S and G type monomer increased gradually, but H type monomer decreased gradually. Furthermore, from branching to milk ripe stage, the ratio of S/G decreased, and the ratio of G type monomer to total lignin monomer increased gradually. 【Conclusion】The main type of lignin monomer of culm in common buckwheat is S-G type. At different growth stages, the content of lignin, total lignin monomer, G, S and H type lignin monomer of high resistance varieties are higher than moderate resistance and lodging susceptible varieties. The S type lignin monomer increased quickly at branching stage, and G type lignin monomer increased in a great deal from branching to maturity stage.
    TILLAGE & CULTIVATION·PHYSIOLOGY & BIOCHEMISTRY·AGRICULTURE INFORMATION TECHNOLOGY
    Progresses in Research of Evaluation Index System and Its Method on Arable Land Intensification: A Review
    SHI ShuQin, CAO YuQing, WU WenBin, YANG Peng, CAI WeiMin, CHEN YouQi
    Scientia Agricultura Sinica. 2017, 50(7):  1210-1222.  doi:10.3864/j.issn.0578-1752.2017.07.004
    Abstract ( 396 )   HTML ( 4 )   PDF (584KB) ( 591 )   Save
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    Arable land intensification is a necessary choice to face the multiple pressure of socio-economic development, limits of land use quantity, population growth and technology improvement. Started with the interactive framework among arable land intensification, agricultural sustainable utilization, ecological protection and grain security, this paper summarized the current indicator system of arable land intensification, proposed the evaluation index system of intensive cultivated land use based on interactive framework, and reviewed the existing technology, method and their related achievements. Based on above analysis, the next hotspot and development trend of future study were proposed. The result showed that arable land intensification, agricultural sustainable utilization, ecologic-environment and grain security play a role of mutual improvement mainly or with the dual influence of positive or negative. The current indicator system of arable land intensification can be considered mainly in different view of frequency indices, input indices, output indices, potential indices, gap indices, comprehensive indices, intensity indices, energy indices, etc. The evaluation index system of intensive cultivated land use based on interactive framework included some basic indexes such as the average labor input, average fertilizer input, average pesticide input, irrigation index, etc. Indexes which affecting agricultural sustainable utilization mainly included food security factor, non-farming index, cultivated land area per capita, etc. And indexes strongly correlated with grain safety included multiple cropping index, stable production index, grain yield per hectare, per-cultivated land production, etc. Nevertheless, the further choice and revision of the indexes should be considered according to the different study region and scale in specific condition. Within all the research method relating those indices, the paper found that frequency indices were the hotspot of remote sensing focused on arable land intensification. Potential indices were researched mainly by model simulation. On the contrary with the abundant research achievements of yield gap, harvest gap got little concern in those gap indices today. In addition, comprehensive measuring model of intensity also was the main and common method to appraise the arable land intensification. It is noteworthy that some other related research results can be utilized further to appraising the arable land intensification. The type of arable land intensification indices demonstrates the development trend of diversified characteristic. Besides, the comprehensive integration of new method and new technology should push forward in the future.
    Effects of BPDS-Fe(Ⅱ) on the Difference in Tolerance to Iron Deficiency of Maize Seedlings Under Different Ammonium/Nitrate Ratios
    XU JianQin, CHEN XuLei, YU FuTong
    Scientia Agricultura Sinica. 2017, 50(7):  1223-1233.  doi:10.3864/j.issn.0578-1752.2017.07.005
    Abstract ( 309 )   HTML ( 5 )   PDF (1474KB) ( 349 )   Save
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    【Objective】Iron deficiency in crop production is a very common problem in both developing and developed regions. And screening of Fe-efficient genotypes has become more and more meaningful and promising. In this study, two maize inbred lines Wu312 and Ye478 were used to investigate the effects of BPDS-Fe(Ⅱ) on the phenotypes and physiological traits in maize. This work aimed at unraveling the difference in tolerance to iron deficiency between these two maize inbred lines and their mechanisms. 【Method】BPDS (4,7-diphenyl-l,10-phenanthrolinedisulfonic acid) was used to chelate ferrous iron supplied with two different NH4+-N/NO3--N ratios. Phenotypes were observed and some physiological traits were measured, including SPAD value of young leaves, dry matter weights of shoot and root, active Fe concentration in young leaves, and Fe, Mn, Cu, Zn concentrations in shoots. SPAD value was determined by SPAD-502 plus. Samples were cut into pieces and digested by HNO3-H2O2. Trace elements were determined by ICP-AES. Leaf samples of 2.00 g were soaked in 20 mL HCl (mol·L-1) and determined by ICP-AES.【Result】Root exudation was generated in two inbred lines and the solution pH decreased from 6.0 to approximately 4.0 during the last 24 hours when supplied with NH4NO3. Compared with the treatment without iron, shoot dry weights of Wu312 and Ye478 in the treatment of 40 μmol·L-1 BPDS-Fe(Ⅱ) were decreased by 31% and 64%, respectively; root dry weight of Wu312 showed no significant difference; and root dry weight of Ye478 was declined by 63%. The ratios of R/S of both inbred lines were out of range (normal value, 0.20-0.28). Solution pH has decreased by no more than one unit and plants turned to become healthy when supplied with 0.5 mmol·L-1 NH4+-N and 3.5 mmol·L-1 NO3--N. Ye478 has the ability to sustain more dry weights in shoots and roots, higher leaf SPAD value as well as active Fe concentration than Wu312, showing remarkable advantages of growth and staying green. When supplied with 0.1 μmol·L-1 BPDS-Fe(Ⅱ), Wu312 could not compete with Ye478 to produce its fifth leaf, finally turned out to be dead. However, Ye478 produced the fifth leaf and turned to be green obviously. There was no significant difference in shoot Fe concentrations between these two lines. Shoot Fe content was all negatively correlated to Mn, Cu and Zn concentrations in shoots. 【Conclusion】Reduction of the ratio of NH4+-N has improved the solution pH and inhibited root exudation. Ye478 is a Fe-efficient genotype and Wu312 is a Fe-inefficient genotype. The most significant difference of phenotypes between the two inbred lines occurred with 0.1 μmol·L-1 BPDS-Fe(Ⅱ). Dry matter weights of shoot and root, together with leaf SPAD value were identified to be most suitable physiological traits charactering the tolerance to iron deficiency of maize seedlings. R/S ratios and active Fe concentration were found to be good indicators. Possible mechanisms contained the release of protons, preferential allocation of biomass to roots, and the translocation and redistribution of iron.
    PLANT PROTECTION
    Influencing Factors of Conidiospore and Expression Analysis of GATA Transcription Factor Gene Family in Setosphaeria turcica
    FENG ShengZe, LIU XingChen, WANG HaiXiang, ZHAO Jie, ZHAO LiQing, ZHENG YaNan, GONG XiaoDong, HAN JianMin, GU ShouQin, DONG JinGao
    Scientia Agricultura Sinica. 2017, 50(7):  1234-1241.  doi:10.3864/j.issn.0578-1752.2017.07.006
    Abstract ( 316 )   HTML ( 6 )   PDF (433KB) ( 669 )   Save
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    【Objective】 Northern leaf blight of corn is an important leaf disease and its agent is Setosphaeria turcica. The objectives of this study are to determine the main influencing factors in conidium yield and the expression characteristics of GATA transcription factor gene family at conidium formation stage in S. turcica. The research will lay a foundation for further illuminating the molecular mechanism of regulating growth, development and pathogenicity in S. turcica. 【Method】S. turcica wild-type strain 01-23 was used as test material in the study. The main factors which affecting conidium yield was analyzed as follows. The media were lactose casein agar, potato dextrose agar, corn stalk dextrose agar, corn stalk agar, corn leaf dextrose agar, corn leaf agar, corn niblet dextrose agar, corn niblet agar, corn starch dextrose agar, corn starch agar, czapek, minimal medium, and water agar. The carbons were lactose, glucose, sucrose, fructose, and maltose. Nitrogen sources were beef jelly, peptone, NH4Cl, KNO3,  (NH4)2SO4, NH4NO3, which were added in lactose casein agar medium respectively and the content was 5 g in per liter medium. The pHs were 4, 5, 6, 7, 8, and 9. The temperatures were 15, 20, 25, 30, and 35℃. The light intensities were 1 500, 3 000, 6 000, and 9 000 lx, respectively. quantitative real-time PCR (qRT-PCR) technology was employed to analyze the expression characteristics of GATA family members during conidium formation period. 【Result】 The optimum conditions for conidia yield in S. turcica based on single factor method were as follows. The most appropriate medium was maize leaf dextrose agar medium, pH was 8, 25℃, 6 000 lx light intensity (12 hours light and 12 hours dark). In addition, it was found that the addition of KNO3 in lactose casein agar medium significantly increased conidial yield. GATA2 significantly enhanced its expression level while other GATA family members, such as GATA1, GATA3, GATA4, GATA5, cut down their expression level at conidium formation stage compared with mycelium development stage. 【Conclusion】 The optimum culture condition of S. turcica conidium yield was determined based on single factor method in the study. According to the relative expression analysis of 5 genes in GATA family, it was speculated that GATA2 may be involved in the formation of conidia in S. turcica.  
    Cloning, Expression and Anti-Virus Function Analysis of Tomato Resistance-Related Gene SlHin1
    PENG HaoRan, PAN Qi, WEI ZhouLing, PU YunDan, ZHANG YongZhi, WU GenTu, QING Ling, SUN XianChao
    Scientia Agricultura Sinica. 2017, 50(7):  1242-1251.  doi:10.3864/j.issn.0578-1752.2017.07.007
    Abstract ( 365 )   HTML ( 5 )   PDF (2479KB) ( 573 )   Save
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    【Objective】The objective of this study is to clone tomato resistance-related gene SlHin1, to analyze its bioinformatics characters, tissue expression and subcellular localization, evaluate its anti-role in the process of the infection and movement of Tobacco mosaic virus (TMV), and to provide a theoretical basis for tomato resistance breeding. 【Method】 Gene cloning and RT-PCR were combined to isolate a gene which might encode SlHIN1. Bioinformatics tools were applied to analyze both the DNA sequence and the characteristics of encoded protein sequence, MEGA6.0 was used to make multiple sequence alignments between the SlHIN1 amino acid sequences and their homologous sequences, and the phylogenetic tree of homologous species was constructed. The subcellular localization test of SlHIN1 was analyzed by using fusion expression vector pCV-mGFP-C1, then the gene was inserted into the expression vector by Sal I and Bam H I digestion. The real-time fluorescent quantitative RT-PCR (qRT-PCR) was used to analyze to the expression levels of SlHin1 in different tomato tissues. The gene fragment was inserted into the plant expression vector pFGC5941 by Nco I and Bam HI digestion. The SlHIN1 was transiently expressed in the N. benthamiana leaves by agro-infiltration, and the SlHIN1 expressed leaves were inoculated with TMV-GFP. The accumulation of the virions was detected by indirect ELISA to investigate the antiviral function of the gene. Western blot was used to evaluate the expression of SlHIN1 protein.【Result】Using RT-PCR method, the tomato Hin1 was obtained from tomato (Solanum lycopersicon cv. Ailsa Craig) leaves and was designated as SlHin1 (GenBank number: KU195820). SlHin1 was 675 bp in length. It was predicted to encode a protein with 225 amino acid residues, a molecular weight of 26.1 kD and a theoretical isoelectric point of 9.35. SlHin1 contains the LEA-14 domains structure, doesn’t have transmembrane segments and locates on chromosome 10 (Solyc10g081980). Sequence analysis and phylogenetic tree analysis showed that SlHIN1 shared approximately 80% similarity with HIN1 from other solanaceae plants and was close to that of rice and sorghum monocotyledons. Subcellular localization was showed that SlHIN1 distributed on the plasma membrane of the leaf epidermis cell of N. benthamiana, which is consistent with the predicted results. The results of qRT-PCR showed that the SlHin1 was of tissue-specificity, whose expression decreased from tomato roots, leaves to stems. The SlHIN1 was transiently expressed in the N. benthamiana leaves by agro-infiltration, and the SlHIN1 expressed leaves were inoculated with TMV-GFP. After 4 days, there was no green fluorescence observed in the SlHIN1 expressed leaves under UV light, but the green fluorescence could be observed in the control group. With the passage of the inoculation, the sporadic fluorescence on the leaves of the treated group was slightly enlarged after 7 days post inoculation, and the green fluorescence of the control group had spread to the leaf. The spread of virus was inhibited and the time that the virus needed to reach the lobus cardiacus was longer, the content of the virions was less than that of control group through indirect ELISA test result. Western blot analysis showed that there was a specific band at about 26 kD in the nitrocellulose membrane, and no corresponding control was observed in the empty vector, indicating that the SlHIN1 protein was successfully expressed at the injection site.【Conclusion】Hin1 genes are present in all tested species, and the SlHIN1 is localized on the plasma membrane of the leaf epidermis cell of N. benthamiana. Transiently expressing SlHIN1 could inhibit the accumulation of the virions and spread of the virus, which can reflect that it may be involved in the resistance reaction of Solanaceae plants to TMV causing them to have resistance.
    SOIL & FERTILIZER·WATER-SAVING IRRIGATION·AGROECOLOGY & ENVIRONMENT
    Changes of the Characteristics of Soil Organic Carbon and Total Nitrogen After Conversation from Upland to Paddy Field in Black Soil Region of Northeast China
    JIA ShuHai, ZHANG JiaNan, ZHANG YuLing, DANG XiuLi, FAN QingFeng, WANG Zhan, YU Na, ZOU HongTao, ZHANG YuLong
    Scientia Agricultura Sinica. 2017, 50(7):  1252-1262.  doi:10.3864/j.issn.0578-1752.2017.07.008
    Abstract ( 420 )   HTML ( 3 )   PDF (397KB) ( 504 )   Save
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    【Objective】The aims of this article were to analyze the dynamic changes in the contents of soil organic carbon and total nitrogen and their densities, and in the values of 13C and 15N natural abundance, explore the sequestration and stability of soil organic carbon and total nitrogen, and to reveal the evolving rule of soil organic carbon (nitrogen) after conversation from upland to paddy field in black soil region of northeast China. The results would provide a theoretical basis for the reasonable utilization and fertilization in black soil. 【Method】 Combined with field investigation, the upland field (soybean planted for over 60 years) and paddy field with different years (3, 5, 10, 17, 20 and 25 a, soybean was planted in all the fields before their conversation to paddy field) were chosen as research objectives, and stable isotope analysis technology was employed to study the changes of the characteristics of soil organic carbon and total nitrogen after conversation from upland to paddy field. 【Result】 In 25 years of conversation from upland to paddy field, the changes of soil organic carbon and total nitrogen contents in the 0-60 cm soil layers showed the similar change trends of rapid drop in the first 3 years, the soil organic carbon and total nitrogen contents were decreased by 13.60%-43.27% and 10.40%-40.60%, then gradually increased with the extension of plant years between 3 to 25 years of conversation, and showed accumulation in the 20-60 cm soil layers, but increased faster between 3 to 5 years of conversation, increased slower between 5 to 25 years of conversation, between 17 to 25 years of conversation, the soil organic carbon and nitrogen contents in the paddy field all higher than in the upland field; the densities of soil organic carbon and total nitrogen in the 0-60 cm soil layers showed the similar change trends with their contents, they were decreased by 26.53% and 21.89% in the first 3 years, and their densities in the paddy field all higher than in the upland field between 5 to 25 years of conversation, they were increased by 9.87%-21.48% and 10.2%-19.3%. The close relationship was found between the changes of soil organic carbon and total nitrogen after conversation from upland to paddy field, also significant positive relationships were found between soil total nitrogen with organic carbon contents and it density (P<0.01), soil 13C natural abundance value in the 0-60 cm soil layers was significantly increased in the first 3 years, and dropped gradually between 3 to 25 years of the conversation. And soil 13C natural abundance value was lower in the paddy filed for more than 5 years than in the upland field. Soil 15N natural abundance value declined gradually with time during the conversation for 25 years, and it was lower in the paddy field than in the upland field in every year. Soil 13C and 15N natural abundance values increased with soil depth in the same years. It was found a significant negative relationship between soil 13C natural abundance value and soil organic carbon contents in the 0-40 cm soil layers (P<0.01), but there was no significant relationship between 15N natural abundance values and total nitrogen contents. 【Conclusion】 After conversation from upland to paddy field for more than 5 years, paddy field showed stronger ability of carbon (nitrogen) sequestration and accumulation of stable carbon (nitrogen) in the 20-60 cm soil layers. After the conversation less than 5 years, more attention should be paid to supplementation of organic carbon and nitrogen for soil in order to maintain and promote the level of soil carbon and nitrogen.
    Zn-Toxicity Thresholds as Determined by Micro Morphological Endpoints of Barley Roots in Polluted Soils and Its Prediction Models
    HE Jun, TIAN XinZhu, WANG XueDong, LIU Bin, LI Ning, ZHENG Han, MENG Nan, CHEN ShiBao
    Scientia Agricultura Sinica. 2017, 50(7):  1263-1270.  doi:10.3864/j.issn.0578-1752.2017.07.009
    Abstract ( 269 )   HTML ( 8 )   PDF (488KB) ( 424 )   Save
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    【Objective】With the continuous requirements for the management of contaminated soil, intensive studies on the ecological risk assessment of contaminated soil are increasingly important. At present, the toxicity test based on species sensitivity distribution for ecological risk assessment of regional contaminated soil has been becoming more and more important compared with the toxicity test based on single species. The toxicity assay should include not only the test for throughout the life cycle, but also the sensitivity of different test endpoints. Toxicity thresholds of Zn determined with different test endpoints are important for evaluation of the environmental risk of Zn in polluted soils. Root ecology plays an important role in the ecological risk assessments and the derivation of toxicity thresholds of heavy metals in polluted soils. The Zn toxicity thresholds (ECx, x=10, 50) of barley as determined by total root length, number of root tips, root surface area and the average diameter of barley root and the quantitative relationships between the ECx and soil properties were studied with the aim to provide basic data for environmental risk evaluation of Zn polluted soils. 【Method】 Eight kinds of field soils with various properties were collected around China, Zn (ZnCl2) was applied and the treated soils were used in a pot experiment after 4 weeks of incubation. Different micro morphological endpoints of barley roots were measured with root scanner (STD1600 Epson), the Zn toxicity thresholds (ECx, x=10, 50) of barley were determined based on the dose-responsive curve with Log-logistic model, the prediction models of Zn toxicity thresholds were developed based on the total Zn concentrations and soil properties (e.g. pH, CEC and org-C etc).【Result】The results indicated that toxicity thresholds of Zn varied significantly among the different micro morphological endpoints of barley roots. The average EC10 and EC50 determined with total root length, number of root tips, root surface area and the average diameter of the root were 228, 295, 335, 261 mg·kg-1 and 702, 779, 837, 739 mg·kg-1, respectively. The smallest ECx were observed with the test of number of root tips while the maximum ECx were observed with endpoint of root surface area, which indicated that the growth of root tips is the most sensitive endpoint among the root micro morphological endpoints of barley when it is stressed by Zn pollution in soil. Meanwhile, the variation coefficient of EC10 (43.4%) was generally higher than that of EC50 (21.6%). A positive correlation was found between ECx (x=10, 50) and the background Zn contents, soil properties (pH, OC contents), and correlation coefficient of pH between ECx reached was very significant (P<0.01).【Conclusion】It was found that the growth of root tips is more sensitive than other root micro morphological endpoints when stressed by Zn pollution in soil. The toxicity thresholds of Zn to plant can be predicted very well based on soil pH, OC, and soil Zn background concentration.
    Effects of Fertilization on Bacterial Community Under the Condition of Continuous Soybean Monoculture in Black Soil in Northeast China
    GAO ShengChao, GUAN DaWei, MA MingChao, ZHANG Wei, LI Jun, SHEN DeLong
    Scientia Agricultura Sinica. 2017, 50(7):  1271-1281.  doi:10.3864/j.issn.0578-1752.2017.07.010
    Abstract ( 386 )   HTML ( 5 )   PDF (662KB) ( 525 )   Save
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    【Objective】The objective of this study is to investigate the effects of fertilization on structure and composition of bacterial community in soybean continuous cropping in Chinese Mollisols, and the main environmental factors causing changes of bacterial communities, focusing on the analysis of the differences in the treatment of inoculated nodules. This study would provide a theoretical support for the efficient planting of soybean and the reduction of nitrogen fertilizer.【Method】Based on a 5-year fertilization experiment of soybean continuous cropping, four different fertilization treatments were sampled, including CK (no fertilizer), PK (phosphorus and potassium inorganic fertilizer), NPK (nitrogen, phosphorus and potassium inorganic fertilizer) and PK+5821 (phosphorus and potassium inorganic fertilizer with Bradyrhizobiumjaponicum 5821). High-throughput sequencing (Illumina Hiseq) and quantitative PCR of 16S rRNA gene were used to analyze bacterial communities in soil samples in different fertilization treatments. Correlation analysis of the bacterial community structure and environmental factors was followed.【Result】Compared with CK, fertilization significantly increased soybean yield and concentration of soil nutrients, it was found that single application of chemical fertilizer decreased soil pH. Inoculation with B. japonicum5821 significantly increased the gene copy number of soil bacteria, and soil bacterial abundance. The analysis of bacterial communities at phylum and class level showed that Proteobacteria, Actinobacteria and Acidobacteria were dominant bacteria, occupying more than 70% of all phyla; Fertilization significantly reduced the relative abundance of Actinobacteria in the phylum analysis, which is consistent with the level of class. Diversity analysis showed that the indexes (richness and diversity) of CK treatment were different from those in other three treatments. The principal coordinate analysis (PCoA) showed that the bacterial community structure in the 3 fertilization treatments (PK, NPK and PK+5821) clustered together on the PC1 axis, and the CK treatment was separated, which indicated that fertilization significantly changed the soil bacterial community composition. Redundancy analysis (RDA) showed that total nitrogen (F=3.2, P=0.002) had the greatest effect on the soil bacterial community structure, explaining 24% of the community changes. The order of contribution rate was total nitrogen>available phosphorus>available K>organic matter>pH. Spearman correlation analysis also showed that all the five soil physiochemical characteristics were closely related with different dominant bacteria.【Conclusion】Fertilization changed the bacterial community structure in continuous soybean cropping, and total nitrogen was the main environmental factor impacting the bacterial abundances and communities. Inoculation with B. japonicum 5821could not only significantly improve soil quality and increase soybean yield, but also reduce the application of chemical nitrogen fertilizer. This study could provide important information for the efficient planting of soybean and the reduction of nitrogenous fertilizer.
    HORTICULTURE
    The QTL Analysis of Fruit and Seed Associated Traits in Watermelon Based on CAPS Markers
    CHI YingYing, GAO Peng, ZHU ZiCheng, LUAN FeiShi, LI GuiYing, YU Peng
    Scientia Agricultura Sinica. 2017, 50(7):  1282-1293.  doi:10.3864/j.issn.0578-1752.2017.07.011
    Abstract ( 426 )   HTML ( 7 )   PDF (3245KB) ( 791 )   Save
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    【Objective】 Cleaved amplified polymorphic sequence (CAPS) markers are useful tools for detecting single nucleotide polymorphisms (SNPs). The aim of this study is to convert SNP sites into CAPS markers based on high-throughput re-sequencing data in watermelon for linkage map construction and quantitative trait locus (QTL). 【Method】 A genetic map was constructed using a BC1P1 segregating population consists of 225 individuals, which F1 was obtained to backcross with P1 from female parent (garden female parent) and male parent (PI186490). The center and edge brix, center and edge flesh firmness, 100-seed-weight and seed coat color were investigated, respectively, then the obtained data were analyzed by SPSS19. Two inbred lines which are female and male parents, were re-sequenced and analyzed by Perl self-compiled script for CAPS marker development. It was found that 90% and 88% of the assembled sequences of the two parental materials could map to the reference watermelon genome, respectively. With the published genome as a reference, the obtained data were assembled with BWA, and explored for the SNP by SAMTOOLS. The sequence of the SNP loci was extracted by in house perl script and then input into SNP2CAPS to transform into CAPS markers. The 450 CAPS restriction sites were selected evenly on the markers whole-genome. CAPS primers were designed 100-500 bp around the mutation by Primer Premier 6.Screening polymorphic CAPS markers for each plant within 225 BC1P1 populations were genotyped. Finally QTL analysis of fruit and seed associated traits in watermelon was done by using QTL IciMapping and Windows QTL CartographerV2.5 softwares. 【Result】 A total of 751 532 SNPs were detected, 450 pairs of CAPS were designed with 7 restriction enzymes among which 200 pairs of primers were polymorphic. An initial CAPS-based genetic linkage map was constructed with the BC1P1 population which spanned 1 376.95 cM with 11 linkage groups, 15 QTLs were detected and the rate of contribution explained 5.25%-74.59%. Among the additive loci there were 3 loci for center brix (CTSS2.1, CTSS2.2, CTSS8.1), 1 for edge brix (ETSS2.1), 3 for center flesh firmness (CFF6.1, CFF6.2, CFF8.1), 2 for edge flesh firmness (EFF6.1, EFF6.2), 4 for seed coat color (SCC8.1, SCC8.2, SCC8.3, SCC8.4), and 2 for 100-seed-weight (SHW6.1, SHW9.1). 【Conclusion】 A total of 751 532 SNPs were detected. Phenotypic contribution rate of 15% or more has five QTL (CTSS8.1, SCC8.1, SCC8.2, SCC8.3, SCC8.4) which are center brix and seed coat color. The research will provide a scientific basis for further fine positioning and cloning the genes controlling superior traits of fruit in watermelon.
    Establishment of an Efficient CRISPR/Cas9-Mediated Gene Editing System in Banana
    HU ChunHua, DENG GuiMing, SUN XiaoXuan, ZUO CunWu, LI ChunYu, KUANG RuiBin, YANG QiaoSong, YI GanJun
    Scientia Agricultura Sinica. 2017, 50(7):  1294-1301.  doi:10.3864/j.issn.0578-1752.2017.07.012
    Abstract ( 662 )   HTML ( 22 )   PDF (1697KB) ( 1932 )   Save
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    Objective The objective of this study is to establish a CRISPR/Cas9-mediated gene editing system in order to find a new way for revealing gene function and breeding of banana. Method According to the Musa genomic (A genome) sequence of phytoene desaturase gene 8 (MaPDS), online tools ZiFiT Targeter Version 4.2 was used to determine the appropriate designing of CRISPR/Cas9 target sites, one of the output target sites was selected for designing the sgRNA. The sgRNA and U6a promoter sequences were linked by overlapping PCR to produce U6a-sgRNA expression cassette, then the fragment was cloned into the binary pYLCRISPR/Cas9 multiplex genome targeting vector system based on Golden Gate Cloning, a pYLCRISPR/Cas9 vector carrying sgRNA cassette named pYLCRISPR/Cas9-sgRNA was generated. The plant expression binary vector, containing a Cas9 (Cas9p) expression cassette, driven by the maize ubiquitin promoter (Pubi), and the sgRNA with target sequence of MaPDS was transcribed under the control of the U6a promoter from rice. The constructed plasmid was then transformed into EHA105 strain of Agrobacterium tumefaciens, which was subsequently used to infect embryogenic cell suspensions (ECS) of banana to obtain resistant plantlets. PCR amplifications were carried out using primer pairs flanking the designed target site. The PCR products were sequenced for detection of mutation.ResultMaPDS was selected as the target of Cas9 endonuclease, and a sgRNA was designed as the target site for CRIPSR/Cas9. The sgRNA with target sequence was firstly inserted into the sgRNA expression cassette by using overlapping PCR, then the cassette was combined with the pYLCRISPR/Cas9 in a single plant binary vector. The results of identification by enzyme digestion and sequencing showed that the pYLCRISPR/Cas9-sgRNA vector aiming at MaPDSa Cas9 expression cassette driven by Pu was successfully constructed, and the vector harbors bi, and a sgRNA expression cassette under the U6a promoter. Using this system and Agrobacterium-mediated transformation,129 independently transformed lines with visible albino, chimerism mutant phenotypes and normal phenotypes were regenerated from ECS after transformation, resistance screening, resistant embryo induction, germination and rooting process. The ratio of albino and chimerism phenotype occurred in transgenic banana was more than 55% (71 out of 129).Albino phenotype in these transgenic plants indicates the loss of PtoPDS function. To further verify whether the loss of green in the transgenic banana was caused by generation of mutations in the MaPDS by the CRISPR/Cas9 system, 33mutant phenotypes and 14 normal phenotypes from 129 independent transgenic plants were randomly selected for sequencing. The results confirmed that all of these transgenic plants with albino phenotype contained mutants in the MaPDS, insertion (a Aor T) and displacement were found in the target sites of MaPDS in the mutants. And it was also found the displacement in downstream of the target sites of MaPDS in the experiments, no mutation was detected in the normal phenotypes of transgenic and control plants.Conclusion The CRISPR/Cas9 system was successfully applied to knock out the endogenous phytoene dehydrogenase (PDS) gene in banana. The results demonstrate that the Cas9/sgRNA system can be exploited to targeted mutagenesis and effectively create knockout mutations in banana.
    Frost Hardiness Assessment of Floribunda Roses by Means of Electrical Impedance Tomography
    JIAO MeiLing, GONG RuiJuan, QIAN Ji, ZHANG Gang
    Scientia Agricultura Sinica. 2017, 50(7):  1302-1316.  doi:10.3864/j.issn.0578-1752.2017.07.013
    Abstract ( 283 )   HTML ( 2 )   PDF (3190KB) ( 353 )   Save
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    【Objective】 The objective of this experiment is to study whether the electrical impedance tomography (EIT) is feasible to assess the frost hardiness of floribunda roses (Rosa hybrida Hort. ‘Floribunda’), and with the view to provide a new reference method for rapid and non-destructive detection of ornamental plant for frost hardiness. 【Method】 Three perennial floribunda roses ‘Hongmao’, ‘Rouqingsishui’ and ‘Xianjing’ were used to measure EIT and electrical impedance spectroscopy (EIS) parameters of one-year-old stems under natural conditions of low temperature as well as artificial freezing, and the correlations of frost hardiness assessed by means of EIT and EIS parameters were compared with that of the traditional electrolyte leakage (EL) method. Meanwhile, the correlation analysis of four EIS parameters specific extracellular resistance (re), specific intracellular resistance (ri), relaxation time (t) and distribution coefficient of t (y) of stems for three varieties of floribunda roses and EIT reconstructed values were studied.【Result】 The order of frost hardiness of the three floribunda roses varieties was: ‘Xianjing’>‘Rouqingsishui’>‘Hongmao’. Stems of the three varieties could achieve a clear EIT imaging at different temperatures and EIT reconstructed value showed a decreasing trend as temperature decreased. Frost hardiness calculated by EIT reconstructed value had a significant correlation (r=0.92) with that assessed by EL method, however, the results were lower than the EL method. EIS parameters changed during cold acclimation, on the whole, the extracellular resistance (re) rose, while the relaxation time (t) dropped. Under natural conditions of low temperature, re and EIT reconstructed values of ‘Hongmao’ and ‘Xianjing’ positively correlated (r>0.72), and t and EIT reconstructed value of ‘Rouqingsishui’ and ‘Xianjing’ positively correlated (r>0.63) as well, indicating that all of them can be used to characterize the state of cell and tissue.【Conclusion】 Under freezing temperature test, after frost damage EIT reconstructed values of stems in floribunda roses reduced significantly, and could be calculated with Logistic equation to get the frost hardiness, but the results were lower than those of EL method. Under natural conditions of low temperature, two EIS parameters extracellular resistance (re) and relaxation time (t) had correlations with EIT reconstructed values. After freezing test, the frost hardiness of the floribunda roses could be assessed by the EIT reconstructed values of stems.
    FOOD SCIENCE AND ENGINEERING
    Cumulative Risk Assessment of Dietary Sulfur Dioxide Residues of Consumers
    HU GuiXian, LAI AiPing, YUAN YuWei, ZHANG ZhiHeng, ZHAO ShouPing, ZHU JiaHong, WANG Qiang
    Scientia Agricultura Sinica. 2017, 50(7):  1317-1325.  doi:10.3864/j.issn.0578-1752.2017.07.014
    Abstract ( 386 )   HTML ( 4 )   PDF (390KB) ( 414 )   Save
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    【Objective】The aim of the study is to assess the cumulative risk of dietary sulfur dioxide residues, result of the research will provide a scientific basis for the guidelines of sulfur dioxide in fruits or vegetables and also its threshold line in agricultural products. 【Method】 There were 3 299 samples of foods collected from market and field, then the residues of sulfur dioxide were determined according to GB/T 5009.34-2003 (Determination of sulphite in foods). There were 408 samples used as references, which including starch, inebriant, mushrooms and maconochie. According to the mean value of sulfur dioxide in samples, per capita consumption and weight, the point evaluation method was used in the research to assess the cumulative exposure risk of dietary sulfur dioxide for different age groups in China.【Result】The results showed that, 4 of 27 kinds of samples, mean pickled vegetables, canned vegetables (only bamboo shoot and sour pickled cabbage), dried fruit and edible starch all had higher values of sulfur dioxide than their threshold line ruled by GB2760-2014 (National Standards for Food Safety Standards for the Use of Food Additives), with their values were 148.92, 147.36, 191.21, and 41.57 mg·kg-1, respectively, and they were 2.9, 1.5, 1.9 and 1.4 times as high as their threshold line correspondingly, while the assessment showed that the risk was still within the acceptable range of human body because of their low consumption. Although the risk of dietary exposure to sulfur dioxide in vegetables was higher than other food categories, but its risk quotient was only 0.124, still less than 1, at a safe level, will not have a health risk to consumers. In addition, risk quotient based on the average residue level were all less than 1 in different age groups. However, at the confidence point P95 and P97.5, the risk quotient of four age groups (2-3 years, 4-6 years, 7-10 years and 11-13 years old), were higher than or similar to 1, means dietary risk of sulfur dioxide causing a certain threat to health.【Conclusion】It was recommended that the consumption of pickled vegetables, canned vegetables (only bamboo shoot and sour pickled cabbage), dried fruit and edible starch should be properly controlled, especially for high risk population such as children groups aged from 2 to 13 years old, who should control their diet intake of sulfur dioxide including pickled food to reduce dietary risk. The results showed that the safety evaluation of sulfur dioxide on agricultural products should be carried out on products without guidelines of preservative containing sulfur dioxide, especially for some fresh vegetables. The authors suggested that systematic revision of the limits of sulfur dioxide in agricultural products or revision of the use of food additives also should be carried out with concerning about the agricultural demand, background value or other objective factors.
    ANIMAL SCIENCE·VETERINARY SCIENCERE·SOURCE INSECT
    Effect of Exogenous Protease on in vitro Dry Matter Digestibility and Enzymatic Hydrolysate Gross Energy of Diets for Broilers
    ZHANG LiLan, CHEN Liang, ZHONG RuQing, ZHANG HongFu
    Scientia Agricultura Sinica. 2017, 50(7):  1326-1333.  doi:10.3864/j.issn.0578-1752.2017.07.015
    Abstract ( 313 )   HTML ( 4 )   PDF (381KB) ( 781 )   Save
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    【Objective】 The objective of the present study was to determine the effect of exogenous protease supplementation on the in vitro dry matter digestibility (DMD) and enzymatic hydrolysate gross energy (EHGE) using a computer-controlled simulated digestion system (CCSDS) and obtain the optimum protease supplementation into a corn-soybean meal-based diet for broilers. It would provide a reference for the establishment of in vitro method for efficacy evaluation of exogenous enzyme.【Method】 A single factor completely randomized design with 8 treatments was adopted and each treatment contained 5 replicates with 1 digestion tube per replicate was adopted in the present study. The control diets for starter and finisher phrases (24.29% and 21.72% of crude protein, respectively, dry matter basis) of broilers were formulated to meet or exceed the nutrient requirements for broilers, and the test diets were supplemented with 0, 15 000, 75 000, and 150 000 PROT·kg-1 exogenous protease (90 000 PROT·g-1 ) into the control diets for starter and finisher phrases of broilers, respectively. The DMD, in vitro gross energy digestibility (GED) and EHGE of the diets were determined using a CCSDS (SDS-II). The correlation between DMD or EHGE and protease supplementation (PS) of diets were also analyzed. 【Result】 The results showed as follow: 1) The in vitro total tract DMD, GED and EHGE of diets increased as the level of exogenous protease supplementation in the control diet for starter and finisher phrases of broilers increased (linear, P<0.01). 2) The in vitro gastric DMD and GED of diets decreased as the level of exogenous protease supplementation in the control diet for starter phrases of broilers increased (linear, P<0.01). However, the in vitro gastric DMD and GED of diets were not affected by the exogenous protease supplementation in the finisher control diet (P>0.05). 3) A linear regression analysis, taking into account protease supplementation (g·kg-1), explained the DMD (%) of starter and finisher diets for DMD = 2.56 PS - 0.82 PS × PS + 73.90 (R2 = 0.82, RSD = 0.42, P<0.001) and DMD = 3.56 PS – 1.46 PS × PS + 75.20 (R2 = 0.70, RSD = 0.60, P<0.001), respectively. A linear regression analysis, taking into account protease supplementation (g·kg-1), explained the EHGE (MJ·kg-1) of starter and finisher diets for EHGE = 0.75 PS - 0.30 PS × PS + 15.02 (R2 = 0.89, RSD = 0.07, P<0.001) and EHGE = 0.61 PS - 0.30 PS × PS + 15.67 (R2= 0.79, RSD = 0.06, P<0.001), respectively. There was greatest EHGE when protease supplementation was  112 500 PROT·kg-1 in a starter diet and 91 800 PROT·kg-1 in a finisher diet, respectively. 【Conclusion】 In conclusion, the in vitro gastrointestinal DMD, GED and EHGE were increased by the protease supplementation into a corn-soybean diets for broilers. However, the effect of protease supplementation on nutrition digestibility in different intestinal segments was not simple consistent with a dose-response pattern. The 112 500 PROT·kg-1 of protease supplementation in a starter diet and 91 800 PROT·kg-1 of protease supplementation in a finisher diet for broilers may improve more effectively in the present study.
    GPNMB Affects Melanin Synthesis in the Melanocytes via MITF to Regulate the Downstream Pigmental Genes
    ZHAO BingLing, LI YaNan, CHEN TianZhi, LIU Ying, CHANG LuCheng, FAN RuiWen, XUE LinLi, WANG HaiDong, DONG ChangSheng
    Scientia Agricultura Sinica. 2017, 50(7):  1334-1342.  doi:10.3864/j.issn.0578-1752.2017.07.016
    Abstract ( 270 )   HTML ( 3 )   PDF (2418KB) ( 662 )   Save
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    【Objective】 The objective of this study is to explore whether GPNMB affects melanin synthesis in the melanocytes by regulating the levels of MITF and its downstream pigmentation-related genes, further clarify the specific mechanism of GPNMB in the melanin genesis in the melanocytes to provide a theoretical foundation.【Method】Firstly, the mouse GPNMB nucleic acid sequences was searched from NCBI. After analysis of the sequences of GPNMB and the lentiviral expression vector, the Sal I and Xba I restriction sites was selected. Then, the GPNMB full-length primers containing SalI and XbaI restriction sites was designed and the GPNMB full-length sequence containing the restriction site was cloned, and finally, the T vector was ligated and sequenced. The constructed T vector plasmid was extracted and doubly digested, followed by ligating with the lentiviral expression vector containing green fluorescence protein (GFP) and then sequenced. A large number of endotoxin-free GPNMB lentivirus eukaryotic expression vectors were collected according to the instruction of the plasmid midiprep system. Furthermore, mouse melanocytes was cultured in vitro and transfected using Lipofectamine 2000 reagent according to the manufacturer’s guidelines when it was in the logarithmic growth phase. Forty-eight hours after transfection, the morphological changes and green fluorescent protein expression levels of melanocytes was observed. Then, the cells were harvested and the transfection efficiency and melanin content of melanocytes were tested. The relative expression levels of PMEL, MITF, TYR, TYRP1, TYRP2 and OA1 were qualitatively and quantitatively analyzed by real-time quantitative PCR (qRT-PCR) and Western blot. 【Result】 The morphological characteristics of melanocytes in the experimental group and empty vector group had no obvious change compared with the normal group. The results of green fluorescent protein (GFP) in both the experimental group and the empty vector group showed that the transfection efficiency in 5 μg DNA / well was the highest. GPNMB mRNA and protein levels in the experimental group were extremely significantly higher than the empty vector group and normal group. Compared with the empty vector group, the melanin content in the experimental group increased by 1.34 times (P<0.01). The qRT-PCR results showed that the relative expression levels of PMEL, TYRP1 and TYRP2 mRNA in experimental group was 1.59 (P<0.05), 2.35 (P<0.01) and 1.60 times (P<0.01) of that in empty vector group, respectively; However, the relative expression level of MITF mRNA was significantly reduced by 2.25 times (P<0.01); TYR and OA1 mRNA increased 1.65 and 1.50 times, respectively, but not significantly. Furthermore, the western blot results showed that TYRP2 protein was significantly increased by 4.35 times (P<0.01); TYR protein was significantly increased by 1.23 times (P<0.01); OA1 protein was significantly increased by 1.31 times (P<0.05); By contrast, MITF protein was significantly reduced by 1.59 times (P<0.01); TYRP1 protein had no significantly change.【Conclusion】Over-expression of GPNMB increased the expression of PMEL, TYR, TYRP1, TYRP2 and OA1, but reduced the expression of MITF. It was speculation that GPNMB may affect the melanin synthesis by regulating the expression of PMEL, TYR, TYRP1, TYRP2 and OA1 in a MITF-independent fashion.
     
    RESEARCH NOTES
    Genetic Variation of p20 of the Severe and Mild Strains of CTV in the Sweet Orange and Pummelo
    WANG YaFei, RUAN Tao, ZHOU Yan, WANG XueFeng, WU GenTu, SUN XianChao, ZHOU ChangYong, QING Ling
    Scientia Agricultura Sinica. 2017, 50(7):  1343-1350.  doi:10.3864/j.issn.0578-1752.2017.07.017
    Abstract ( 243 )   HTML ( 5 )   PDF (400KB) ( 232 )   Save
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    【Objective】 In order to clarify the genetic variation of p20 of the severe and mild strains of Citrus tristeza virus (CTV) in sweet orange and pummelo, seven CTV strains were studied. 【Method】The CTV p20 populations were established by RT-PCR, cloning and sequencing, and the phylogenetic tree was constructed with MEGA6. The genetic structure and mutation level of the CTV populations were analyzed by DNAStar. Haplotype diversity, nucleotide diversity and neutral test of the CTV populations were analyzed by DnaSP. 【Result】 Seven CTV p20 populations consisted of 162 sequences with 11 haplotypes were established. One or more haplotypes were found in each population. Sequence analysis showed that the original nucleotide sequence identity of the haplotypes from different populations was 88.2%-100.0%, the corresponding amino acid sequence identity was 92.3%-100.0% and the lowest amino acid sequence identity was found between CT23-1 and CT9-2. There were 50 sequences of haplotypes PeraIAC-4, CT22 and CT9-1, the corresponding nucleotide sequence identity was 100.0%, of which were dominant haplotypes and close to the standard strain T36. PeraIAC possessed the most abundant haplotype diversity (0.800±0.051) and CT22 had the lowest haplotype diversity (0.170±0.102). Compared with haplotype diversity (0.170-0.552) and nucleotide diversity (0.00032-0.05919) of the populations from pummelo, the populations from sweet orange had richer haplotype diversity (0.513-0.800) and nucleotide diversity (0.04208-0.05677). Phylogenetic analysis showed that the population structure of the isolates from sweet orange was complex, and the haplotypes detected in the populations from sweet orange were related to the standard strains T30, T36, VT and T3. CT31-2, which is close to the strain T3, was the most distant to the dominant haplotype in the phylogenetic tree, and the identity of the corresponding nucleotide sequences was 88.3%. The results of neutrality test showed that the populations from sweet orange tended to be balanced or contracted, while the populations from pummelo tended to be expanded except for CT23. The Tajima’s D values, Fu and Li’s D* values and Fu and Li's F* values of the TR-514Y, CT31 and CT23 populations were positive and significantly different, while those of the CT9 population were negative and significantly different. Recombination analysis of each population showed that no recombination occurred in any group. The population variation analysis showed that the percentage of mutant clones was 0-30.8% and the base mutation frequency was 0-7.706×10-4. The highest percentage of mutant clones (30.8%), the highest mutation frequency (7.706×10-4), the most base mutations (11) and the most mutation sites (7) happened in p20 populations of CTV severe strains from sweet orange. The percentage of mutated clones and mutation frequency of CTV mild populations from sweet orange were significantly lower than those of CTV severe populations from sweet orange. The percentage of mutated clones and mutation frequency of CTV mild populations from pummelo were slightly lower than those of severe populations from pummelo. The analysis of base mutation types showed that the base mutation types were mainly base substitution, and substitution from A to G was dominant. The sole insertion of A was detected between the 156 and 157 sites of CT3 and no deletion was found in p20 populations. 【Conclusion】 The variations of severe and mild populations of CTV p20 from sweet orange and pummelo were different. CTV p20 populations from sweet orange had more complex population structure and higher variation level than those from pummelo, and the severe populations of CTV p20 had higher variation level than those of mild populations.
    Cloning, Prokaryotic Expression and Preparation of the Polyclonal Antibody Against CYP6CY3 from Aphis gossypii
    WEI YuanJie, WANG YaMei, HUANG LiNa, LIU Ning, ZHAO Jie, AI XinYu, LIU XiaoNing
    Scientia Agricultura Sinica. 2017, 50(7):  1351-1360.  doi:10.3864/j.issn.0578-1752.2017.07.018
    Abstract ( 237 )   HTML ( 2 )   PDF (2513KB) ( 368 )   Save
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    【Objective】The objective of this study is to clone and express CYP6CY3 in Escherichia coli to obtain the recombinant protein, and then to immunize mice for the preparation of polyclonal anti-CYP6CY3 antibody, which provides a basis for further analysis of the role of CYP6CY3 in different tissues from insecticide-resistance Aphis gossypii. 【Method】The open reading frame (ORF) of CYP6CY3 from A. gossypii was cloned by RT-PCR and 3′-RACE, and analyzed by the bioinformatics and phylogenetic relationship. And then it was expressed in E. coli (DE3). The recombinant protein was purified by gel extraction. The polyclonal antibody of CYP6CY3 was prepared by immunizing Kunming white mice with the purified protein. The antibody titer was detected by ELISA. The specificity of the antibody was monitored by Western blot and immunohistochemistry. 【Result】The ORF of CYP6CY3 contained 1 266 bp, encoding 421 amino acids, with the predicted molecular weight of 48.8 kD, the theoretical isoelectric point of 8.99, and there was no signal peptide sequence. As a kind of hydrophilic protein, the amino acid sequence included W×××R, AG××T, E××R, P××F×PE/DRT and F××G×××C×G five characteristic motifs of P450 family. The analysis of homologous sequences by NCBI Blastx, cotton aphid CYP6CY3 amino acid sequence shared 81% identity with the pea aphid (Acyrthosiphon pisum) (XP_001948581.1) , 79% amino acid identity with the wheat aphid (Diuraphis noxia) (XP_015379193.1) , 76%, green peach aphid (Myzus persicae) (AHB52749.1), respectively. The amino acid sequences of four species of aphids have completely conserved sequence E××R in the spiral K and heme binding site consensus sequence F××G×××C×G. The phylogenetic tree was constructed using MEGA5 software, the results showed that A. gossypii, A. pisum, D. noxia and M. persicae were clustered into one group, suggesting that they likely developed from a common ancestral gene. The relative molecular weight of the CYP6CY3 fusion protein was about 66 kD. The antibody titer was determined as high as 1﹕409 600 dilution ratio by ELISA. Western blot and immunohistochemistry analysis showed that the antibody could specifically bind both the recombined expression protein and the endogenous CYP6CY3 from the A. gossypii.【Conclusion】The ORF of CYP6CY3 from A. gossypii was cloned, and the polyclonal antibody against CYP6CY3 was prepared by protein immunoassay, which will lay a foundation for further study on the function of CYP6CY3, and the role in the resistance of A. gossypii.