Scientia Agricultura Sinica

QTL Mapping and Integration as well as Candidate Genes Identification for Plant Height in Rapeseed (Brassica napus L.)

ZHANG JiangJiang, ZHAN JiePeng, LIU QingYun, SHI JiaQin, WANG XinFa, LIU GuiHua, WANG HanZhong

Scientia Agricultura Sinica. 2017, 50(17): 3247-3258. doi:10.3864/j.issn.0578-1752.2017.17.001

Abstract ( 444 )

PDF (870KB) ( 1131 )

Save

References | Related Articles | Metrics

【Objective】In order to reveal the genetic architecture and candidate genes for plant height in rapeseed, QTLs were mapped in multiple environments and were integrated with previously reported plant height QTLs and then aligned with the plant height genes, which will provide a basis for the molecular improvement of plant height in rapeseed. 【Method】 The BnaZNF₂population of 184 individuals derived from the elite rapeseed cultivar Zhongshuang11 (de novo sequencing) and No.73290 (re-sequencing) was used as the experimental material. First, the BnaZNF₂ population was subjected to genotype analysis and a high-density linkage map of 803 molecular markers was constructed using Joinmap 4.0. Second, the F_2:3and F_2:4family of BnaZNF₂ population were planted and phenotyped at two locations (Wuhan and Xining) for successive two years (2010 and 2011). Then QTL mapping was conducted by the composite interval mapping method incorporated into WinQTLCart 2.5 software, using the genotype of BnaZNF₂ population and the plant height phenotype of its F_2:3and F_2:4family. 【Result】 After integration of QTLs detected in two locations over two years, a total of 5 consensus QTLs (qPH.A2-1, qPH.A2-2, qPH.C2-1, qPH.C3-1, qPH.C3-2) were obtained, which were distributed on A2, C2 and C3 chromosomes and, explained 2.6%-55.6% of the phenotypic variance. A major QTL on the C2 chromosome, qPH.C2-1, was only detected repeatedly in Xining and its LOD value, additive effect and R²(23.4, -16.0 and 55.6%, respectively) were largest among all of the reported plant height QTLs. Based on the physical map of rapeseed, all of the currently and previously reported plant height QTLs in rapeseed were integrated and then aligned with the plant height genes, which revealed a relatively completed genetic architecture map consisting of 183 QTLs in rapeseed and 287 candidate genes in rapeseed. Of these, a total of 18 QTL cluster were commonly detected in different studies, which were distributed on A1, A2, A3, A6, A7, A9, C6 and C7 chromosomes. In addition, the physical positions of the five QTL detected in the current study were all not overlapped with those of the previously detected plant height QTL, which should be novel. A total of 15 homologues of plant height genes were found within the physical intervals of qPH.A2-2, qPH.C3-1 and qPH.C3-2, of which 11 homologues showed sequence variations between the two parents, which were chosen as the candidates for further study. 【Conclusion】 QTL mapping and integration identified five QTL for plant height in rapeseed, which were all novel. The effect of the major QTL on the C2 chromosome was larger than those of the previously reported plant height QTL, which also showed the strong interaction with the environment. The integration of the reported plant height QTLs and the alignment with the plant height genes systematically revealed the genetic architecture and candidate genes for plant height in rapeseed. By bioinformatics analysis, a total of 11 candidates were identified within the physical intervals of three plant height QTLs detected in the current study.

Genome-Wide Identification and Expression Analysis of WRKY Transcription Factor Under Abiotic Stress in Beta vulgaris

KONG WeiLong, YU Kun, DAN NaiZhen, YANG ShaoZong, BAO ManZhu, HUANG XiangRong, FU XiaoPeng

Scientia Agricultura Sinica. 2017, 50(17): 3259-3273. doi:10.3864/j.issn.0578-1752.2017.17.002

Abstract ( 355 )

PDF (8919KB) ( 542 )

Save

References | Related Articles | Metrics

【Objective】 WRKY transcription factor is a kind of transcription factor which plays an important role in plant response to biotic, abiotic stress, plant growth and development. Based on the sugar beet genome information, the WRKY family genes (BvWRKYs) were identified, tissue-specific expression profiles and expression pattern under salt and heat stresses were analyzed. The aim of the study is to provide reference for the WRKY gene function research, and lay a foundation for ornamental beet (B. vulgaris) and other Dianthus ornamental plant genetic engineering research. 【Method】 A total of 75 Arabidopsis WRKY proteins were used as references, according to WRKY conserved protein sequence (PF03106), the WRKY genes of B. vulgaris were identified by hmm and BLAST homology searches, and the chromosome location, phylogeny, gene structure, conserved domain, conserved element were also analyzed by MapInspect, GSDS2.0, MEGA5.0, DNAMAN5.0, WebLogo 3 and MEME bioinformatics tools. The specificity of WRKY genes expression and expression pattern under salt stress and heat stress of B. vulgaris were analyzed by RNA-seq and qRT-PCR analysis. 【Result】 B. vulgaris WRKY gene family contained 40 members, 39 were unevenly distributed on 9 chromosomes, 1 on the random fragment. According to the WRKY conserved domain features and the evolution analysis with Arabidopsis thaliana, 40 members were divided into three classes: Ⅰ, Ⅱ, and Ⅲ. ClassⅠhad 9 members, class Ⅱ had 26 members and class Ⅲ had 5 members. According to the evolutionary relationship, class Ⅱ further divided into five subclass: Ⅱa (1), Ⅱb (4), Ⅱc (9), Ⅱd (5) and Ⅱe (7). Genetic structure analysis showed that exon and intron number of B. vulgaris WRKY genes had high variability (2-7 exons), even within the same subgroup. Conserved element analysis showed that within the same class or subclass had the same conserved elements. WRKY conserved domain analysis revealed two mutations in the WRKY domain: WRKYGKK and WRKYGEK. Each WRKY was expressed in at least two tissues, 30 WRKYs were expressed in leaf, 40 WRKYs were expressed in inflorescence, 36 WRKYs were expressed in young leaf, 38 WRKYs were expressed in root, 39 WRKYs were expressed in seedling, and 36 WRKYs were expressed in seed. The expression levels of WRKY genes were also different. All WRKY genes were divided into two types: low expression genes and high expression genes. Such as: BvWRKY23, BvWRKY3, BvWRKY11, BvWRKY7, BvWRKY6, BvWRKY26, BvWRKY4, BvWRKY40, BvWRKY24, BvWRKY2 and BvWRKY28 were highly expressed in all tissues; BvWRKY38, BvWRKY13, BvWRKY36, BvWRKY35, BvWRKY5 and BvWRKY34 were low expressed in all tissues. BvWRKY21, BvWRKY20, BvWRKY22, BvWRKY32, BvWRKY33 and BvWRKY34 genes were up-regulated under heat stress, and BvWRKY1, BvWRKY6, BvWRKY19, BvWRKY31 and BvWRKY33 were up-regulated under salt stress. In addition, BvWRKY33 had a significant response to both heat and salt stress. 【Conclusion】 B. vulgaris WRKY proteins are highly conserved, the length of the gene sequence and the number of introns varied widely, all WRKY genes showed a variety of expression patterns in different tissues, some WRKY genes responded to heat or salt stress, which play an important role in stress physiological regulation.

Analysis on Gene Feature and Function of the Subfamily Members Containing START Domain Only in Arabidopsis thaliana

YAN QingDi, ZHAO YaLin, ZHANG Hao, GAO Jing, GAO MengZhu, WANG Qian, WANG Rui, WANG FengRu, DONG JinGao

Scientia Agricultura Sinica. 2017, 50(17): 3274-3285. doi:10.3864/j.issn.0578-1752.2017.17.003

Abstract ( 342 )

PDF (3008KB) ( 468 )

Save

References | Related Articles | Metrics

【Objective】 The objective of this study is to analyze the promoter elements and expression feature of the genes coding the subfamily containing the START domain (the lipid/sterol-binding StAR-related lipid transfer protein domains) only in Arabidopsis thaliana, expound the relationship between the cis-acting element and the gene function, and the results of study will provide an important theoretical basis for clarifying the biological function of the genes, analyzing the plant growth and development mechanism and promoting or suppressing plant growth better. 【Method】 Using bioinformatics method to analyze the genetic relationship of 6 members and all the elements contained in their respective promoter regions. The function of the promoter elements of subfamily were analyzed and classified, and the expression features and the regulation factors of these subfamily members were predicted. Real-time PCR method was employed to determine the expression characteristics of subfamily members, clarify the relationship between the test results and the conclusions of promoter analysis, thus providing scientific research ideas and direction for analysis of the function and mechanism of genes. The relationship between the gene promoter element and function was confirmed after analysis of the gene mutation type surface. 【Result】 There are 6 members belong to the subfamily containing START domain only in A. thaliana, divided into three branches, every branch has a pair of genes, each pair of genes has a promoter containing high transcription element 5′ UTR Py-rich stretch. The promoter regions of the 6 subfamily members contain multiple common response elements, such as light, hormone and stress, and also have specific response elements for their own. The real-time PCR results of the genes expression level showed that the expression level of At3g13062 was significantly higher than the same branch gene At1g55960, At3g23080 was significantly higher than the same branch gene At4g14500, At1g64720 was significantly higher than the same branch gene At5g54170. All the subfamily members have higher expression levels at seedling stage, but less expression levels at ageing stage, and have its own specific expression of space and time characteristics. In the gene promoter analysis of the family containing START domain structure only, it was found that each gene promoter has a cis-acting element Skn-1 motif which was specially expressed in endosperm, implied that the family genes may be involved in regulation of endosperm development. Seeds analysis of At3g23080 and At4g14500 T-DNA insertion mutant showed that At3g23080 and At4g14500 expression decreased and caused a decline in the seed vigor. 【Conclusion】 There are 6 members in the subfamily containing START domain only. Six members of the family may be play an important role in the response process to light, hormones and stress. the 6 family members have a close kinship, but their expression characteristics of time and space are different and carry out their respective functions in different development periods of A. thaliana. There is a direct relationship between gene promoter elements and the gene function, and the promoter region with endosperm expressed specially element Skn1 motif in At3g23080 and At4g14500, so the decreased expression of At3g23080 or At4g14500 caused a decline in seed vigor.

Advances in AquaCrop Model Research and Application

SUN ShiJun, ZHANG LinLin, CHEN ZhiJun, SUN Juan

Scientia Agricultura Sinica. 2017, 50(17): 3286-3299. doi:10.3864/j.issn.0578-1752.2017.17.004

Abstract ( 738 )

PDF (510KB) ( 1334 )

Save

References | Related Articles | Metrics

AquaCrop is a kind of new crop model developed by FAO in 2009. It is widely used in agricultural fields, because it need fewer parameters, and can provide users more simple interface, compared with other similar crop models. According to the principle and characteristics of the AquaCrop model, some further discussions were developed on the application of this model at domestic and abroad. Analysis shows that, the AquaCrop model has achieved remarkable results in irrigation strategy, scenario simulation under climate change and joint application with other models. However, at present, the model is not perfect enough, because of the following reasons. Firstly, the lack of verification of the conservative parameters of the model usually results in lower simulation accuracy. Secondly, the objective existence of spatial variability of soil, which is commonly suitable and helpful so much when applied at single experimental station, but when applied to larger area, some more stations and data are needed. Thirdly, less research on crop growth in rain-fed areas was conducted, and its non-conservative parameters are difficult to be obtained accurately. Finally, the physiological modules, nutrient modules and interaction modules of water and nutrient are not perfect, and the crop genetic varieties and pests are not included, which result in lower simulation accuracy under severe stress conditions. It is concluded that, when the model is applied, the conservative parameters should be modified by using many years' data of crop, and non-conservative modeling parameters should be corrected by combining multi-year data at a single site with multi-sites data in the experimental region. Currently, scholars and researches are supposed to strengthen the research in rain-fed areas, enlarge the scope of application of the model, and related designers should develop more sub-modules of AquaCrop model to increase the modeling accuracy and broaden its application scope.

Effects of Continuous Flooding on Cadmium Absorption and Its Regulation Mechanisms in Rice

CHEN JiangMin, YANG YongJie, HUANG QiNa, HU PeiSong, TANG ShaoQing, WU LiQun, WANG JianLong, SHAO GuoSheng

Scientia Agricultura Sinica. 2017, 50(17): 3300-3310. doi:10.3864/j.issn.0578-1752.2017.17.005

Abstract ( 535 )

PDF (421KB) ( 661 )

Save

References | Related Articles | Metrics

【Objective】The objective of this experiment is to study the effects of continuous flooding on cadmium (Cd) uptake in rice. By analysis of alteration of soil available Cd, plant Cd content and Cd uptake related gene expression under continuous flooding condition in different Cd accumulation rice varieties, further to reveal the effects and detail regulation mechanism on Cd uptake with continuous flooding management in rice. 【Method】 Pot experiments (exogenous 1.5 mg·kg^-1CdCl₂ ) were carried out by using rice varieties of Fupin36 (FP36, high Cd accumulation) and Zhongjiazao 17 (ZJZ17, low Cd accumulation). Plantlets were treated by continuous flooding management at early tillering stage and sampled at middle tillering stage. Cd content of plant root and shoot, soil available Cd, Fe and Mn content, root plaque Cd, Fe and Mn content and Cd uptake related gene expression of rice were analyzed. The same treatment was continued until mature stage, plantlets and milled rice were harvested to determine Cd content and yield traits. 【Result】 Compared with the control, the results showed that continuous flooding sharply reduced Cd contents of FP36 and ZJZ17 under pollution condition at middle tillering stage, the root Cd content decreased by 39.5% and 33.9%, shoot Cd content decreased by 62.1% and 71.7%, respectively. Continuous flooding also significantly reduced Cd content of rice root, shoot and milled rice at mature stage. The management reduced root, shoot and milled rice Cd content of FP36 by 36.4%, 43.7% and 36.8%, respectively, it also showed a decrease of 62.5%, 61.5% and 55.4% in ZJZ17. Furthermore, the soil available Cd of FP36 and ZJZ17 significantly decreased by 12.1% and 17.7%, and root plaque Cd content decreased by 52.2% and 43.1% under continuous flooding treatment, respectively. While soil available Fe content (23.7% and 10.3%) and Mn content (24.5% and 43.9%) of FP36 and ZJZ17 significantly increased. Root plaque Fe (83.1% and 81.5%) and Mn content (41.5% and 27.7%) were also elevated under continuous flooding. Simultaneously, the relative expression of OsNramp1 (58.3% and 58.0%) and OsLCD (21.6% and 17.8%) genes were also down-regulated by continuous flooding in rice roots.【Conclusion】Continuous flooding decrease Cd uptake by decreasing soil available Cd content and down-regulating Cd uptake genes expression of OsNramp1 and OsLCD in rice.

Comprehensive Evaluation and Indicators of the Drought Resistance of Different Genotypes of Fagopyrum tataricum at Seedling Stage

LU ZhiJuan, ZHANG YongQing, ZHANG Chu, LIU LiQin, YANG ChunTing

Scientia Agricultura Sinica. 2017, 50(17): 3311-3322. doi:10.3864/j.issn.0578-1752.2017.17.006

Abstract ( 383 )

PDF (489KB) ( 702 )

Save

References | Related Articles | Metrics

【Objective】Fagopyrum tataricum not only has rich nutritional and medicinal values, but also has great physiological characteristics such as relatively high tolerance and strong adaptation ability to cold and barren conditions. There is a significant difference in resistance to drought stress among different genotypes of F. tararicum. Study of drought resistance characteristics of F. tataricum, screening of the materials of high tolerance genotypes and identification of indicators of drought resistance, and building a mathematic model for appraising the drought resistance not only can provide a foundation for evaluating the drought resistance and selection of genotypes, but also can provide a theoretical basis for germplasm breeding in cold areas of Loess Plateau. 【Method】Multiple indices of seedlings, including agronomic traits and physiological characteristics, were measured in the normal supply and drought stress with nine F. tataricum genotypes. Subordination function method, principal component analysis and clustering analysis were used to synthetically evaluate the drought resistance of different F. tataricum genotypes. Moreover, stepwise regression analysis was used to establish the optimal regression equation that could forecast and identify the drought resistance ability of F. tataricum. 【Result】Drought stress has an obvious effect on the indexes of F. tataricum at seedling stage. Results of difference of testing indices showed that the overground index, root dry weight, root activity, root morphology index, soluble protein contents, relative water content, chlorophyll content and leaf fluorescence parameters under drought stress were obviously decreased compared with the control group. Compared with the control group, drought stress not only resulted in the decreases of aboveground and root characteristics, root activity, soluble protein contents, leaf relative water and chlorophyll content, Fm and Fv/Fm, but also resulted in the increase of the activities of superoxide dismutase (SOD) and peroxidase (POD), the contents of malonaldehyde (MDA) and soluble sugar, and leaf Fo. Besides, the root-shoot ratio of drought tolerance genotypes showed a rising trend, while the intermediate and drought-intolerant genotypes showed a declining trend. The principal component analysis turned 21 single indices into three independent comprehensive indices (accumulative contribution of 87.30%). The principal component 1 mainly reflected the information of biomass, root morphology, and leaf fluorescence parameters etc. The principal component 2 mainly reflected the information of root activity, root enzyme activity and osmotic adjustment substances etc. While the principal component 3 mainly reflected the information of aboveground morphology, partial leaf and root physiological characteristics. Nine F. tataricum genotypes were divided into three categories by clustering analysis: drought-tolerant type, middle type, and drought-intolerant type. In order to predict the drought resistance of each genotype and build a mathematical evaluation model, D values and drought tolerance index of various indexes were taken as the dependent variable and independent variable, respectively, for the stepwise regression analysis. By establishing an optimal regression equation, eight influential indices for drought resistance, including plant height, stem diameter, root-shoot ratio, root activity, main root length, MDA content, Fo, and leaf relative water content, were selected. The predictive values of drought tolerance of nine F. tataricum had a significantly correlation with D values (R²=0.988**), which showed a certain accuracy and efficiency to forecast the F. tataricum drought-resistance by using this equation. And the identification work could become easier when measuring these indexes selectively. 【Conclusion】Drought stress has significant influence on each index during seeding stage of F. tataricum. According to the cluster trend diagram, the tested categories can be divided into three types: drought tolerance type (including Diqing kuqiao, Xinong 9909, Qitai farmers), intermediate type (including Guangku 1, Qianku 6 and Yunqiao 1), and non drought-intolerance type (including Duoyuan kuqiao, Heifeng 1 and Xiqiao 1). The study selected the nine indexes including plant height, stem diameter, root-shoot ratio, root activity, main root length, MDA content, Fo, and leaf relative water content, which could rapidly identify the drought resistance of F. tataricum, and the predictive values of drought tolerance is significantly correlated with D values (R²=0.988**).

Transcriptome Analysis of Creeping Bentgrass (Agrostis stolonifera) Infected with Rhizoctonia solani

SHI Yi, NIU KuiJu, MA HuiLing

Scientia Agricultura Sinica. 2017, 50(17): 3323-3336. doi:10.3864/j.issn.0578-1752.2017.17.007

Abstract ( 339 )

PDF (2489KB) ( 342 )

Save

References | Related Articles | Metrics

【Objective】The objective of this study is to reveal the gene expression pattern of creeping bentgrass (Agrostis stolonifera) after been inoculated with Rhizoctonia solani, by comparing the gene expression level between plant with disease and without disease during gene transcription, and to identify the key genes of turfgrass responding to pathogen infection. 【Method】A. stolonifera which was grown for 14 days, was inoculated with R. solani. Leaf samples of plant with or without disease were collected after 3 days. High-throughput sequencing technology and Trinity software were used for obtaining the transcriptome. Different expression genes were screened with |log₂ (fold change)| ＞1, q-value＜0.005 as threshold and the assembly was used as reference. Bioinformatics software was used to analyze the transcriptome difference. iTAK was used for transcription factors. All the DEGs were blasted with plant resistance gene database to find the R protein. Mapman software was used to analyze the signaling pathway. 【Result】 Using the high-throughput transcriptome sequencing, 125 253 092 were obtained. 466 761 transcripts were assembled by Trinity software. More than half of them were longer than 700 bp and N₅₀=1 100 bp. CD-HIT was used to choose 334 212 transcripts as Unigene with the average length of 573 bp and N₅₀=791 bp. By comparing the A. stolonifera transcripts with disease and without disease, 7 937 up-regulated genes and 1 570 down-regulated genes were obtained. Among the up-regulated genes, 296 of them are transcription factors (TFs), while 142 of down-regulated genes can be defined as TFs. Those TFs were classified as 58 families, including 54 zinc-finger protein containing TFs C2H2, which was the most, then 22 C3H TFs. A total of 451 of different expression genes (DEGs) can be annotated as plant R-protein, which can be classified into 33 families. Among these families, NBS-LRR containing resistance protein, LRR-like receptor protein kinase, ABC-2 type transporter, U-box domain containing protein kinase, and heat shock protein gene, these 5 families showed the most variation. A bunch of up-regulated DEGs can be mapped in plant biotic response pathway and enriched in pathogen recognition, ROS eliminate, signaling transport, programmed cell death, pathogenesis-related protein and so on, while the down-regulated genes were enriched in plant growth and development pathways. The expression level of random selected DEGs was tested with qRT-PCR, and they were consistent with the result of RNA-seq analysis. The random selected genes included 12 C2H2 TFs, 10 C3H TFs and 12 R-protein genes.【Conclusion】The inoculation of pathogen caused great expression difference of A. stolonifera transcriptome. Most of the TFs, R-protein and defense-related genes were up-regulated to suppress pathogen growth. The inherent resistance of A. stolonifera to R. solani has been generated with the co-action of all these genes.

Correlational Analyses Between Dwarfing of Plant Height Induced by Wheat dwarf virus (WDV) Infection and Gibberellin Metabolism

WU HuiJuan, LIU Yan, WANG XiFeng

Scientia Agricultura Sinica. 2017, 50(17): 3337-3343. doi:10.3864/j.issn.0578-1752.2017.17.008

Abstract ( 308 )

PDF (809KB) ( 517 )

Save

References | Related Articles | Metrics

【Objective】 In recent years, wheat dwarf virus disease transmitted by leafhopper (Psammotettix alienus) is becoming one of the severe virus disease on triticeae crops in the northwest of China. Infected plants may be severely dwarfed and reduced effective tillering so that it causes serious production loss. The objectives of this study are to clear the correlation between dwarfing of plant height induced by Wheat dwarf virus (WDV) infection and gibberellin metabolism and provide a solid basis for the control of the disease. 【Method】The cultivar Yangmai 12 was used as experimental material. Leafhoppers were fed on viruliferous wheat containing WDV and then transferred to healthy seedling at single-leaf stage (3 heads/plant). The leaves were collected as samples at different time points according to experimental requirements and the seedlings treated with non-viruliferous leafhoppers as control. To guarantee the accuracy of the experiment, all the infected samples were validated by PCR. The plant GA3 ELISA Kit was used to assay the GA3 contents of both healthy and WDV-infected wheat leaves at 21 days post-infection (dpi). Viruliferous treatments were divided into two parallel groups, one was treated with spraying of exogenous GA3 at 50 mg·L^-1 and the other treated with water at 7 dpi. The spraying of GA3 solution was carried out 4 times every 7 days. Meanwhile, the control group consisted of similar size seedlings were treated with non-viruliferous leafhoppers. The phenotype was analyzed by statistical analysis of the plant heights after application of exogenous GA3 at 50 mg·L^-1. According to the coding sequence of ent-kaurene synthase-like 3 (KSL3) of Aegilops tauschii for reference, the complete coding sequence of KSL3 of Yangmai 12 was cloned with the primer pair (KSL3-F: 5′-ATGATGGTGAATCCGCCGC-3′, KSL3-R: 5′-TTAATGGTTGATCTTTGTTT-3′) and its structure was analyzed with BLAST. The total RNA of infected plants at 7, 14 and 21 dpi were prepared and reversed transcription into cDNA. The primers were designed following the obtained sequence of TaKSL3 (TaKSL3-F: 5′-GAGACATGTGCCATGGCGTTC-3′, TaKSL3-R: 5′-CGTGTCACTC AGATCGGTGGAG-3′). With EF-1α as the reference gene, RT-qPCR was used to analyze the expression level of gene related to GA metabolism pathway.【Result】The GA3 content of infected plants was reduced by 28.9% at 21 dpi by indirect ELISA and the height of infected plants increased by 35.9% owing to application of exogenous GA3 at 50 mg·L^-1 compared with control. Using homology-based cloning, the complete coding sequence of Yangmai 12 KSL3 was obtained, which is the key enzyme in gibberellin synthesis pathway. The sequence of KSL3 has the length of 1 827 bp, encoding 608 amino acids. By BLAST analysis, its DNA sequence was found to have a similarity of 85.2% compared with KSL3 of A. tauschii. Particularly, by qRT-PCR analysis, the expression level of KSL3 compared to healthy plants significantly decreased to 35.7% at 14 dpi and 9.6% at 21dpi, respectively.【Conclusion】 It was concluded that WDV could interfere the biosynthesis of GA leading to the decreased content of GA, which was responsible for the development of dwarf symptom that can be relieved by exogenous GA. This study will provide a solid basis for further research on pathogenesis of WDV and control of the disease.

Subcellular Localization and Expression Analyses of IP-L Protein Interacting with ToMV Coat Protein

PENG HaoRan, PU YunDan, ZHANG YongZhi, XUE Yang, WU GaiXia, QING Ling, SUN XianChao

Scientia Agricultura Sinica. 2017, 50(17): 3344-3351. doi:10.3864/j.issn.0578-1752.2017.17.009

Abstract ( 271 )

PDF (1308KB) ( 693 )

Save

References | Related Articles | Metrics

【Objective】Previous studies showed that the coat protein (CP) of ToMV could interact with protein L (IP-L) in Nicotiana benthamiana. The objectives of this study are to understand the collocation of ToMV CP and IP-L, investigate the expression of IP-L in different tissues of tobacco, and investigate the IP-L expression in tomato leaves in response to ToMV infection.【Method】The ToMV CP and IP-L target fragments were isolated from the recombinant pGBKT7-CP and pGADT7-IP-L recombinant vectors by digestion with corresponded enzymes. Then the plant expression vectors pPZP-IP-L-N-EGFP and pPZP -CP-N-DsRed and prokaryotic expression vector pEGX-IP-L were constructed. The plant expression vectors were transformed into Agrobacterium tumefaciens EHA105 by heat shock method. The transient expression of ToMV CP and IP-L in tobacco epidermal cells was observed under the fluorescent microscope. Real-time quantitative RT-PCR (qRT-PCR) was used to analyze the expression level of IP-L in different tobacco tissues. The recombinant plasmid pEGX-IP-L was transformed into E. coil BL21 to express the soluble GST-IP-L protein in the optimized condition. GST-IP-L protein was purified with high-affinity GST resin to immunize rabbit for preparing anti-IP-L antibody in a rabbit. The title was determined by enzyme-linked immunosorbant assay (ELISA). The specificity of anti-IP-L antibody and the expression of IP-L in tobacco leaves infected by ToMV were detected by Western blot method and verified it by qRT-PCR.【Result】The ToMV CP was found in cell cytoplasm, cell membrane and chloroplasts, but IP-L only present in leaf epidermal cells membrane. They both co-localized in the plasma membrane. The relative expression of IP-L in the tobacco leaves has the specific high expression, significantly higher than that in stem, root and flower. The soluble GST-IP-L protein with molecular weight 42.8 kD was successfully expressed in E. coil BL21 induced with 0.3 mmol·L^-1 IPTG at 30℃. About 4.2 mg fusion protein was purified and used to immunized rabbit. Anti-IP-L antibody with the title of 1/6 400 was obtained. Western blot analysis showed that the antibody could specially bind with IP-L. After the tobacco leaves were infected by ToMV for 1, 3 and 7 d, western blot analysis showed that IP-L was significantly up-regulated in leaves after 7 days of ToMV infection. qRT-PCR also got the same results, the expression level of IP-L was significantly (more than 200%) higher than that in healthy controls. 【Conclusion】The ToMV CP and IP-L were co-located in the plasma membrane of the tobacco epidermis cell. IP-L specifically expressed in the tobacco leaves at high level. The high title and specificity antibody of tomato’s IP-L were successfully achieved. The results of qRT-PCR and Western blot with the polyclonal antibody showed that the expression of IP-L in tobacco leaves was increased by the infection of ToMV.

Effects of Nitrogen Fertilizer Rates on Rhizosphere Soil Characteristics and Yield After Anthesis of Wheat

AN TingTing, HOU XiaoPan, ZHOU YaNan, LIU WeiLing, WANG Qun, LI ChaoHai, ZHANG XueLin

Scientia Agricultura Sinica. 2017, 50(17): 3352-3364. doi:10.3864/j.issn.0578-1752.2017.17.010

Abstract ( 420 )

PDF (474KB) ( 621 )

Save

References | Related Articles | Metrics

【Objective】 Making the relationship between rhizosphere soil properties and wheat grain yield or grain nitrogen(N) accumulation (GNA) clear could help managing N fertilizer application, improving N use efficiency and reducing environment pollution. 【Method】A field experiment with four N fertilizer treatments (0 kg N·hm^-2, CK; 150 kg N·hm^-2, N150; 240 kg N·hm^-2, N240; 300 kg N·hm^-2, N300) was conducted in 2014-2015 and 2015-2016, soil samples including rhizosphere soil (R) and bulk soil (B) in 0-20 cm and 20-40 cm depths were collected at anthesis, milking and maturity stages. Soil NH₄⁺-N, NO₃^--N, urease, saccharase and wheat biomass including root, stem, leaf, spike biomass and their N content were measured, and the relationships between rhizosphere soil characteristics and grain yield and GNA were analyzed. 【Result】(1) Results of the experiment showed that in comparison with CK, wheat grain yields of three N fertilizer treatments increased by 99%, 130% and 107% for the two annual average, respectively. Wheat root, stem, leaf and spike biomass and aboveground nitrogen accumulation (ANA) increased with the N application rates increasing, while nitrogen recovery efficiency (NRE) declined, and their differences among the four treatments were significant. (2) Over the periods of studies, soil NH₄⁺-N concentration, NO₃^--N concentration, soil sacharase and urease activities (0-20 cm excluded) of both rhizosphere and bulk soil in 0-20 cm and 20-40 cm depths showed a decreasing trend. The average NH₄⁺-N and NO₃^--N concentration of four fertilizer treatments in rhizosphere soil was significantly lower than that in bulk soil. In comparison with bulk soil in 0-20 cm depth, rhizosphere soil NH₄⁺-N concentration of four treatments in the two years reduced by 29%, and by 22% for NO₃^--N concentration; and rhizosphere soil NH₄⁺-N concentration reduced by 34% and by 14% for NO₃^--N concentration in 20-40 cm depth. Rhizosphere soil sacharase activities in 0-20 cm depth increased by 29% than that of bulk soil, and by 15% for urease activities; while increased by 33% for sacharase activities and by 13% for urease activities in 20-40 cm. (3) Pearson correlation analysis showed that soil inorganic-N (NH₄⁺-N + NO₃^--N), urease, and saccharase (2016 year GNA excluded) in rhizosphere soil and bulk soil were all significantly and positively correlated with wheat grain yield and GNA. 【Conclusion】All of these results indicated that wheat rhizosphere soil available N concentration was less than the bulk soil after anthesis stage, while rhizosphere soil enzyme activity was higher than the bulk soil. Grain yield and GNA were all significantly and positively correlated with rhizosphere and bulk soil. The variations of rhizosphere and bulk soil characteristics could affect wheat grain yield.

Nitrogen Effect and Emission Reduction Potential of Rice Planting in Jilin Province by GIS

YAN Li, FENG GuoZhong, LAN Chang, GAO Qiang

Scientia Agricultura Sinica. 2017, 50(17): 3365-3374. doi:10.3864/j.issn.0578-1752.2017.17.011

Abstract ( 245 )

PDF (2576KB) ( 454 )

Save

References | Related Articles | Metrics

【Objective】The differences of nitrogen fertilizer effect in rice planting regions were studied to strengthen precision nitrogen management, to increase crop yield and fertilizer use efficiency, and to reduce nitrogen emission from farmland.【Method】Rice yield and effect of applied nitrogen fertilizer in different rice planting regions in Jilin Province were studied to explore the differences of nitrogen effect and emission reduction potential in different regions with no nitrogen fertilizer treatment (N₀P₂K₂) as the control, three levels of nitrogen fertilizer application treatments including (0.5N₂P₂K₂), (N₂P₂K₂), (1.5N₂P₂K₂) were designed in “soil testing and formulated fertilization” field experiment carried out in 2005-2013 in Jilin Province. 【Result】The results showed that rice yields in different regions in Jilin Province were variant significantly, the highest was in the western region, and the lowest was in the eastern region. The yield in western region averaged 7.6 t·hm^-2with no nitrogen fertilizer application, yield differences of which reached 2.1 t·hm^-2and 2.2 t·hm^-2 on an average, respectively, compared to that in the central and eastern regions. With nitrogen fertilizer application, minimum increasing yield rate in the central and eastern regions was 29.8% (the maximum was 59.5%), which was significantly higher than 12.6% in the western region (the maximum was 29.4%). Agronomic utilization ratios of nitrogen fertilizer in the central and eastern region were 12.2-19.7 kg·kg^-1 and 12.5-19.5 kg·kg^-1, far higher than 8.8-13.1 kg·kg^-1in the western region. Function relation between the nitrogen fertilizer rate and net income was established by maximum economic income(MRTN) method to calculate the best nitrogen application rate. The optimal nitrogen rate in the western region, central region and eastern region was 114.9, 128.9, and 134.1 kg·hm^-2, respectively, which reduced by 25.6, 18.3, and 5.3 kg·hm^-2, respectively, compared with the recommended fertilizer rate. With no significant differences in rice yield, nitrogen fertilizer rate decreased in three regions, especially in western and central regions. By calculating nitrogen reduction cost and yield income, economic benefit was increased in all regions, especially in the western region. Yearly nitrogen input reduction in western, central and eastern regions in Jilin Province was 4 378, 7 064 and 604 t respectively; nitrogen emission reduction was 98.2, 158.6, and 13.6 t, respectively, by optimal fertilizer amount input with no obvious yield differences. 【Conclusion】Measures should be made to control the nitrogen fertilization rate in western region; total emission reduction potentials in central region is the largest because of the planting areas; fertilizer application rate in the eastern region is moderate presently, rice yield can be improved by cooperating with other management measures to reduce the limitation of natural factors.

Genome-Wide Association Studies for Flowering Time in Brassica rapa

GAO BaoZhen, LIU Bo, LI ShiKai, LIANG JianLi, CHENG Feng, WANG XiaoWu, WU Jian

Scientia Agricultura Sinica. 2017, 50(17): 3375-3385. doi:10.3864/j.issn.0578-1752.2017.17.012

Abstract ( 439 )

PDF (2546KB) ( 791 )

Save

References | Related Articles | Metrics

【Objective】To identify the genetic loci or candidate genes for flowering time regulation in Brassica rapa for improvement of pre-mature bolting resistance of B. rapa. 【Method】 In this study, 116 B. rapa germplasm accessions were selected to evaluate flowering time variations in greenhouse and open-field, respectively. Total genomic DNA was extracted with 1.2x re-sequenced depth. Filtering, mapping with reference by Pooled Mapping was conducted to obtain a genomic high quality SNP set. Then the population structure and linkage disequilibrium (LD) were analyzed using SNP set after condition filtering. In total 2000 SNP points were selected from all SNPs randomly to conduct phylogenetic tree analysis using PhyML software with maximum likelihood method. all high quality SNPs were used to conduct genomic linkage disequilibrium analysis with Haploview software. Genome-wide association study (GWAS) for flowering time variations was then conducted based on software TASSEL, GAPIT and R. According to the position of strong association signals and LD block, the candidate signals for flowering time were identified. Eventually, flowering time candidate genes in B. rapa were predicted by gene colinearity relationship between A. thaliana and B. rapa, and gene function annotation.【Result】The 116 B. rapa accessions showed extensive variations in flowering time. Significant variation was also observed between greenhouse and open-field environments. The distribution of flowering time under open-field was partial normal, while the flowering time distributed evenly under greenhouse. Phenotypes of flowering time were significantly correlated between different environments, indicating that genetic effect played a crucial role in regulation of flowering time. A total of 1.03 million SNPs covering genome-wide were generated by biotechnology analysis. Population structure showed that accessions from each sub-group were clustered, and had a close relationship with geographic origin in phylogenetic tree. The linkage disequilibrium decay across genome-wide was 2.3 kb, demonstrating that there were frequent recombinations and variants in 116 B. rapa accessions. A total of 54 strong signals (P＞4) were detected using mixed linear model and 87 (P＞5) using general linear model under two different environments. Thirty-three strong signals (27 loci under greenhouse, 19 loci under open-field) were saved after considering LD block (r²＞0.33), including 13 co-identified SNPs. Based on genome colinearity between A. thaliana and B. rapa, and gene function annotation, 14 candidate genes were predicted. Three candidate genes, FUL, PHYB, FPF1, were co-identified under greenhouse and open-field environments. FT1, a key gene involved in flowering time regulation was also identified under open-field condition.【Conclusion】 Correlation analysis of flowering time under different environments indicated that genetic control is a decisive effect on flowering time. A total of 33 significant associated SNPs controlling flowering time were identified by GWAS. By combining LD block, genome colinearity between A. thaliana and B. rapa, and gene annotation, 14 flowering time candidate genes were predicted.

Cloning and Expression Analysis of Copper and Zinc Superoxide Dismutase Cu/Zn-SOD Gene Family from Luffa cylindrical

ZHU HaiSheng, LIU JianTing, CHEN MinDong, LI YongPing, WANG Bin, ZHANG QianRong, YE XinRu, LIN Hui, WEN QingFang

Scientia Agricultura Sinica. 2017, 50(17): 3386-3399. doi:10.3864/j.issn.0578-1752.2017.17.013

Abstract ( 369 )

PDF (2273KB) ( 673 )

Save

References | Related Articles | Metrics

【Objective】The aim of this study was to clone the Cu/Zn-SOD gene family from Luffa cylindrical, investigate their sequence characteristics and analyze their expression in luffa browning. These findings will provide a scientific basis for further revealing the mechanism of luffa browning and lay a practical foundation for the genetic improvement of luffa. 【Method】 The cDNA sequences of Cu/Zn-SOD gene family were obtained by transcriptome sequencing and RT-PCR. The bioinformatics methods were used to analyze the putative amino acid sequence, and quantitative real-time PCR (qRT-PCR) method was used to study the expression of Cu/Zn-SOD gene family in different tissues and browning conditions. The superoxide dismutase enzyme activity was measured by NBT deoxidization method. The total phenols was measured by folin-cioncaleuc method. 【Result】Three cDNAs of Cu/Zn-SOD were cloned from luffa fruit, in turn being named LcCu/Zn-SOD1, LcCu/Zn-SOD2 and LcCu/Zn-SOD3. The cDNA sequence of LcCu/Zn-SOD1 was 758 bp in length, containing a 456 bp opening reading frame(ORF), encoded a polypeptide of 152 amino acids. The cDNA sequence of LcCu/Zn-SOD2 was 799 bp in length, containing a 471 bp ORF, encoded a polypeptide of 157 amino acids. The cDNA sequence of LcCu/Zn-SOD3 was 1 011 bp in length, containing a 663 bp ORF, encoded a polypeptide of 221 amino acids. They shared over 90% identity with the homologous proteins from Cucumis melo, Cucurbita pepo and Cucumis sativus. The bioinformatics analysis showed that three proteins were hydrophilic protein without signal-peptide and transmembrane region, and the Wolf Psort protection indicated that they were located in the cytoplasm. The expression of LcCu/Zn-SODgene familywas the highest in root and the lowest in flower. During post-harvest storage, the expression of LcCu/Zn-SOD1 and LcCu/Zn-SOD3 was up-regulated in the early, and then decreased. The expression levels of three genes were overall down-regulated in fresh-cut luffa fruit. Correlation analysis showed that the expression level of LcCu/Zn-SOD1 showed a extremely significant positive correlation with SOD activity, and the expression level of LcCu/Zn-SOD3 showed a significant positive correlation with SOD activity during fresh-cut and post-harvest storage. SOD activity was significantly and negatively correlated with total phenols content during fresh-cut conditions. LcCu/Zn-SOD1 and LcCu/Zn-SOD3 play an important role in regulating the activity of SOD, and the expression of LcCu/Zn-SOD1 and LcCu/Zn-SOD3 may influence the activity of SOD and the process of luffa browning.【Conclusion】Three cDNAs of Cu/Zn-SOD were firstly obtained and characterized from luffa fruit, LcCu/Zn-SOD1 and LcCu/Zn-SOD3 may play an important role in luffa browning process.

Evaluation of Frozen Fruit Quality of Different Pear Cultivars

WANG Yang, WANG WenHui, JIA XiaoHui, TONG Wei, WANG ZhiHua, YANG XiaoLong

Scientia Agricultura Sinica. 2017, 50(17): 3400-3412. doi:10.3864/j.issn.0578-1752.2017.17.014

Abstract ( 401 )

PDF (846KB) ( 730 )

Save

References | Related Articles | Metrics

【Objective】 In order to find out the key indicators and select suitable pear cultivars for freezing, different frozen pears were selected as the research objects to analyze their frozen quality. 【Method】 There were 13 quality indicators and sensory evaluation of fifty-nine frozen pear cultivars were evaluated, in which, the quality indicators included peel L^*, flesh L^*, a^*, b^*, h, soluble solid content (SSC), titratable acid (TA) content, the ratio of soluble solid content to titration acid content (SSC/TA), juice yield, pH, stone cell content, alcohol content and juice leakage. The correlations of 13 quality indicators and sensory evaluation were analyzed and the principal components analysis (PCA) was used to screen the key indicators. Then the fuzzy evaluation method was used to rank the pear cultivars based on their suitability for frozen.【Result】Correlation analysis showed that considerable variations were found in different frozen quality indicators and sensory evaluation among tested pears. Peel L^* showed a positive correlation with juice leakage rate (P＜0.05), flesh L^* was significantly correlated with flesh a^*andb^*(P＜0.01), and flesh h showed a negative relationship with flesh a^*(P＜0.01). In addition, the sensory evaluation was significantly correlated with the flesh L^*, TA content, SSC/TA, Stone cell content and juice leakage rate (P＜0.01). The PCA results showed that the eigenvalues were greater than 0.9 and the cumulative proportion was 74.257% of the first four principal components. The first principal component was named taste factor (TF), it contained 37.272% information of the total amount. TF was mainly determined by SSC content, the TA content and the pH. The second principal component was named browning factor (BF), it contained 19.687% information of the total amount. BF was mainly determined by flesh a^*and flesh h. The third principal component was named roughness factor (RF), it contained 10.074% information of the total amount. RF was mainly determined by stone cell content. The fourth principal component was named appearance factor (AF), it contained 7.224% information of the total amount. AF was mainly determined by peel L^*. Among the 13 quality indicators, SSC, TA content, pH, flesh a^*andh, stone cell content, peel L^*, together with juice leakage rate contributed the most, so these seven factors were selected as the key indicators for the evaluation of frozen pear qualities. Based on fuzzy evaluations, the cultivars of Jianbali, Shatangli, Ruan'erli, Balixiang, Okusankichi, Baiba Lixiang, Hongnanguo and Reli were selected as the suitable ones (Huangjin Duima was removed because of its high stone cell content). However, the Xianghuang, Hongmacha, Housui, Huanghua, Lshiiwase, Okusankichi, Sumuli, Waseaka and Xinli No.7 were not suitable cultivars to make frozen pears because of the lower SSC and TA contents. Sensory evaluation indicated that the suitable frozen pears had high SSC and TA content, light browning. This was basically consistent with the results of PCA and fuzzy evaluation. 【Conclusion】Based on the quality evaluation, price and market share of 59 pears cultivars, ussurian pear is selected as the most suitable ones to make frozen pear, except for some cultivars with higher stone cell content and lower TA content.

Saponion Profiles and Antioxidant Activity, α-Glucosidase Inhibitory Activity of Momordica charantia of Different Varieties

LIU HuiJuan, ZHANG MingWei, ZHANG RuiFen, ZHANG Yan, WEI ZhenCheng, MA YongXuan, LIU Lei, DENG YuanYuan

Scientia Agricultura Sinica. 2017, 50(17): 3413-3421. doi:10.3864/j.issn.0578-1752.2017.17.015

Abstract ( 382 )

PDF (490KB) ( 843 )

Save

References | Related Articles | Metrics

【Objective】 The content and composition of saponion of 13 different M. charantia varieties as well as the antioxidant activity and inhibitory effect of α-glucosidase were studied. 【Method】 Perchloric acid-vanilin-glacial method was used to determine saponin content, while HPLC method was used to measure the contents of 7 saponin components. In addition, their antioxidant activity was evaluated by oxygen radical absorbance capacity (ORAC). The 4-nitrophenyl-2-β-D-glucopyranoside method was used to measure the α-glucosidase inhibitory activity and to analyze the relationship between the components and the corresponding activity. 【Result】There is a significant difference in contents of saponin in 13 different varieties of M. charantia.The content ranges of saponin were 0.52-1.20 g/100g DW, with an average value being 0.79 g/100g DW, and the coefficient of variability being 21.65%. The average contents of components were as follows: momorcharaside A 5.32 μg·g^-1DW, momordicoside A 25.42 μg·g^-1 DW, karaviloside XI 3.96 μg·g^-1DW, momordicoside F2 66.95 μg·g^-1DW, momordicoside K 183.70 μg·g^-1DW, (23E)-3β,7β,25-trihydroxycucubita-5,23-dien-19-al 40.13 μg·g^-1 DW, and kuguacin N 3.87 μg·g^-1 DW. ORAC values of the 13 M. charantia varieties varied from 2 747.76 to 15 584.07 μmol Trolox?g^-1, the average value being 8 879.48 mol Trolox·g^-1, the coefficient of variability being 34.91% and the IC₅₀ value of alpha glucosidase varied from 1.55 to 4.96 mg·mL^-1. 【Conclusion】Significant differences in the components of saponin and the antioxidant activity and the inhibitory effect on α-glucosidase of different varieties of M. charantia were detected. Saponin is the main active basis of α-glucosidase activity in M. charantia, but not the main antioxidant substance. (23E)-3β,7β,25-trihydroxycucubita-5,23-dien-19-al is the major active compound.

Application of Gene Engineering Technique in Aflatoxin Biodegradation

JI Cheng, JIA Ru, ZHAO LiHong

Scientia Agricultura Sinica. 2017, 50(17): 3422-3428. doi:10.3864/j.issn.0578-1752.2017.17.016

Abstract ( 492 )

PDF (362KB) ( 1079 )

Save

References | Related Articles | Metrics

Aflatoxin is highly carcinogenic, mutagenic and carcinogenic. It can reduce animal production performance, feed conversion efficiency and the amount of meat, egg and milk yield. Besides, it can increase animal mortality, causing huge economic losses in animal husbandry. In addition, aflatoxin can threaten human’s health through the meat, eggs, milk, food chain. Therefore, prevention and control of aflatoxin has become a global concern. At present, detoxification of aflatoxin in feed mainly by physical adsorption, such as montmorillonite, activated carbon, yeast cell wall, but the physical adsorption can only adsorb aflatoxin, can not reduce the amount of toxin. Moreover, it can cause the secondary pollution to the environment. The adsorbent is unstable and may absorb vitamins and other nutrients in feed, resulting in a decrease of feed quality. The biological degrading method could fully degrading mycotoxins, has strong specificity, no adverse impact on the nutritional value of the feed and the ability to avoid the release toxins again. This method has attracted the attention of many researchers. Nowadays, more and more studies have reported that fungi, bacteria and their enzymes are able to degrade aflatoxin, but there are few reports on degrading aflatoxins using recombinant enzymes. The application of gene engineering technology in aflatoxin biodegradation can be separated and purified from the compound detoxification enzymes by modern molecular biology methods. Because of the complex process of enzyme separation and purification, unstable enzyme activity and harsh enzymatic conditions, it is difficult to be used in practical production. Therefore, people use genetic engineering means to detoxification gene with high activity for gene cloning, heterologous implementation of degrading enzyme gene in prokaryotic or eukaryotic expression engineering strain, which laid a theoretical foundation for the practical production of enzyme in the degradation of mycotoxins. The recent advances in aflatoxin degrading using gene engineering technique and development direction are reviewed in this paper, to provide theoretical and practical bases for using of enzymes in degrading mycotoxins in feed and food.

Effect of Melatonin on Insulin and Gαi/o Expression in Rat Insulinoma Cell Line

ZHAO YiWen, ZHAO Jia, PANG QuanHai

Scientia Agricultura Sinica. 2017, 50(17): 3429-3438. doi:10.3864/j.issn.0578-1752.2017.17.017

Abstract ( 274 )

PDF (2370KB) ( 367 )

Save

References | Related Articles | Metrics

【Objective】To demonstrate the role of melatonin in the circadian secretion of insulin, the effect of melatonin on insulin and Gαi/o expression in rat insulinoma cell line (INS-1) and molecular mechanism were studied. As melatonin can regulate insulin secretion in a circadian manner, maintaining a homeostasis of blood glucose by regulating diurnal glucose metabolism, thus study on the effect of melatonin on expression of insulin may provide a basis for regulation of melatonin in circadian secretion of insulin.【Method】The cryopreserved INS-1 cells were passaged for at least three generations. After the passage of cells, INS-1 cells were cultured in RPMI1640 medium for about 24-48 h. When the cell confluency reached 60%-70%, washed with RPMI1640 without serum and glucose for two times. INS-1 cells were cultured in media supplemented with different concentrations of glucose(0, 10, 20, 30, and 50 mmol·L^-1) and 100 nmol·L^-1 melatonin for 12 h treatment, then observation of morphological changes of INS-1 cells and statistical analysis were performed. The total RNA extracted from INS-1 cells by Easypure RNA kit, measuring the cells total RNA concentration and purity, then the quality of total RNA was measured by formaldehyde denaturing agarose gel electrophoresis. TranScript One-step Removal and Synthesis Supermix were used to synthesize cDNA by reverse transcription. Primer Premier 5.0 primer design software, reference gene sequence of GenBank were used to design primers. qRT-PCR was used to detect the changes of Insulin1, Gαi1, Gαi2, Gαo, PKA and PKCα inmRNA level.【Result】After incubation of INS-1 cells with different concentrations of glucose, it was observed that the growth of cell synapse first increased and then decreased along with the increase of the concentration of glucose in the culture medium. In the 20 mmol·L^-1 glucose treatment group, the synaptic growth of INS-1 cells was increased significantly; in the 20 mmol·L^-1glucose and melatonin co-treatment group, the area of INS-1 cells was increased, but the synaptic growth was not obvious. In the glucose treatment group, the mRNA level of Insulin1 showed a trend of increase and then reduction, especially was increased at 20 mmol·L^-1 (P＜0.05). The mRNA level of Gαi1 showed a trend of reduction and then increase, especially was reduced at 20 mmol·L^-1 (P＜0.05). The mRNA level of Gαi2 and Gαo was not significantly reduced (P＞0.05). Thus, Gαi1 but not Gαi2 and Gαo showed a negative correlation with Insulin1 mRNA level. Compared with 20 mmol·L^-1 glucose treatment group, the mRNA level of Gαi1 was significantly increased (P＜0.05) at 20 mmol·L^-1 glucose and melatonin co-treatment group, the mRNA level of Insulin1 and PKCα was significantly reduced (P＜0.05), whereas the mRNA level of PKA was not significantly reduced (P＞0.05).【Conclusion】Melatonin inhibits the expression of Insulin1 in INS-1 cells and reduces insulin secretion resulting in the adaptive hyperplasia of INS-1 cells. The binding of melatonin to its receptor can promote the increase of Gαi1 mRNA levels and inhibit the expression of PKCα, which leads to the inhibited expression of Insulin1 and decreased insulin secretion, so that blood glucose remains homeostatic during the night.

Table of Content