高粱GRF基因家族鉴定及SbGRF4原核表达分析 Click here to download the full text Citation：陈俊,屈志广,方远鹏,蒋君梅,李向阳,谢鑫.高粱GRF基因家族鉴定及SbGRF4原核表达分析.Journal of Plant Protection,2020,47(4):929-938 Hits: Download times: Author Name Affiliation E-mail CHEN Jun  College of Agriculture, Guizhou University, Guiyang 550025, Guizhou Province, China    QU Zhiguang  College of Agriculture, Guizhou University, Guiyang 550025, Guizhou Province, China    FANG Yuanpeng  College of Agriculture, Guizhou University, Guiyang 550025, Guizhou Province, China    JIANG Junmei  College of Agriculture, Guizhou University, Guiyang 550025, Guizhou Province, China    LI Xiangyang  Key Laboratory of Green Pesticide and Agricultural Bioengineering, Ministry of Education Guizhou University, Guiyang 550025, Guizhou Province, China  xyli1@gzu.edu.cn  XIE Xin  College of Agriculture, Guizhou University, Guiyang 550025, Guizhou Province, China  xiexin2097757@163.com  中文摘要:为研究高粱生长调控因子（growth-regulating factor，GRF）在高粱生长过程中的功能，采用生物信息学分析方法对高粱GRF基因家族进行鉴定，并以高粱BTx623品种为材料，利用PCR技术扩增获得SbGRF4基因cDNA全长序列，构建pET-28a-SbGRF4重组质粒，采用原核表达技术分别对表达菌株、诱导温度以及异丙基-β-D-硫代半乳糖苷（isopropyl-β-D-thiogalactopyranoside，IPTG）诱导浓度进行优化，最后用Western blot技术对原核表达的SbGRF4蛋白进行验证。结果表明，在高粱中共鉴定到10个GRF基因SbGRF1~SbGRF10；亚细胞定位预测结果显示均定位于细胞核中；分布于高粱的6条染色体上，所有家族成员均含有QLQ和WRC保守结构域；系统进化结果显示，高粱GRF蛋白分为Class 2、Class 3、Class 4a、Class 4b四个亚家族；相同亚家族各个成员间外显子和内含子的数量差异较小；同一亚家族的高粱GRF蛋白保守基序具有相似性；克隆到的SbGRF4基因cDNA序列全长为1 221 bp，并成功构建pET-28a-SbGRF4融合表达载体；SbGRF4蛋白的最佳表达菌株为大肠杆菌Escherichia coli JM109（DE3）菌株，最佳诱导温度为25℃，最佳IPTG诱导浓度为0.6 mmol/L；Western blot验证结果显示原核表达的蛋白为SbGRF4。表明SbGRF4蛋白可在大肠杆菌原核表达系统中表达。 中文关键词:高粱  基因克隆  GRF基因家族  SbGRF4  原核表达   Identification of GRF gene family and prokaryotic expression analysis of SbGRF4 in sorghum Author Name Affiliation E-mail CHEN Jun  College of Agriculture, Guizhou University, Guiyang 550025, Guizhou Province, China    QU Zhiguang  College of Agriculture, Guizhou University, Guiyang 550025, Guizhou Province, China    FANG Yuanpeng  College of Agriculture, Guizhou University, Guiyang 550025, Guizhou Province, China    JIANG Junmei  College of Agriculture, Guizhou University, Guiyang 550025, Guizhou Province, China    LI Xiangyang  Key Laboratory of Green Pesticide and Agricultural Bioengineering, Ministry of Education Guizhou University, Guiyang 550025, Guizhou Province, China  xyli1@gzu.edu.cn  XIE Xin  College of Agriculture, Guizhou University, Guiyang 550025, Guizhou Province, China  xiexin2097757@163.com  Abstract:In order to study the function of growth-regulating factor (GRF) in the growth of sorghum, bioinformatics analysis methods were used to identify the sorghum GRF gene family and the full-length cDNA sequence of SbGRF4 gene was amplified by PCR using sorghum BTx623 as the template. Expression strains, induction temperatures, and isopropyl-β-D-thiogalactopyranoside (IPTG) induction concentrations were optimized for SbGRF4 prokaryotic expression. Finally, the expressed SbGRF4 protein was verified by Western blot. The results showed that ten GRF genes (SbGRF1-SbGRF10) were identified in sorghum; the subcellular localization of GRF genes was in the nucleus; all the family members, distributed on six chromosomes of sorghum, contained QLQ and WRC conserved domains. The phylogenetic analysis showed that the sorghum GRF gene family proteins were divided into four subfamilies: Class 2, Class 3, Class 4a, and Class 4b. Gene structure analysis showed that the numbers of exons and introns among members of the same subfamily were various. Motif analysis showed that the same subfamily proteins were similar. SbGRF4 was 1 221 bp in length and the pET-28a-SbGRF4 fusion expression vector was successfully constructed. The best expression strain for SbGRF4 was JM109 (DE3) of Escherichia coli; the optimal induction temperature was 25℃, and the optimal IPTG induction concentration was 0.6 mmol/L. The expressed SbGRF4 protein was verified by Western blot. The results indicated that SbGRF4 protein could be expressed in E. coli prokaryotic expression system. keywords:sorghum  gene cloning  GRF gene family  SbGRF4  prokaryotic expression View Full Text  View/Add Comment  Download reader

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