摘要
为探究高粱抗病基因SbSGT1的功能,以高粱BTx623为材料,利用RNA反转录得cDNA,以cDNA为模板扩增出全长SbSGT1基因,并对其进行生物信息学分析。进一步构建pET-28a-SbSGT1重组质粒进行原核表达,并分别对表达菌株、诱导温度以及异丙基-β-D-硫代吡喃半乳糖苷(IPTG)诱导浓度进行了优化。结果表明,SbSGT1全长1095 bp,编码364个氨基酸,蛋白理论分子大小约为40.45 kDa,蛋白质等电点为5.0。进化树分析表明,SbSGT1与玉米和水稻的亲缘性较高;蛋白质序列分析表明,SbSGT1蛋白为亲水性蛋白,定位在细胞质中;二级结构预测发现其α螺旋和无规则卷曲占比最高,分别达到43.41%和45.88%。原核表达结果表明,SbSGT1最佳表达菌株为大肠杆菌BL21(DE3),最佳诱导温度和IPTG浓度分别为25℃和0.8 mmol·L-1。本研究为进一步探究SbSGT1基因在高粱抗病机理中所发挥的生物学功能奠定了基础。
In order to explore the function of sorghum disease resistant gene SbSGT1 of sorghum,the full-length SbSGT1 gene was amplified from sorghum BTx623 by RNA reverse transcription,and analyzed by bioinformatics.The pET-28 a-SbSGT1 recombinant plasmid was further constructed for prokaryotic expression,and the expression strain,induction temperature and induction concentration of isopropyl-β-D-thiopyranogalactoside(IPTG)were optimized.The results showed that SbSGT1 was 1095 bp in length and encoded 364 amino acids.The theoretical molecular size of SbSGT1 was about 40.45 kDa and the isoelectric point of SbSGT1 was 5.0.Phylogenetic tree analysis showed that SbSGT1 was highly related to maize and rice;protein sequence analysis showed that SbSGT1 was a hydrophilic protein,which was located in the cytoplasm;secondary structure prediction showed thatαhelix and irregular curl accounted for the highest proportion,reaching up to 43.41%and 45.88%respectively.The results of prokaryotic expression showed that the best expression strain of SbSGT1 was Escherichia coli BL21(DE3),and the best induction temperature and IPTG were 25℃and 0.8 mmol·L-1 respectively.This study laid a foundation for the further exploring of the biological function of SbSGT1 gene in sorghum disease resistance mechanism.
作者
陈美晴
陈俊
蒋君梅
王营锴
屈志广
方远鹏
任明见
谢鑫
CHEN Meiqing;CHEN Jun;JIANG Junmei;WANG Yingkai;QU Zhiguang;FANG Yuanpeng;REN Mingjian;XIE Xin(College of Agricultural,Guizhou University,Guiyang,Guizhou 550025;Guizhou Sub-Center of National Wheat Improvement Center,Guiyang,Guizhou 550025)
出处
《核农学报》
CAS
CSCD
北大核心
2020年第9期1933-1942,共10页
Journal of Nuclear Agricultural Sciences
基金
国家自然科学基金资助项目(31801691)
贵州省科技厅农业公关项目(黔科合支撑[2019]2408号)
贵州省高层次留学人才创新创业择优资助项目([2018]02号)
贵州大学大学生创新创业训练计划项目(贵大[国]创字2019[031])。
关键词
高粱
SGT1
基因克隆
原核表达
sorghum
SGT1
gene cloning
prokaryotic expression
