芝麻抗裂蒴基因SiIND1的克隆与原核表达  

Cloning and Prokaryotic Expression of Capsule Indehiscent Gene SiIND1 from Sesame

作　　者：刘艳阳[1] 武轲[1] 梅鸿献[1] 杜振伟[1] 崔承齐[1] 江晓林 郑永战[1] LIU Yanyang;WU Ke;MEI Hongxian;DU Zhenwei;CUI Chengqi;JIANG Xiaolin;ZHENG Yongzhan(Sesame Research Center,Henan Academy of Agricultural Sciences,Zhengzhou 450002,China)

机构地区：[1]河南省农业科学院芝麻研究中心,河南郑州450002

出　　处：《河南农业科学》2020年第5期63-68,共6页Journal of Henan Agricultural Sciences

基　　金：国家特色油料产业技术体系项目(CARS-14);河南省农业科学院优秀青年科技基金项目(2018YQ27);国家自然科学基金青年基金项目(31301359)。

摘　　要：LOC105167765基因属于KANADI(KAN)基因家族,与芝麻裂蒴性有关。从芝麻裂蒴品种豫芝11号和抗裂蒴品种郑芝InD01中分别克隆得到LOC105167765基因cDNA序列SiIND1,并进行序列生物信息学分析和原核表达分析。结果表明,豫芝11号SiIND1基因cDNA序列全长为1320 bp,编码439个氨基酸,推测的蛋白质分子质量为50.0 ku,等电点7.53,编码SHAQKYF类MYB家族的转录因子,具有GARP保守结构域,属于KAN家族。郑芝InD01 SiIND1基因cDNA序列长度为1246 bp,包括1个最大的开放阅读框900 bp,编码299个氨基酸,推测的蛋白质分子质量为32.9 ku,等电点8.37,GARP结构域发生缺失突变,翻译提前终止,可能导致基因功能丧失。将SiIND1连接到原核表达载体pET30a,并转化大肠杆菌BL21,通过IPTG诱导和SDS-PAGE电泳检测,表达的融合蛋白与预测蛋白大小相符合,进一步证实了抗裂蒴品种和裂蒴品种SiIND1基因的蛋白质表达存在差异,抗裂蒴材料SiIND1基因在翻译过程中发生提前终止。LOC105167765 belongs to KANADI(KAN)gene family,which is related with sesame capsule indehiscence.In this study,the capsule dehiscent variety Yuzhi No.11 and capsule indehiscent variety Zhengzhi InD01 were selected as experiment materials,and the SiIND1 full-length cDNAs of LOC105167765 were amplified.Bioinformatics analysis and prokaryotic expression were conducted.The results showed that the whole cDNA sequence of SiIND1 in Yuzhi No.11 was 1320 bp and encoded 439 amino acids.The putative protein of the gene had an isoelectric point of 7.53 and a calculated molecular weight of 50.0 ku.Sequence analysis showed that the protein of SiIND1 in Yuzhi No.11 contained a SHAQKYF class MYB-like DNA binding domain similar to Arabidopsis KAN family members and shared a highly conserved GARP domain.The whole cDNA sequence of SiIND1 in Zhengzhi InD01 was 1246 bp,which contained an ORF of 900 bp and encoded 299 amino acids.The putative protein of the gene had an isoelectric point of 8.37 and a calculated molecular weight of 32.9 ku.Compared to Yuzhi No.11,the SiIND1 gene in Zhengzhi InD01 had a deletion and generated a premature stop codon in GARP domain,which might lead to loss function of this gene.The SiIND1 gene was connected to the prokaryotic expression vector pET30a and transformed into Escherichia coli BL21 to induce expression.After the detection of SDS-PAGE,bands of target protein were identified.It was further confirmed that there was a difference in SiIND1 protein expression size between the capsule dehiscent variety and indehiscent variety,and there was a premature termination of translation in capsule indehiscent variety.

关 键 词：芝麻 抗裂蒴基因 基因克隆 生物信息学分析 原核表达 

分 类 号：S565.3[农业科学—作物学]