谷子糖基转移酶基因SiDDOST的克隆及表达载体的构建  

Cloning of Glycosyltransferase Gene SiDDOST and Construction of Expression Vector in Foxtail Millet

作　　者：李兴杰 王振山 栾孟澳 贾小平[1] LI Xingjie;WANG Zhenshan;LUAN Mengao;JIA Xiaoping(College of Agriculture,Henan University of Science and Technology,Luoyang,Henan 471023,China)

机构地区：[1]河南科技大学农学院,河南洛阳471023

出　　处：《天津农业科学》2024年第10期1-7,共7页Tianjin Agricultural Sciences

基　　金：国家自然科学基金(31471569)。

摘　　要：为探究谷子糖基转移酶基因的生物学功能及调控作用,以谷子为材料,利用RT-PCR技术从谷子农家品种毛毛亮扩增获得1338 bp包含完整编码区的糖基转移酶基因(SiDDOST)cDNA序列,并通过生物信息学方法分析SiDDOST全长序列的特征,利用限制性内切酶NcoⅠ和PmlⅠ,分别将目的基因片段与植物过表达载体pCAMBIA3301双酶切,然后切胶回收基因片段和载体片段,通过T4 DNA连接酶将糖基转移酶基因片段与过表达载体pCAMBIA3301连接,重组表达载体转化大肠杆菌DH5α,经重组表达载体与空载体电泳检测、菌液PCR、双酶切分析验证。结果表明:谷子糖基转移酶基因CDS区为1323 bp,编码441个氨基酸,DDOST蛋白N末端的30个氨基酸范围保守性差,糜子与其他8种植物相比,DDOST蛋白存在3处插入/缺失突变;系统进化分析表明,谷子与糜子、哈希黍、柳枝稷存在较近进化关系;目的基因片段已成功插入表达载体中,转录方向正确。综上,本研究构建的谷子糖基转移酶基因过表达载体为后续在拟南芥、水稻或谷子中进行遗传转化、功能验证奠定了基础。In order to explore the biological function and regulatory role of glycosyltransferase gene in foxtail foxtail foxtail fox Mao Maoliang amplified the 1338 bp cDNA sequence of the glycosyltransferase gene(SiDDOST)containing the complete coding region,and analyzed the characteristics of the full-length sequence of SiDDOST by bioinformatics methods,and the target gene fragment was double-digested with the restriction enzyme Nco I and Pml I,respectively,and the gene fragment and the vector fragment were recovered,and the glycosyltransferase gene fragment was linked to the overexpression vector pCAMBIA3301 by T4DNA ligase,and the recombinant expression vector was transformed into E.coli DH5α.It was verified by electrophoresis detection of recombinant expression vector and empty vector,PCR of bacterial solution,and double enzyme digestion analysis.The results showed that the CDS region of glycosyltransferase gene was 1323 bp and encoded 441 amino acids,the range of 30 amino acids at the N-terminus of DDOST protein was poorly conserved,and there were three insertion/deletion mutations in DDOST protein compared with 8 other plant species.This indicated that the gene fragment of interest had been successfully inserted into the expression vector and the transcription direction was correct.In conclusion,the overexpression vector of foxtail millet glycosyltransferase gene constructed in this study lays a foundation for subsequent genetic transformation and functional verification in Arabidopsis thaliana,rice or foxtail millet.

关 键 词：谷子 糖基转移酶基因 克隆 生物信息学分析 载体构建 

分 类 号：S515[农业科学—作物学]