虎杖转录因子PcMYB1基因的克隆及原核表达  

Cloning and Prokaryotic Expression of Transcription Factor PcMYB1 Gene from Polygonum cuspidatum

作　　者：柳忠玉[1] 雷健 李晓筱 刘婵 覃建兵[1] 许锋[2] 

机构地区：[1]长江大学生命科学学院,湖北荆州434025 [2]长江大学园艺园林学院,湖北荆州434025

出　　处：《河南农业科学》2018年第1期96-102,共7页Journal of Henan Agricultural Sciences

基　　金：国家自然科学基金项目(31670608);长江大学湿地生态与农业利用教育部工程研究中心开放基金项目(KF201511);长江大学校级大学生创新训练计划项目(2017073)

摘　　要：为了探明髓细胞组织增生病毒癌基因同源物(MYB)转录因子在虎杖苯丙烷代谢途径的作用,以虎杖叶片为材料,通过RT-PCR和RACE技术从虎杖中获得MYB转录因子基因,命名为PcMYB1,GenBank登录号为KY495789。序列分析表明,PcMYB1基因的cDNA序列全长1 152 bp,该基因开放阅读框为813 bp,编码270个氨基酸,推测蛋白质分子质量为30.455 ku。PcMYB1编码的氨基酸序列具有MYB类转录因子的共同特征,其N端具有R2、R3两个MYB结构域,同时其C端还存在一个抑制结构域C2基序pdLNL[D/E]LXI[G/S]。系统进化树分析表明,该基因编码的蛋白质与矮牵牛PhMYB4的亲缘关系最近。将该基因片段连接到原核表达载体pET-28a中,构建融合表达质粒pET28a-PcMYB1,转化到E.coli BL21(DE3)中进行表达。SDS-PAGE电泳检测结果表明,表达蛋白与预期大小一致。成功克隆PcMYB1基因的全长序列,构建的原核表达载体pET28a-PcMYB1可用于该基因的功能研究。In order to identify the role of MYB transcription factors in phenylpropanoid pathway of Polygonum cuspidatum,the MYB homologue gene PcMYB1（ GenBank accession No. KY495789） was amplified from the leaf of P. cuspidatum by RT-PCR and RACE methods. The full length cDNA of PcMYB1 is 1 152 bp and the open reading frame is 813 bp,encoding a protein of 270 amino acids with relative molecular mass of 30. 455 ku. Sequence analysis indicated that the deduced amino acids had common characteristics of MYB transcription factors with two MYB domains of R2 and R3 at the N-terminal and a transcriptional repression domain C2 motif pdLNL[D/E]LXI[G/S] at the C-terminal. Phylogenetic tree analysis suggested that the encoded protein had the closest genetic relationship with PhMYB4 from Petunia × hybrida.The recombinant prokaryotic expression vector pET28a-PcMYB1 was constructed by inserting the DNA fragment into the prokaryotic expression vector pET-28 a,and then transformed into E. coli BL21（ DE3）.SDS-PAGE analysis showed that the size of expressed protein was consistent with expectation. In conclusion,the full-length sequence of PcMYB1 gene in P. cuspidatum is obtained,and the prokaryotic expression vector pET28a-PcMYB1 can be used for functional study of Pc MYB1 gene.

关 键 词：虎杖 MYB转录因子 PcMYB1 原核表达 苯丙烷代谢 RACE 

分 类 号：S567.23[农业科学—中草药栽培]