棉花GhMYB42基因的克隆及原核表达

Cloning and Prokaryotic Expression of the GhMYB42 Gene in Cotton

MYB转录因子对棉花的生长发育起到重要作用,而GhMYB42为MYB家族之一的转录因子同样具有一定的研究价值,因此,从棉花中克隆了GhMYB42基因的编码序列,并构建了原核表达载体。利用生物信息学方法对GhMYB42的核苷酸序列及氨基酸序列进行了分析,通过Gataway BP和LR反应,将GhMYB42基因的编码序列构建到原核表达载体pGEX-4T-1上,通过设置不同的IPTG诱导条件来确定IPTG诱导蛋白的最佳条件,最后利用Western Blot鉴定重组蛋白。结果显示,GhMYB42(XP_016732693.1)全长序列1508 bp,编码区长792 bp,编码263个氨基酸,预测分子量约为29.534 ku,等电点为5.18。氨基酸序列比对分析发现,MYB转录因子的序列相似率为80.62%,且GhMYB42蛋白N末端含有2个串联的SANT结构域,是一个R2R3转录因子。进化树分析结果显示,陆地棉MYB42蛋白与陆地棉中另一MYB蛋白(XP_012439547.1)相似性最高并在一个分支上。蛋白诱导时由于各个试验梯度结果差别不明显,因此,选择的条件为IPTG的终浓度0.2 mmol/L,温度37℃,时间3 h,蛋白溶解的温度和时间为37℃诱导3 h。Western Blot结果表明,重组蛋白的大小正确,最终成功获得了大小为55.54 ku的GhMYB42重组蛋白,后续将对该重组蛋白进行纯化及深入研究转录因子GhMYB42的功能。

MYB transcription factors play an important role in the growth and development of cotton,and GhMYB42 is one of the transcription factors of the MYB family,which also has certain research value,so this study cloned the coding sequence of GhMYB42 gene from cotton and constructed a prokaryotic expression vector.The nucleotide sequence and amino acid sequence of GhMYB42 were analysed using bioinformatics methods,and the coding sequence of the GhMYB42 gene was constructed into the prokaryotic expression vector pGEX-4T-1 by the Gataway BP and LR reactions,and the optimal conditions for the induction of the protein by IPTG were determined by setting up different IPTG-inducing conditions,and finally the recombinant protein was identified by Western Blot.The results showed that the full-length sequence of GhMYB42(XP_016732693.1)was 1508 bp,the coding region length was 792 bp,the coding area was 263 amino acids,the predicted molecular weight was about 29.534 ku,and the isoelectric point was 5.18.Amino acid sequence comparison analysis revealed that the sequence similarity of the MYB transcription factors was 80.62%,and that the N-terminus of the GhMYB42 protein contained two tandem SANT structural domains,making it an R2R3 transcription factor.Phylogenetic tree analysis showed that the MYB42 protein in Gossypium hirsutum had the highest homology with another MYB protein(XP_012439547.1)in Gossypium hirsutum and was on one branch.Since the results of each experimental gradient were not significantly different during protein induction,it chose the conditions for IPTG to be 0.2 mmol/L at a final concentration of 0.2 mmol/L,a temperature of 37℃for 3 h,and a temperature and time for protein solubility to be induced at 37℃for 3 h.The Western Blot results showed that the size of the recombinant protein was correct,and finally the GhMYB42 recombinant protein with a size of 55.54 ku was successfully obtained,and it will purify the recombinant protein and further study the function of the transcription factor GhMYB42.