番茄SlMYB86基因的原核表达及缺氮胁迫下的表达分析

Prokaryotic Expression and Expression Analysis of Tomato SlMYB86 Gene under Nitrogen Deficiency

MYB转录因子参与植物细胞形态与模式建成、次级代谢的调控以及生物和非生物胁迫应答等反应。该研究采用RT-PCR方法扩增了番茄SlMYB86基因,并进行了聚类分析和保守域序列分析,构建原核表达载体和诱导纯化蛋白,利用qRT-PCR和Western blot检测SlMYB86在缺氮复氮下的表达水平,为深入探究番茄MYB86转录因子在缺氮胁迫下的功能奠定基础。结果表明:(1)番茄SlMYB86与番茄SlMYB26在进化树上属于同一分支,亲缘关系较近,且SlMYB86含有2个Myb_DNA-binding保守结构域,属于R2R3-MYB型转录因子。(2)qRT-PCR分析发现,SlMYB86基因在番茄根和叶中均有表达,缺氮胁迫下SlMYB86表达较对照显著增加。(3)成功构建pET-28a-SlMYB86原核表达载体并转化E.coli BL21(DE3),SDS-PAGE和Western blot结果表明,SlMYB86蛋白的最佳诱导条件为0.5 mmol/L的IPTG、37℃诱导8 h;目的蛋白相对分子量大约为41 kD,与预期大小一致,并获得较高纯度的SlMYB86原核蛋白。(4)将纯化的SlMYB86蛋白免疫小白鼠获得抗体,利用该抗体进行Western blot分析发现,番茄中SlMYB86蛋白在缺氮胁迫后表达上调,表明番茄SlMYB86基因参与了缺氮胁迫的应答。

MYB transcription factors are involved in the formation of plant cell morphology and pattern,the regulation of secondary metabolism and the response to biological and abiotic stress,while the expression of tomato MYB(SlMYB86)under nitrogen deficiency stress has not been reported.In this study,the tomato SlMYB86 gene was amplified by RT-PCR,cluster analysis and conserved domain sequence analysis were carried out,prokaryotic expression vector and induced purified protein were constructed,and the expression level of SlMYB86 under nitrogen deficiency and nitrogen re-supply was detected by qRT-PCR and Western blot,which laid a foundation for further exploring the function of tomato MYB86 transcription factor under nitrogen deficiency stress.The results show that:(1)tomato SlMYB86 and tomato SlMYB26 belong to the same branch in the evolutionary tree,with close genetic relationship,and SlMYB86 contained two Myb_DNA-binding conserved domains,belonging to R2R3-MYB transcription factor.(2)qRT-PCR analysis showed that SlMYB86 gene was expressed in tomato roots and leaves,and the expression of SlMYB86 was significantly higher than that of the control under nitrogen deficiency stress.(3)The prokaryotic expression vector pET-28a-SlMYB86 was constructed and transformed into E.coli BL21(DE3).The results of SDS-PAGE and Western blot showed that the best induction conditions of SlMYB86 protein were 0.5 mmol/L IPTG and 37℃for 8 hours.The relative molecular weight of the target protein is about 41 kD,which is consistent with the expected size,and SlMYB86 prokaryotic protein with high purity is obtained.(4)Mice were immunized with the purified SlMYB86 protein to obtain the antibody.Western blot showed that the expression of SlMYB86 protein in tomato was up-regulated after nitrogen deficiency stress,indicating that tomato SlMYB86 gene was involved in the response to nitrogen deficiency stress.