番茄转录因子基因SlWRKY6的克隆与原核表达分析

Cloning and Prokaryotic Expression Analysis of a WRKY Transcription Factor Gene SlWRKY6 in Solanum lycopersicum

该研究基于番茄基因组数据库SGN(Sol Genomic Network)信息,利用RT-PCR从栽培番茄‘M82’(Solanum lycopersicum)中成功克隆到番茄SlWRKY6基因(登录号:Solyc02g080890),通过qRT-PCR方法和原核表达初步验证其生物学功能。结果表明:(1)生物信息学分析显示,番茄SlWRKY6基因ORF全长1653 bp,编码550个氨基酸,其蛋白结构含有1个WRKYGQK保守结构域和C2H2锌指结构域,属于IIb类;其基因启动子上游1500 bp含有多个激素响应元件和非生物胁迫响应元件。(2)进化树分析显示,SlWRKY6与潘那利番茄SpWRKY31-X1(NP_001352691.1)的相似性最高,且定位于细胞核内。(3)qRT-PCR结果显示,SlWRKY6基因在番茄根、茎、叶中均有表达,在叶中的表达量最高,且受盐和干旱诱导表达。(4)SDS-PAGE及Western blot结果显示,pET-30a-SlWRKY6重组蛋白的大小约66 kDa,与预期大小一致。(5)原核表达分析显示,重组菌E.coli BL21∷pET-30a-SlWRKY6在不同浓度盐(NaCl)和干旱(Mannitol)胁迫下生长速度显著低于对照菌E.coli BL21∷pET-30a,且在400 mmol/L NaCl、800 mmol/L甘露醇胁迫条件下最为显著;滴板实验初步验证SlWRKY6转录因子能提高重组菌E.coli BL21∷pET-30a-SlWRKY6在ABA和pH 9(NaOH)胁迫的耐受性;在400 mmol/L NaCl、pH 5(HCl)、800 mmol/L甘露醇胁迫条件下耐受能力降低。研究表明,SlWRKY6转录因子可能通过参与ABA途径来响应非生物胁迫。

According to the data of Sol Genomic Network,we obtained the full-length of SlWRKY6(Solyc02g080890)from cultivated tomato variety M82 by RT-PCR and verified its biological function by prokaryotic expression.The results showed that:(1)an open reading frame(ORF)of SlWRKY6 was obtained from Solanum lycopersicum,which is 1653 bp long and encodes 550 putative amino acids with a WRKYGQK conserved domain and C2H2 zinc finger domain,belonging to IIb subgroup.The upstream of the SlWRKY6 promoter contains several hormone response elements and abiotic stress response elements.(2)Phylogenetic tree analysis showed that SlWRKY6 shared a high degree of sequence similarity with SpWRKY31-X1(NP_001352691.1)from Solanum pennellii and localized to the nucleus.(3)qRT-PCR results showed that the pattern of SlWRKY6 was highly expressed in leaves and also induced by salt and drought stress.(4)SDS-PAGE and Western blot analysis showed that the molecular weight of SlWRKY6 was approximately 66 kDa.(5)Prokaryotic expression analysis showed that the growth rate of recombinant E.coli BL21∷pET-30a-SlWRKY6 was significantly lower than that of the control E.coli BL21∷pET-30a under different concentrations of salt(NaCl)or drought(mannitol)stress,especially under 400 mmol/L NaCl and 800 mmol/L mannitol stress.In addition,the drip plate test showed that SlWRKY6 gene could enhance the tolerance of E.coil to salt,drought and ABA stresses.Taken together,the transcription factor SlWRKY6 might respond to abiotic stress by participating in the ABA pathway.