丹参小分子热激蛋白SmHSP21.8基因克隆、诱导模式和原核表达

Cloning,induction pattern and prokaryotic expression of a small heat shock protein SmHSP21.8 gene from Salvia miltiorrhiza

为揭示小分子热激蛋白在丹参抵御逆境胁迫和有效成分积累的分子机制,根据橙根丹参转录组测序结果并结合RT-PCR技术在丹参中克隆获得一个小分子热激蛋白基因,其开放阅读框长度为585 bp,编码194个氨基酸,根据该基因编码的蛋白分子质量命名为SmHSP21.8。根据氨基酸多重序列比对和系统进化树分析,SmHSP21.8属于小分子热激蛋白的内质网亚族,并含有内质网小分子热激蛋白N端保守基序DPFR-I/V-LE-H/Q-x-P。构建原核表达载体pMAL-c2X-SmHSP21.8,并转化至大肠杆菌BL21感受态细胞,经诱导,成功表达出目的蛋白。时空表达分析表明,该基因在花中表达量最高,有显著的组织特异性。经38℃、PEG6000、脱落酸(abscisic acid,ABA)和吲哚-3-乙酸(indole-3-acetic acid,IAA)的诱导,该基因的相对表达量均有提高,表明SmHSP21.8参与了丹参幼苗对非生物胁迫高温、干旱,以及外源激素ABA和IAA响应的过程,为进一步研究小分子热激蛋白参与丹参响应逆境的分子机制奠定了基础。

In order to reveal the molecular mechanism of the small heat shock proteins(sHSPs)involved in stress resistance and active ingredients accumulation in Salvia miltiorrhiza,a small heat shock protein gene was cloned from Salvia miltiorrhiza by reverse transcription PCR according to the transcriptome data of orange root Salvia miltiorrhiza.The gene is named SmHSP21.8 based on the molecular weight of the protein,and it contains an open reading frame of 585 bp,which encodes 194 amino acids.The results of phylogenetic analysis and amino acid sequence alignment showed that SmHSP21.8 protein belongs to the endoplasmic reticulum(ER)subfamily,and contains a conserved endoplasmic reticulum-specific DPFR-I/V-LE-H/Q-x-P motif at N-terminus.The prokaryotic expression vector pMAL-c2X-SmHSP21.8 was constructed and transformed into E.coli BL21 competent cells.The recombinant protein was successfully expressed after inducted.Temporal and spatial expression analysis showed that SmHSP21.8 gene was the highest expressed in flowers and had significant tissue specificity.The relative expression of the gene was significantly increased in seedlings after induction by 38℃,PEG6000,abscisic acid(ABA),and indole-3-acetic acid(IAA),indicating that SmHSP21.8 gene may be involved in abiotic stress such as high temperature and drought,as well as the response to exogenous hormones ABA and IAA.These results lay the foundation for further research on the molecular mechanism of small heat shock proteins involved in adversity stress.