茶树CssHSP18.1基因克隆及表达分析

Cloning and Expression Analysis of CssHSP18.1 Gene in Camellia Sinensis

sHSPs基因家族可编码一类小分子的热激蛋白,广泛分布于植物中,具有分子伴侣的功能,在植物抵抗逆境胁迫中起着重要作用。通过基因克隆的方法,获得1个茶树CssHSP18.1基因的开放阅读框(Open reading frame,ORF),其全长480 bp,编码159个氨基酸。生物信息学分析表明,CssHSP18.1蛋白含有1个典型HSP20结构域,相对分子质量约为18.25 kDa,等电点为5.68,偏酸性,与栎和苹果亲缘关系最近,无信号肽与跨膜结构。RT-qPCR分析表明,CssHSP18.1在甘露醇(D-Mannitol)处理下表达量低于对照组;γ-氨基丁酸(GABA)能促进该基因的表达,在处理后1 h时表达量达到峰值;吲哚乙酸(IAA)和聚乙二醇(PEG 6000)处理后,CssHSP18.1在0.5 h时表达量最高,即GABA、IAA、PEG 6000均可诱导CssHSP18.1的表达。为获得CssHSP18.1可溶性蛋白,构建了pET-28a-CssHSP18.1重组质粒进行原核表达,并分别对表达菌株、诱导温度以及IPTG(异丙基-β-D-硫代吡喃半乳糖苷)诱导浓度进行优化。结果显示,CssHSP18.1蛋白最佳表达菌株为BL21(DE3),最佳诱导温度和IPTG浓度分别为30℃和1.2 mmol·L-1。最后,采用Western blot对表达的CssHSP18.1蛋白进行验证。本研究为进一步揭示CssHSP18.1基因的生物学功能提供依据。

The sHSPs gene family encodes a class of small molecular heat shock proteins,which are widely distributed in plants,functioned as molecular chaperones,and play an important role in plant resistance to stresses.In this study,the open reading frame(ORF)of CssHSP18.1 gene cDNA was obtained by gene cloning,which is 480 bp in length and encodes 159 amino acids.Bioinformatics analysis showed that CssHSP18.1 protein contained a typical HSP20 domain.Its molecular weight and isoelectric point are about 18.25 kDa and 5.68 respectively.Phylogenetic tree analysis showed that CssHSP18.1 has the closest relationship with quercus and apple.It was predicted that CssHSP18.1 protein was does not have signal peptide and transmembrane structure.RT-qPCR analysis showed that the expression of CssHSP18.1 under D-Mannitol treatment was lower than that in the control group.GABA could enhance the expression of CssHSP18.1 with its peak at 1 h after GABA treatment.The expression of CssHSP18.1 was upregulated upon IAA and PEG 6000 treatments,and reached the peaks at 0.5 h.Thus,GABA、IAA、PEG 6000 could induce the expression of CssHSP18.1.To obtain CssHSP18.1 soluble protein,a recombinant plasmid pET-28a-CssHSP18.1 was constructed and expressed in prokaryotic system.The expression strains,induction temperatures and induction concentrations of IPTG(isopropyl-β-D-thiopyranogalactoside)were optimized.The results showed that the best expression strain of CssHSP18.1 protein was BL21(DE3),and the best induction temperature and IPTG concentration were 30℃and 1.2 mmol·L-1 respectively.Finally,western blot was used to verify the expression of CssHSP18.1 protein.This study provided a basis for further study on the biological function of CssHSP18.1 gene.