新塔花查耳酮合成酶基因克隆及表达分析

Cloning and expression analysis of chalcone synthase gene from Ziziphora bungena

目的克隆新塔花Ziziphora bungena黄酮类化合物生物合成途径中关键酶查耳酮合成酶(chalcone synthase,CHS)基因,对其进行组织表达特异性分析,构建原核表达载体并进行重组蛋白诱导表达。方法结合新塔花转录组数据设计特异性引物,通过RT-PCR克隆新塔花查耳酮合成酶zbCHS基因,对其进行生物信息学分析,通过RT-PCR进一步分析其组织表达特异性,构建原核表达载体pET28a-chs,并转化至BL21(DE3)感受态细胞,经异丙基-β-D-硫代半乳糖苷(IPTG)诱导重组蛋白的表达。结果zbCHS基因长1226 bp,包含一个长度为1176 bp的开放阅读框,编码319个氨基酸,其理论相对分子质量为42848.37。该编码蛋白定位于细胞质中,不存在跨膜区及信号肽,为非分泌蛋白,有1个糖基化位点和31个磷酸化位点。α-螺旋是该蛋白多肽链中大量的结构元件,散布于整个肽链之中。系统进化树分析表明,新塔花zbCHS基因与丹参、藿香、紫苏等亲缘关系最为接近。RT-PCR结果显示zbCHS基因在各组织中均有所表达,在叶中表达量较高,花相对表达量次之,根、茎中表达量相对较低。原核表达载体pET28a-chs在BL21(DE3)感受态细胞表达系统中,经IPTG诱导后,有明显的重组蛋白表达,其相对分子质量为42800,与预测相符合。结论通过对zbCHS基因的全长cDNA克隆、组织表达特异性分析和原核表达载体的构建,为进一步研究zbCHS基因在新塔花黄酮类化合物生物合成与基因调控提供依据,最终为该药材品质的提升奠定基础。

Objective To clone the key gene of chalcone synthase(zbCHS)in flavonoid biosynthesis pathway of Ziziphora bungena,analyze tissue-specific expression of the chalcone synthase,construct the prokaryotic expression vector and induce the recombinant protein to express.Methods Based on the transcriptome data of Zb in the previous study,the full-length cDNA of ZbCHS was cloned by RT-PCR and bioinformatics analysis was performed.The RT-PCR was used to analyze the tissue-specific expression of ZbCHS.The prokaryotic expression vector pET28a-CHS was constructed,transformed into BL21(DE3)competent cells and the expression of recombinant protein was induced by IPTG.Results The size of zbCHS gene was 1226 bp,containing an open reading frame(ORF)of 1176 bp and encoding 319 amino acids.The theoretical molecular weight of ZbCHS protein was 42848.37.The encoded protein was located in the cytoplasm without a transmembrane region and signal peptide.It was a non-secretory protein with one glycosylation site and 31 phosphorylation sites.Theα-helix was a large number of structural elements in that polypeptide chain,which was scattered throughout the peptide chain.Phylogenetic analysis indicated that the sequence of the amino acids was most closely related to Salvia miltiorrhiza,Agastache rugosa,and Perilla frutescens.of Real-Time PCR showed that zbCHS gene was expressed in all tissues,with a higher expression level and a relatively lower expression level in roots and stems.Prokaryotic expression vector pET28a-CHS was expressed in BL21(DE3)competent cell expression system after induction by IPTG,and its molecular weight was 42800,which was consistent with the prediction.Conclusion Through the full-length cDNA cloning of ZbCHS gene,tissue-specific expression analysis and prokaryotic expression vector construction,we can provide the basis for further study on the biosynthesis and gene regulation of ZbCHS gene in Z.bungena,and finally lays the foundation for the improvement of the quality of this herb.