粉葛查尔酮合成酶基因PtCHS的克隆与植物表达载体构建

查尔酮是植物体形成的一类次级代谢产物,在植物花色形成、育性及抵抗胁迫中发挥重要作用。前期研究发现粉葛与野葛中总黄酮的含量和葛根素的含量差异较大,而查尔酮合成酶是黄酮类化合物合成中的首个关键酶。为了研究野葛与粉葛中的查尔酮合成酶(CHS)基因是否存在差异性,利用同源克隆的方法,根据已报道的野葛CHS基因序列设计引物,从粉葛中克隆CHS基因,对其进行蛋白相对分子质量、二级结构及亚细胞定位预测等生物信息学分析并构建植物表达载体。结果显示,成功克隆到粉葛CHS基因,将其命名为PtCHS,该基因cDNA序列长1187 bp,包含一个长1179 bp完整的ORF框,推导编码389个氨基酸,预测蛋白相对分子质量为42.8 kD,理论等电点为5.96,无跨膜结构域,为稳定的亲水性蛋白,二级结构主要由α-螺旋、延伸链、β-转角和无规则卷曲组成,同时亚细胞定位预测结果显示蛋白位于细胞质;氨基酸序列多重比对发现,粉葛的查尔酮合成酶基因(PtCHS)所编码的氨基酸与已报道的野葛CHS基因编码的氨基酸同源性为100%,与大豆、赤豆、菜豆及木豆的氨基酸同源性均在95%以上;蛋白互作预测分析得出,CHS与CHI、C4H及REDUCTASE发生互作的可能性较大;用PtCHS与植物表达载体pBI121构建重组子pBI121-PtCHS,为葛根查尔酮合成酶基因的功能验证提供重要的参考依据。

Cloning and Construction of Plant Expression Vector of Chalcone Synthase Gene PtCHS in Pueraria montana var.thomsonii

Chalcone is a secondary metabolite formed by plants,which plays an important role in plant flower formation,fertility,and resistance to stress.Previous studies have found that the contents of total flavonoids and puerarin in Pueraria montana var.thomsonii and Pueraria lobata(Willd.)Ohwi were quite different and chalcone synthase was the first key enzyme in the synthesis of flavonoids.In order to study whether the chalcone synthetase(CHS)genes are different between Pueraria lobata(Willd.)Ohwi and Pueraria montana var.thomsonii,a homologous cloning method was used to design primers based on the reported CHS gene sequence,and the CHS gene was cloned from Pueraria montana var.thomsonii.Bioinformatics analysis was performed on the relative molecular mass,secondary structure and subcellular localization of the protein,and a plant expression vector was constructed.The results showed that the Pueraria montana var.thomsonii CHS gene was successfully cloned and named PtCHS.The cDNA sequence of the gene was 1187 bp in length and contained a complete ORF frame of 1179 bp in length.The deduced encoding was 389 amino acids.The predicted molecular weight of the protein is 42.8 kD,theoretical isoelectric point is 5.96,don't have transmembrane domain,and is a stable hydrophilic protein.The secondary structure was mainly composed of alpha helix,extension chain,beta corner and irregular curl,and the prediction results of subcellular localization showed that the protein is located in the cytoplasm;Multiple alignments of amino acid sequences found that the chalcone synthase gene(PtCHS)encoded by Pueraria montana var.thomsonii is 100%homologous to the amino acid encoded by the reported Pueraria lobata(Willd.)Ohwi CHS gene,and it is identical to soybean,Vigna angularis,Phaseolus vulgaris Linn and Cajanus cajan.The amino acid homology of beans is more than 95%.Protein interaction prediction analysis showed that CHS has a greater possibility of interaction with CHI,C4H and REDUCTASE.PtCHS and expression vector pBI121 were used to constructe recombinant pBI121-PtCHS,which provides significant reference for functional verification of puerarin chalcone synthase gene.