三叶崖爬藤查耳酮异构酶基因克隆与外源诱导表达及酶活性分析

Cloning and exogenous induction of expression of the chalcone isomerase gene from Tetrastigma hemsleyanum and enzymatic activity analysis

目的克隆三叶崖爬藤Tetrastigma hemsleyanum查耳酮异构酶(chalcone isomerase,CHI)基因,探究CHI基因对三叶崖爬藤黄酮合成途径的调控作用。方法分析三叶崖爬藤转录组,获得三叶崖爬藤CHI基因序列,并进行生物信息学分析。采用实时荧光定量PCR(qRT-PCR)测定三叶崖爬藤根、茎、块茎、老叶、幼叶及吲哚-3-乙酸(3-indoleacetic acid,IAA)、茉莉酸甲酯(methyl jasmonate,MeJA)、脱落酸(abscisic acid,ABA)、水杨酸(salicylic acid,SA)和酵母提取物(yeast extract,YE)等诱导条件下CHI基因的表达量,利用分光光度法测定黄酮含量和CHI酶活性变化,并对其进行相关性分析。结果转录组数据分析结果表明,三叶崖爬藤CHI基因全长为1096 bp(GenBank序号:OQ588006),含有1个长为714 bp的开放阅读框,编码237个氨基酸。三叶崖爬藤CHI蛋白分子式为C1148H1819N285O348S5,等电点(PI)为5.28,相对分子质量为25340,编码的氨基酸全都在膜外,不具跨膜结构,也不存在信号肽。三维空间结构分析表明,三叶崖爬藤CHI蛋白呈明显的倒置三明治状折叠结构,属于Chalcone-3超基因家族。进化树分析结果表明,三叶崖爬藤CHI蛋白序列与葡萄科植物亲缘关系最近。qRT-PCR结果表明,三叶崖爬藤CHI基因在三叶崖爬藤根、茎、块茎、老叶和幼叶等不同组织中均表达。诱导分析结果表明,5种外源诱导物质均提高了三叶崖爬藤黄酮含量,ABA诱导后CHI基因表达水平在72 h达到最高,其余诱导处理CHI基因表达量均在96 h达到最高,CHI酶活性均在96 h达到最高。相关性分析结果表明,三叶崖爬藤黄酮含量与CHI基因表达量不存在显著相关性,与CHI酶活性存在极显著正相关(R 2=0.453)。结论成功克隆了三叶崖爬藤CHI基因,该基因在三叶崖爬藤各组织中均表达。IAA、MeJA、ABA、SA和YE均促进CHI基因表达及酶活性的上升,在转录和翻译水平上调控三叶崖爬藤黄酮合成。

Objective To clone chalcone isomerase(CHI)gene of Tetrastigma hemsleyanum,and investigate the regulatory role of CHI genes on T.hemsleyanum flavonoid synthesis pathway.Methods The T.hemsleyanum transcriptome was analyzed to obtain T.hemsleyanum CHI gene sequences,and bioinformatics analysis was performed.Real-time fluorescence quantitative PCR(RT-qPCR)was used to determine the expression of CHI genes in roots,stems,tubers,old leaves,young leaves and indole-3-acetic acid(IAA),methyl jasmonate(MeJA),abscisic acid(ABA),salicylic acid(SA)acid(SA)and yeast extract(YE)induction conditions,and the changes of flavonoid content and CHI enzyme activity were determined by spectrophotometry and its correlation was analyzed.Results The transcriptome data analysis showed that the full length of T.hemsleyanum CHI gene was 1096 bp(GenBank number:OQ588006),which contained an open reading frame of 714 bp and encoded 237 amino acids.The molecular formula of T.hemsleyanum CHI protein was C1148H1819N285O348S5,PI value was 5.28,relative molecular mass was 25340.All amino acids encoded were outside the membrane,without transmembrane structure,and there was no signal peptide.Three-dimensional structural analysis showed that T.hemsleyanum CHI protein had an obvious inverted sandwich-like folded structure and belonged to the Chalcone-3 supergene family.The results of evolutionary tree analysis showed that T.hemsleyanum CHI protein sequence was most closely related to the Vitaceae family.RT-qPCR results showed that the CHI gene was expressed in different tissues of T.hemsleyanum.The results of induction analysis showed that all five exogenous inducing substances increased the content of T.hemsleyanum flavonoids,the highest CHI gene expression level was reached at 72 h after ABA induction,the highest CHI gene expression was reached at 96 h for the rest of the induction treatments,and all CHI enzyme activities reached their maximum at 96 h.Correlation analysis showed that there was no significant correlation between flavonoid content and CHI gene expression,and there was a highly significant positive correlation between flavonoid content and CHI enzyme a ctivity(R 2=0.453).Conclusion The CHI gene was successfully cloned and expressed in all the tissues of T.hemsleyanum.IAA,MeJA,ABA,SA,and YE promote the CHI gene expression and enzyme activity and regulate trichothecene flavonoid synthesis at the transcriptional and translational levels.