怀玉山三叶青黄酮醇合酶基因克隆和表达分析

Cloning and expression analysis of flavonol synthase gene of Tetrastigma hemsleyanum Diels et Gilg in Huaiyushan

【目的】对怀玉山三叶青黄酮醇合酶基因进行克隆和表达分析,为进一步揭示怀玉山三叶青黄酮醇合酶的生物学功能奠定基础。【方法】怀玉山三叶青黄酮醇合酶基因的核心片段通过其转录组数据库进行筛选,怀玉山三叶青黄酮醇合酶基因利用逆转录PCR进行克隆,序列分析采用生物信息学进行分析,器官表达采用实时荧光定量PCR(qRT-PCR)进行比较。【结果】怀玉山三叶青黄酮醇合酶基因cDNA总长度为987 bp,G+C含量为47.92%。怀玉山三叶青黄酮醇合酶由329个氨基酸组成,分子量37578.03 Da,理论等电点5.39,为亲水性蛋白;其二级结构中α-螺旋、β-折叠和无规则卷曲的比例分别占27.66%、21.88%和50.46%。怀玉山三叶青黄酮醇合酶主要存在内质网中,在进化上与山甜菜(Nekemias grossedentata)、河岸葡萄(Vitis riparia)和葡萄(Vitis vinifera)的亲缘关系较近,尤其是与山甜菜(N.grossedentata)黄酮醇合酶在进化上具有最高的亲缘关系。qRT-PCR分析结果表明,怀玉山三叶青黄酮醇合酶基因的表达在2个栽培种中存在器官特异性表达,怀玉2号在根中的相对表达量最高,怀玉1号在茎中的相对表达量最高。【结论】怀玉山三叶青黄酮醇合酶具有典型黄酮醇合酶的结构特征,氨基酸序列及核酸序列与同源物种相似度高,在进化上高度保守,且怀玉山三叶青黄酮醇合酶基因的表达存在组织器官差异性。

【Objective】In order to provide theoretical basis for revealing the biological function of flavonol synthase of Tetrastigma hemsleyanum Diels et Gilg in Huaiyushan,the flavonol synthase gene of T.hemsleyanum Diels et Gilg in Huaiyushan was cloned and analyzed by expression.【Method】The core fragment of flavonol synthase gene was screened from the transcriptome database of plantlets of T.hemsleyanum Diels et Gilg in Huaiyushan.The flavonol synthase gene of T.hemsleyanum Diels et Gilg in Huaiyushan was cloned by reverse transcription PCR(RT-PCR)technique,sequenced by bioinformatics method and analyzed in organ expression by real-time fluorescence quantitative PCR.【Result】The total length of flavonol synthase gene cDNA of T.hemsleyanum Diels et Gilg in Huaiyushan was 987 bp and the content of G+C was 47.92%.The flavonol synthase of T.hemsleyanum Diels et Gilg in Huaiyushan was made up of 329 amino acids with molecular weight of 37578.03 Da and isoelectric point of 5.39,which was hydrophilic protein.The secondary structure was composed ofα-helix(27.66%),β-lamella(21.88%),irregular curl(50.46%).The flavonol synthase of T.hemsleyanum Diels et Gilg in Huaiyushan mainly existed in the endoplasmic reticulum.The evolution of flavonol synthase of T.hemsleyanum Diels et Gilg in Huaiyushan was closely related to Nekemias grossedentata,Vitis riparia and V.vinifera,especially to flavonol synthase of N.grossedentata,they had the highest phylogenetic relationship.The results of qRT-PCR showed that the expression of flavonol synthase gene was organ specific in the two cultivars of T.hemsleyanum Diels et Gilg in Huaiyushan.Huaiyu No.2 had the highest expression in the root and Huaiyu No.1 had the highest expression in the stem.【Conclusion】The flavonol synthase of T.hemsleyanum Diels et Gilg in Huaiyushan has typical structural characteristics of flavonol synthase.Its amino acid sequence and nucleic acid sequence are highly similar to homologous species and highly conserved in evolution.There are tissue organ differences in the expression of flavonol synthase gene of T.hemsleyanum Diels et Gilg in Huaiyushan.