甜叶菊中新KAH基因的克隆及表达模式分析

生物技术通报 ›› 2017, Vol. 33 ›› Issue (4): 104-107.

甜叶菊中新KAH基因的克隆及表达模式分析

朱静雯, 郭书巧, 束红梅, 巩元勇, 蒋璐, 倪万潮

江苏省农科院经济作物研究所，南京 210014

收稿日期:2016-09-12 出版日期:2017-04-25 发布日期:2017-04-25
作者简介:朱静雯，女，博士，研究方向：植物次生代谢物；E-mail：jingwenstudy@126.com
基金资助:
江苏省农业三新工程［SXGC（2015）213］，江苏省农业科学院科技服务项目［KF（16）1042］

Cloning and Expression Analysis of New KAH Genes from Stevia rebaudian

ZHU Jing-wen GUO, Shu-qiao, SHU Hong-mei, GONG Yuan-yong, JIANG Lu, NI Wan-chao

Institute of Industrial Crops，Jiangsu Academy of Agricultural Science，Nanjing 210095

Received:2016-09-12 Published:2017-04-25 Online:2017-04-25

摘要/Abstract

摘要： 为进一步揭示甜菊糖苷生物合成途径的分子调控机制，探究甜菊糖苷在甜叶菊叶片中积累差异原因。从不同甜叶菊材料中分别克隆关键分支点酶KAH的编码基因，并对获得的核苷酸序列进行生物学功能分析和预测。结果显示，最终共获得6条高度同源的核苷酸序列，个别关键碱基的突变造成序列开放读码框位置和大小的显著差异，共预测获得7个不同KAH编码基因。KAH1、KAH2同时存在于SR1、SR3、鑫丰3号、谱星1号和守田3号中，KAH3存在于江甜1号和守田3号中，KAH4存在于江甜3号中，KAH5、KAH6、KAH7均存在于SR2中且SrKAHs在甜菊糖苷积累多的品种和组织部位中表达更高。7个KAH基因均含有P450保守结构域且均属于CYP716家族成员；亚细胞定位预测KAH2和KAH4定位于细胞质其他5个均定位在叶绿体中。甜叶菊中KAH编码基因的数目、氨基酸长度及在亚细胞结构的定位差异或许是导致甜叶菊不同品种和组织中SrKAHs表达和功能差异的主要原因并最终糖苷的积累差异。

关键词: 甜叶菊, 甜菊糖苷, KAH, CYP716

Abstract: This work is to further reveal the molecular mechanism of the biosynthesis of steviol glycosides（SGs）and analyze the reasons caused the different accumulation of SGs in stevia leaves. Different stevia materials were utilized to clone the coding genes of kaurenoic acid hydroxylase（KAH）at the key point of a branch pathway，and the biological function of the obtained nucleic acid sequences were analyzed and predicted. Finally six highly homologous nucleotide sequences were cloned. The key base mutations of nucleotide sequences caused significant differences in the location and size of the open reading frame（ORF），and totally 7 different KAH genes were identified. Both KAH1 and KAH2 were found in SR1，SR3，Xinfeng 3，Puxing 1 and Shoutian 3，KAH3 was cloned in Jiangtian 1 and Shoutian 3，KAH4 were found in Jiangtian 3，and KAH5，KAH6 and KAH7 all existed in SR2. Furthermore，SrKAHs showed the highest expression in stevia varieties and tissues with high accumulation of SGs. P450 conserved domains were in the all of them，and further evolution analysis and amino acid homology results showed that they belonged to the CYP716 family. And the predicted results of subcellular location showed KAH2 and KAH4 localized in cytoplasm while the other five were localized in the chloroplast. Differences of gene numbers，amino acid length and location in subcellular structures of KAH genes in Stevia maybe cause the different expressions and functions of SrKAHs，finally resulting the different accumulation of SGs.

Key words: Stevia rebaudiana, steviol glycosides, KAH, CYP716

朱静雯, 郭书巧, 束红梅, 巩元勇, 蒋璐, 倪万潮. 甜叶菊中新KAH基因的克隆及表达模式分析[J]. 生物技术通报, 2017, 33(4): 104-107.

ZHU Jing-wen GUO, Shu-qiao, SHU Hong-mei, GONG Yuan-yong, JIANG Lu, NI Wan-chao. Cloning and Expression Analysis of New KAH Genes from Stevia rebaudian[J]. Biotechnology Bulletin, 2017, 33(4): 104-107.

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