‘全红’杨PdHY 5转录因子基因的克隆及功能分析  

Cloning and Functional Analysis of a Transcription Factor Gene PdHY 5 in Populus deltoides‘Quanhong’

作　　者：付妍妍 赵志明 徐立人 王进茂[1] FU Yanyan;ZHAO Zhiming;XU Liren;WANG Jinmao(College of Forestry,Hebei Agricultural University,Baoding,Hebei 071000,China;School of Biological Sciences and Technology,Beijing Forestry University,Beijing 100083,China)

机构地区：[1]河北农业大学林学院,河北保定071000 [2]北京林业大学生物科学与技术学院,北京100083

出　　处：《西北植物学报》2022年第11期1809-1817,共9页Acta Botanica Boreali-Occidentalia Sinica

基　　金：河北省重点研发计划(21326301D)。

摘　　要：HY5(ELONGATED HYPOCOTYL 5)转录因子在花青素的生物合成方面具有重要的作用。该研究以‘全红’杨叶片为材料,克隆了PdHY 5基因,对其进行生物信息学分析;并通过农杆菌介导的叶盘法转化烟草,对其进行功能分析。结果显示:(1)PdHY 5基因开放阅读框(ORF)长度为510 bp,共编码169个氨基酸。多序列比对和系统进化分析表明,‘全红’杨PdHY5蛋白具有bZIP家族蛋白的保守结构域,与毛果杨PtHY5蛋白亲缘关系最近。(2)成功构建过表达载体pNP1302-35S-PdHY 5,经潮霉素筛选获得了4个转基因烟草株系(S1~S4)。(3)qRT-PCR结果表明,PdHY 5基因在4个过表达株系中的表达量显著高于野生型(WT),且S4株系的表达量最高,S1最低;同时过表达株系的花青素生物合成途径关键基因CHS、F3H及FLS的表达水平较WT均显著上调。(4)叶片花色素苷的相对含量在转基因烟草株系S2、S3、S4中较WT显著上调,分别增加了128.23%、97.36%和134.20%。研究表明,过表达PdHY 5基因调控了烟草本身花青素生物合成途径中结构基因的表达,从而促进了花青素的积累。Transcription factor HY5(ELONGATED HYPOCOTYL5)played an important role in anthocyanin biosynthesis.In this study,we firstly cloned PdHY 5 gene from the Populus deltoides‘Quanhong’and analyzed it by bioinformatics methods,we then transformed PdHY5 gene into tobacco by Agrobacterium-mediated leaf disc method for functional analysis.The results showed that:(1)the open reading frame(ORF)of PdHY 5 was 510 bp and encoded 169 amino acids.Comparison of amino acid sequences and phylogenetic analysis indicated that PdHY5 contained a conserved domain of bZIP family and had a closest relationship with PtHY5.(2)An over-expression vector pNP1302-35S-PdHY 5 was successfully constructed,and 4 tobacco transgenic lines(S1-S4)were obtained by hygromycin screening.(3)qRT-PCR results showed that the expression of PdHY 5 gene in the 4 overexpression lines was significantly higher than that of WT(wild type),with the highest expression of S4 and the least of S1.What s more,the expression levels of several key genes participated in anthocyanin biosynthesis,such as CHS,F3H,and FLS in overexpression lines were significantly up-regulated compared with WT.(4)The relative content of anthocyanin was significantly higher in transgenic tobacco lines S2,S3 and S4 compared with WT,increasing by 128.23%,97.36%and 134.20%,respectively.This study indicated that overexpression of PdHY 5 gene regulated the expression of structural genes in the anthocyanin biosynthesis pathway of tobacco itself,thereby promoting the accumulation of anthocyanins.

关 键 词：‘全红’杨 PdHY5转录因子 基因克隆 生物信息学 功能分析 

分 类 号：Q785[生物学—分子生物学] Q786Q789