橡胶树F3’H基因克隆及其功能分析  

Cloning and Functional Verification of Rubber Tree F3’H Gene

作　　者：范月婷 辛士超[2] NAYCHI Koko 畅娇 黄天带[2] 黄华孙[2] 华玉伟[2] FAN Yueting;XIN Shichao;NAYCHI Koko;CHANG Jiao;HUANG Tiandai;HUANG Huasun;HUA Yuwei(College of Tropical Crops,Hainan University,Haikou,Hainan 570228,China;Rubber Research Institute,Chinese Academy of Tropical Agricultural Sciences/Key Laboratory of Biology and Genetic Resources of Rubber Tree,Ministry of Agriculture and Rural Affairs,Haikou,Hainan 571101,China)

机构地区：[1]海南大学热带作物学院,海南海口570228 [2]中国热带农业科学院橡胶研究所/农业农村部橡胶树生物学与遗传资源利用重点实验室,海南海口571101

出　　处：《热带作物学报》2020年第9期1733-1740,共8页Chinese Journal of Tropical Crops

基　　金：国家自然科学基金项目(No.31770717)。

摘　　要：从橡胶树叶片转录组数据库中调取花青素合成途径中的关键酶类黄酮3’-羟化酶(F3’H)基因序列信息,通过RT-PCR扩增得到2个橡胶树F3’H基因,分别命名为HbF3’H1和HbF3’H2。通过荧光定量PCR分析发现,只有HbF3’H1基因在不同发育时期的橡胶树叶片和嫩茎中的表达水平与花青素的合成积累趋势完全一致。HbF3’H1所编码的蛋白属于P450超家族,具有保守的F3’H结构域,而且HbF3’H1基因的启动子中包含多种环境效应元件,说明HbF3’H1基因的表达受环境因子调控。通过农杆菌转化烟草发现,过表达HbF3’H1基因的烟草花瓣大量累积花青素,其颜色较非转基因烟草显著加深,同时,荧光定量PCR发现HbF3’H1表达水平与转基因烟草花瓣颜色呈正相关,说明HbF3’H1基因表达促进花青素的累积。本研究为阐明橡胶树花青素代谢途径奠定基础。Based on the transcriptome database of rubber tree(Hevea brasiliensis)leaf tissues,we retrieved the nucleotide sequences of flavonoid 3’-hydroxylase(F3’H)gene,which is the key enzyme in anthocyanin biosynthesis pathways.Two rubber tree F3’H genes,HbF3’H1 and HbF3’H2 were amplified by RT-PCR.The expression levels of the HbF3’H genes were analyzed by quantitative real-time PCR and the expression of HbF3’H1 were shown to be completely correlated with the anthocyanin contents in the deffirent developmental stage of leaves and stems.Amino acid sequence analysis results showed that HbF3’H1 belonged to the super-family of cytochrome P450,and consisted of typical F3’H conserved domains.In the promoter region of HbF3’H1 gene,we observed multiple environment response elements,which revealed the possible role of HbF3’H1 in environment stress resistance in rubber trees.HbF3’H1 was over-expressed in tobacco(Nicotiana tabacum‘Samsun NN’)by Agrobacterium tumefaciens mediated transformation for further functional verifications.Compared with the wild type control,the petals of transgenic tobacco lines exhibited enhanced red color pigmentation,which was positively correlated with the expression levels of HbF3’H1.The results demonstrated that HbF3’H1 played an important role in the biosynthesis of anthocyanin.The study would lay a foundation for understanding the anthocyanin biosynthesis pathways in rubber trees.

关 键 词：橡胶树 花青素 类黄酮3’-羟化酶 转基因烟草 

分 类 号：S794.1[农业科学—林木遗传育种]