甘蓝型油菜酪氨酸代谢关键基因FAH的克隆、功能鉴定和表达分析  

Cloning,Functional Identification and Expression Analysis of FAH,a Key Gene for Tyrosine Metabolism in Brassica napus L.

作　　者：支添添 周舟 陈纪鹏[1] 韩成云[2] ZHI Tian-tian;ZHOU Zhou;CHEN Ji-peng;HAN Cheng-yun(College of Life Science and Resources and Environment,Yichun University,Key Laboratory of Crop Growth and Development Regulation,Yichun University,Yichun 336000;College of Chemistry and Bioengineering,Yichun University,Yichun 336000)

机构地区：[1]宜春学院生命科学与资源环境学院,宜春学院江西省作物生长发育调控重点实验室,宜春336000 [2]宜春学院化学与生物工程学院,宜春336000

出　　处：《生物技术通报》2023年第10期115-127,共13页Biotechnology Bulletin

基　　金：国家自然科学基金项目(31760301);江西省科技厅重点研发计划(20212BBF63014);江西省教育厅科技项目(GJJ190872)。

摘　　要：克隆甘蓝型油菜(Brassica napus L.)酪氨酸代谢关键基因FAH,对其进行功能验证和表达分析,为进一步解析FAH在甘蓝型油菜中的作用和功能提供理论依据。以甘蓝型油菜‘westar’为试材,克隆与拟南芥AtFAH同源性最高的甘蓝型油菜FAH基因BnaA06g38260D(BnaA06FAH)和BnaC05g49430D(BnaC05FAH),通过生物信息学分析其亲缘关系,构建过表达载体转化拟南芥突变体sscd1进行功能验证。克隆BnaA06FAH和BnaC05FAH启动子序列,利用PlantCare在线数据库分析启动子调控元件,构建启动子和GUS的融合载体,通过GUS组织化学染色分析其表达模式。结果显示,BnaA06FAH和BnaC05FAH与AtFAH的氨基酸序列相似性分别为93.11%和92.40%,2个基因过表达都可以完全抑制拟南芥突变体sscd1在短日照下模拟病斑的形成,暗示BnaA06FAH和BnaC05FAH都与AtFAH功能相似。BnaA06FAH和BnaC05FAH启动子除具有所必需的TATA-box和CAAT-box等基本顺式作用元件外,都含有多个与光诱导、激素响应和逆境胁迫响应元件以及多种与抗病相关的顺式作用元件,但与BnaA06FAH相比,BnaC05FAH与拟南芥AtFAH相同的顺式作用元件更多;GUS活性检测表明,BnaC05FAH启动子驱动的GUS基因的表达比BnaA06FAH强,并且两者驱动表达的组织部位不完全相同。因此,BnaA06FAH和BnaC05FAH启动子的作用部位和作用强度都存在明显差异。BnaA06FAH和BnaC05FAH都能够调控拟南芥sscd1模拟病斑的形成,但两者启动子驱动下游基因的强度和部位不同。The objective of this work is to clone the tyrosine metabolism key gene FAH in Brassica napus L.,identify its function and analyze its expression,so as to provide theoretical evidence for further understanding the role and function of FAH in B.napus L.Two FAH genes BnaA06g38260D(BnaA06FAH)and BnaC05g49430D(BnaC05FAH),which was the highest protein homology to AtFAH in Arabidopsis,were cloned from the B.napus L.variety‘westar',the phylogenetic relationship was analyzed by bioinformatics,and the overexpression vector was constructed and transformed into Arabidopsis mutant sscd1 for function verification;the promoter sequences of two FAH genes BnaA06FAH and BnaC05FAH were cloned.PlantCARE was used to analyze the promoter cis-acting elements,and the fusion expression vector of gene promoter and GUS was constructed to analyze its expression patterns.As results,the amino acid sequence similarity of BnaA06FAH,BnaC05FAH and AtFAH was 93.11% and 92.40% respectively,the overexpressions of BnaA06FAH and BnaC05FAH completely inhibited the mimic lesion in sscd1 under short-day condition,suggesting both BnaA06FAH and BnaC05FAH had high structural and functional similarity with AtFAH.BnaA06FAH and BnaC05FAH promoters had the core elements of eukaryotic promoter TATA-box and CAAT-box,and also contained several cis-acting elements related to stress,hormone,stress response and a variety of cis-acting elements related to disease resistance,however,BnaC05FAH promoter shared more cis-acting elements with Arabidopsis AtFAH than with BnaA06FAH.GUS activity assays indicated BnaC05FAH promoter drove the expression of GUS gene stronger than BnaA06FAH,and the site of GUS gene expression drove by two different promoters were not completely consistent.Conclusively,there were differences in the sites and intensity where BnaA06FAH and BnaC05FAH promoters functioned.The results showed that both BnaA06FAH and BnaC05FAH played a role in regulating lesion mimic in the sscd1 mutant,but the expression of gene downstream and the expression site drov

关 键 词：甘蓝型油菜 FAH 功能鉴定 酪氨酸降解 

分 类 号：S565.4[农业科学—作物学]