甘蓝型油菜BnFAD2-C5基因启动子及内含子在表达水平的功能分析

摘要

富含油酸的菜籽油具有重要的经济价值,使得高油酸育种及油酸形成机制成为热点。油酸脱氢酶基因(FAD2基因)是控制油酸含量的关键酶基因。本文针对BnFAD2-C5基因展开研究,根据油菜和甘蓝的同源性,克隆了1257 bp启动子序列,利用 GUS 和 GFP 作为报告基因分别构建含有不同片段长度的启动子和内含子的缺失载体并转化拟南芥,经GUS染色检测发现–319~–1 bp为该研究中最小启动子；采用Western blot技术分析启动子和内含子不同区域的功能,发现BnFAD2-C5启动子区域–1257~–1020 bp和–319~–1 bp能够诱导报告基因在转基因拟南芥种子发育中期高效表达,BnFAD2-C5内含子具有增强启动子转录水平的功能,该功能主要由+631~+1033 bp区域调控。%High oleic rapeseed breeding and the formation mechanism of oleic acid have become a central issue after finding the important economic value of rapeseed oil with high oleic acid. The fatty acid dehydrogenase gene (FAD2) is a key enzyme gene to control oleic acid content, but the regulation ofFAD2 gene is not well understood. According to the homology between rapeseed and oleracea, theBnFAD2-C5 promoter sequence of 1257 bp was cloned. Promoter and intron ofBnFAD2-C5gene were analyzed usingβ-glucuronidase (GUS) reporter and green fluorescent protein (GFP) reporter system to construct deleted vectors and trans-formArabidopsis thaliana.Deletion analysis ofBnFAD2-C5 promoter through GUS stainning revealed that –319 to –1 bp was the minimum promoter region. And deletion analysis ofBnFAD2-C5 promoter and intron through GFP reporter system using western technique showed that –1257 to –1020 bp and –319 to –1 bp regionsofBnFAD2-C5 promoter could induce expression of reporter genes effectively in transgenicArabidopsisseed in the mid stage of seed development, whileBnFAD2-C5intron could confer the enhancement of promoter’s function and the intron-mediated enhancement region was mainly located in +631 to +1033 bp.