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作物学报 ›› 2016, Vol. 42 ›› Issue (10): 1471-1478.doi: 10.3724/SP.J.1006.2016.01471

• 作物遗传育种·种质资源·分子遗传学 • 上一篇    下一篇

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

刘睿洋,刘芳,张振乾,官春云   

  1. 湖南农业大学农学院 / 国家油料改良中心湖南分中心, 湖南长沙 410128
  • 收稿日期:2016-02-16 修回日期:2016-05-09 出版日期:2016-10-12 网络出版日期:2016-06-06
  • 通讯作者: 官春云, E-mail: guancy2011@yahoo.com.cn
  • 基金资助:

    本研究由湖南省科技创新项目(CX2013A012)和国家重点基础研究发展规划项目(2015CB150200)资助。

Functional Analysis of BnFAD2-C5 Promoter and Intron at Expression Level in Brassica napus

LIU Rui-Yang,LIU Fang,ZHANG Zhen-Qian,GUAN Chun-Yun   

  1. College of Agronomy, Hunan Agricultural University / National Oilseed Crops Improvement Center in Hunan, Changsha 410128, China
  • Received:2016-02-16 Revised:2016-05-09 Published:2016-10-12 Published online:2016-06-06
  • Contact: 官春云, E-mail: guancy2011@yahoo.com.cn
  • Supported by:

    This study was supported by Graduate Innovation Foundation of Hunan (CX2013A012) and the Major State Basic Research Development Program of China (2015CB150200).

摘要:

富含油酸的菜籽油具有重要的经济价值,使得高油酸育种和形成机理的研究成为热点。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GUSGFP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。

关键词: 甘蓝型油菜, BnFAD2-C5基因, 内含子增强效应, 拟南芥

Abstract:

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 of FAD2 gene is not well understood. According to the homology between rapeseed and oleracea, the BnFAD2-C5 promoter sequence of 1257 bp was cloned. Promoter and intron of BnFAD2-C5 gene were analyzed using β-glucuronidase (GUS) reporter and green fluorescent protein (GFP) reporter system to construct deleted vectors and transform Arabidopsis thaliana. Deletion analysis of BnFAD2-C5 promoter through GUS stainning revealed that –319 to 1 bp was the minimum promoter region. And deletion analysis of BnFAD2-C5 promoter and intron through GFP reporter system using western technique showed that –1257 to –1020 bp and –319 to –1 bp regionsof BnFAD2-C5 promoter could induce expression of reporter genes effectively in transgenic Arabidopsis seed in the mid stage of seed development, while BnFAD2-C5 intron could confer the enhancement of promoter′s function and the intron-mediated enhancement region was mainly located in 631 to 1033 bp.

Key words: Brassica napus, BnFAD2-C5 gene, intron-mediated enhancement, Arabidopsis thalina

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