中国农业科学 ›› 2018, Vol. 51 ›› Issue (24): 4603-4614.doi: 10.3864/j.issn.0578-1752.2018.24.002

• 作物遗传育种·种质资源·分子遗传学 • 上一篇    下一篇

GT和GATA转录因子对甘蓝型油菜BnA5.FAD2BnC5.FAD2启动子功能的调控

刘芳1(),肖钢2,官春云1()   

  1. 1 湖南农业大学农学院/国家油料改良中心湖南分中心,长沙 410128
    2 湖南省水稻油菜抗病育种重点实验室,长沙 410128
  • 收稿日期:2018-07-13 接受日期:2018-09-25 出版日期:2018-12-16 发布日期:2018-12-16
  • 基金资助:
    国家重点基础研究发展计划“973”计划(2015CB150200)

Regulation of GT and GATA Transcription Factors on Promoter Function of BnA5.FAD2 and BnC5.FAD2 Genes in Brassica napus

LIU Fang1(),XIAO Gang2,GUAN ChunYun1()   

  1. 1 College of Agriculture, Hunan Agricultural University/National Oilseed Crops Improvement Center in Hunan, Changsha 410128
    2 Hunan Provincial Key Laboratory of Rice and Rapeseed Breeding for Disease Resistance, Changsha 410128
  • Received:2018-07-13 Accepted:2018-09-25 Online:2018-12-16 Published:2018-12-16

摘要:

目的 脂肪酸去饱和酶2基因(FAD2)是控制油菜中油酸含量的重要基因,通过研究GATA和GT转录因子与BnA5.FAD2BnC5.FAD2启动子的互作关系及其转录调控机制,为高油酸油菜育种提供分子理论基础。方法 通过缺失启动子片段及生物信息学分析,预测BnA5.FAD2BnC5.FAD2启动子区潜在顺式作用元件;从PlantTFDB转录因子数据库中筛选候选转录因子,通过实时荧光定量PCR(qRT-PCR)检测转录因子的表达规律,并与BnA5.FAD2BnC5.FAD2表达规律对比,进一步筛选候选转录因子;利用酵母单杂交验证转录因子与启动子序列的互作情况;将转录因子与BnA5.FAD2BnC5.FAD2启动子序列共转入拟南芥,通过Western blot检测报告基因GFP的蛋白表达情况,分析转录因子对于启动子功能的影响。结果 单独敲除BnA5.FAD2BnC5.FAD2启动子中的GT或GATA顺式作用元件后,GFP蛋白丰度均下降,表明GT和GATA顺式作用元件在调节基因转录中起重要作用。酵母单杂交结果显示,GATA家族转录因子(Bna010243-1Bna026124-1Bna026124-2)和GT家族转录因子(Bna010915Bna013749)可以与BnA5.FAD2BnC5.FAD2启动子相互作用。Western blot结果显示,当GATA家族转录因子与含有GATA元件的启动子片段共转化拟南芥时,GFP蛋白含量明显高于无转录因子时,当敲除GATA元件时,GFP蛋白含量无明显变化;当GT家族转录因子与含有GT元件的启动子片段共转化拟南芥时,GFP蛋白含量有明显变化,当敲除GT元件时,GFP蛋白含量无明显变化。结论 GATA家族的转录因子Bna010243-1、Bna026124-1和Bna026124-2可以与BnA5.FAD2BnC5.FAD2启动子中GATA元件相互作用,增强基因的表达水平。GT家族的转录因子Bna010915可以与BnA5.FAD2BnC5.FAD2启动子中的GT元件发生互作,正向调节基因表达;Bna013749通过与BnA5.FAD2BnC5.FAD2启动子中的GT元件发生互作,负向调节基因表达。

关键词: 甘蓝型油菜, GATA转录因子, GT转录因子, BnA5.FAD2启动子, BnC5.FAD2启动子

Abstract:

【Objective】 Fatty acid desaturase 2 gene (FAD2) is an important gene for controlling oleic acid content in rapeseed. And in this paper, the interaction between GATA family and BnA5.FAD2 and BnC5.FAD2 gene promoters or GT family transcription factors and BnA5.FAD2 and BnC5.FAD2 gene promoters was studied to make sure the transcription regulation mechanism to provide the theoretical basis for high oleic acid breeding. 【Method】 The potential cis-elements were predicted by deletion of promoter fragments and bioinformatics analysis, and candidate transcription factors were screened from PlantTFDB transcription factor database. Real-time PCR (qRT-PCR) was used to detect the expression pattern of transcription factors to further screen candidate transcription factors by comparison with BnA5.FAD2 and BnC5.FAD2 gene expression patterns. Transcription factors were co-transformed with BnA5.FAD2 and BnC5.FAD2 gene promoters into yeast and verified interaction relationship between promoter and transcription factors by yeast one-hybrid. The transcription factors were transformed into Arabidopsis thaliana with BnA5.FAD2 and BnC5.FAD2 gene promoter sequence, and the reporter gene GFP was detected by western blot to analyze the effects of transcription factors on promoters. 【Result】 After knocking out the GT or GATA cis-elements from BnA5.FAD2 and BnC5.FAD2 gene promoters respectively, the GFP protein abundance decreased, which indicate that GT and GATA cis-elements play important roles in regulating gene transcription. Yeast one-hybrid results showed that the GATA family transcription factors (Bna010243-1, Bna026124-1, Bna026124-2) and the GT family transcription factors (Bna010915 and Bna013749) can interact with BnA5.FAD2 and BnC5.FAD2 gene promoters. Western blot analysis showed that when the GATA family transcription factor and the promoter fragment containing GATA elements were co-transformed into Arabidopsis thaliana, the GFP protein content was significantly higher than that of no transcription factor. When the GATA element was knocked out, the GFP protein content did not change significantly. When the GT family transcription factor and the promoter fragment containing the GT element were co-transformed into Arabidopsis thaliana, the GFP protein content changed significantly. When the GT element was knocked out, the GFP protein content did not change significantly.【Conclusion】 The GATA family transcription factors Bna010243-1, Bna026124-1, Bna026124-2 can interact with the GATA elements in the BnA5.FAD2 and BnC5.FAD2 gene promoters to enhance gene expression levels. The GT family transcription factor Bna010915 interacts with the GT elements in the BnA5.FAD2 and BnC5.FAD2 gene promoters to positively regulate gene expression; Bna013749 interacts with the GT elements in the BnA5.FAD2 and BnC5.FAD2 gene promoters to negatively regulate gene expression.

Key words: Brassica napus, GATA transcription factor, GT transcription factor, BnA5.FAD2 promoter, BnC5.FAD2 promoter