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园艺学报 ›› 2015, Vol. 42 ›› Issue (10): 1931-1943.doi: 10.16420/j.issn.0513-353x.2015-0388

• 蔬菜 • 上一篇    下一篇

芥菜开花整合子SOC1启动子的克隆及其与FLC、SVP蛋白互作的研究

陈 娇*,赵夏云*,鲜登宇*,马关鹏,谢 婷,王志敏,宋 明**,汤青林**   

  1. 西南大学园艺园林学院,南方山地园艺学教育部重点实验室,重庆市蔬菜学重点实验室,重庆 400715
  • 出版日期:2015-10-25 发布日期:2015-10-25
  • 基金资助:

    国家自然科学基金项目(31000908);国家重点基础研究发展计划(‘973’)项目(2012CB113900);重庆市自然科学基金项
    目(2011BA1002);中央高校基本科研业务费专项(XDJK2012B020)

Promoter Isolation of Flowering Signal Integrator SOC1 Gene and Its Interactions with FLC and SVP Proteins in Brassica juncea

CHEN Jiao*,ZHAO Xia-yun*,XIAN Deng-yu*,MA Guan-peng,XIE Ting,WANG Zhi-min,SONG Ming**,and TANG Qing-lin**   

  1. College of Horticulture and Landscape Architecture,Southwest University;Key Laboratory of Horticulture Science for Southern Mountainous Regions,Ministry of Education;Key Laboratory of Olericulture,Chongqing 400715,China
  • Online:2015-10-25 Published:2015-10-25

摘要:

为了深入研究芥菜开花整合子SOC1基因的表达调控机制,利用染色体步移法从芥菜‘QJ’中克隆了SOC1编码区上游782 bp的启动子,并构建SOC1基因启动子的酵母表达载体pAbAi-SOC1,与蛋白表达载体pGADT7-FLC、pGADT7-SVP共转化酵母Y1HGold菌株。酵母单杂交表明:芥菜FLC和SVP蛋白均能与SOC1的启动子相互作用。进一步分析发现:SOC1启动子含3个CArG-box表达调控基序。分别亚克隆这3个基因片段(SOC1-1、SOC1-2和SOC1-3),并再次构建酵母重组质粒pAbAi-SOC1-1、pAbAi-SOC1-2和pAbAi-SOC1-3,与pGADT7-FLC、pGADT7-SVP分别融合到Y1HGold菌株。融合菌株均能在相应SD/-Leu/AbA培养基上生长,说明SOC1-1、SOC1-2和SOC1-3都能被芥菜FLC、SVP蛋白识别并结合。再次构建SOC1-1、SOC1-2、SOC1-3的CArG-box删除突变体及A-T互换突变体,则均不能与FLC、SVP蛋白互作。由此说明:SOC1-1、SOC1-2和SOC1-3的3个CArG-box基序确实能特异性识别FLC、SVP,发生DNA—蛋白相互作用。这为利用启动子调控SOC1基因的转录表达等深入研究奠定了理论基础。

关键词: 芥菜, SOC1启动子, FLC, SVP, CArG-box, DNA&mdash, 蛋白互作

Abstract:

In order to clarify the expression regulation mechanism of the flowering signal integrator SOC1 gene,a 782 bp promoter of SOC1 gene was cloned from Brassica juncea‘QJ’via genome walking kit. Yeast promoter-reporter vector pAbAi was constructed with SOC1 promoter and transformed into Y1HGold strain. Then protein vectors,pGADT7-FLC and pGADT7-SVP,were transformed into Y1HGold(pAbAi-SOC1)strain,respectively,to test the interactions of SOC1 promoter with FLC and SVPvia yeast one hybrid system. The results showed that Brassica juncea FLC and SVP proteins could interact with SOC1 promoter,respectively. Further analysis indicated that SOC1 promoter had three CArG-box moitfs,which probably regulated the DNA-protein interactions. Thus,three fragments of SOC1 promoter were subcloned respectively and named SOC1-1,SOC1-2 and SOC1-3,each of which had a single CArG-box. The plasmids,pAbAi-SOC1-1,pAbAi-SOC1-2 and pAbAi-SOC1-3,were also constructed,and then transformed into Y1HGold strain and fused with pGADT7-FLC and pGADT7-SVP plasmids,respectively. All the zygote strains could grow on the SD/-Leu/AbA plates,suggesting that SOC1-1,SOC1-2 and SOC1-3 could be targeted by Brassica juncea FLC and SVP proteins,respectively. To verify the specificity of the interactions,we additionally constructed three mutants which deleted their whole CArG-box,as well as another three mutants that only exchanged A and T in CArG-box. However,the DNA–protein interactions vanished after CArG-box was deleted or mutated,which suggests that the three CArG-box motifs in SOC1-1,SOC1-2 and SOC1-3 could specifically interact with FLC and SVP proteins. The study provided valuable information for further studies on the transcriptional expression regulation of SOC1 gene in flowering-time control.

Key words: Brassica juncea, promoter of SOC1 gene, FLC, SVP, CArG-box, DNA–protein interaction

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