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作物学报 ›› 2014, Vol. 40 ›› Issue (06): 1011-1019.doi: 10.3724/SP.J.1006.2014.01011

• 作物遗传育种·种质资源·分子遗传学 • 上一篇    下一篇

结球甘蓝雌蕊调控因子SPT与HEC1的克隆及相互作用分析

许俊强,孙梓健,刘智宇,杨朴丽,汤青林,王志敏,宋明*,王小佳*   

  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
  • 收稿日期:2013-07-01 修回日期:2014-03-04 出版日期:2014-06-12 网络出版日期:2014-04-08
  • 通讯作者: 王小佳, E-mail: wxj@swu.edu.cn; 宋明，E-mail: swausongm@163.com, Tel: 023-68251093
  • 基金资助:

    本研究由国家自然科学基金项目(31071802, 31000908), 重庆市自然科学基金重点项目(2011BA1002), 中央高校基本科研业务费专项(XDJK2012B020)和国家重点基础研究发展计划(973计划)(2012CB113900)资助。

Cloning and Interaction between Transcription Factors SPT and HEC1 of Pistil of Brassica oleracea L.var. capitata L.

XU Jun-Qiang,SUN Zi-Jian,LIU Zhi-Yu,YANG Pu-Li,TANG Qing-Lin,WANG Zhi-Min,SONG Ming*,WANG Xiao-Jia*   

  1. Key Laboratory of Horticulture Science for Southern Mountainous Regions, Ministry of Education / Chongqing Key Laboratory of Olericulture / College of Horticulture and Landscape Architecture, Southwest University, Chongqing 400715, China?
  • Received:2013-07-01 Revised:2014-03-04 Published:2014-06-12 Published online:2014-04-08
  • Contact: 王小佳, E-mail: wxj@swu.edu.cn; 宋明，E-mail: swausongm@163.com, Tel: 023-68251093

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摘要：

为研究SPT和HECs及其相互作用对甘蓝雌蕊发育的影响, 以结球甘蓝自交不亲和系E1为材料, 提取雌蕊总RNA, 根据拟南芥中SPT和HEC1基因设计引物, 采用同源克隆的方法克隆SPT基因, 其序列1085 bp, 开放阅读框(ORF) 1062 bp; HEC1基因ORF 696 bp。通过cDNA推导得到的氨基酸序列表明, SPT编码353个氨基酸残基, 预测分子量为37.67 kD, pI为6.83; HEC1编码231个氨基酸残基, 预测分子量为25.26 kD, pI为10.2。分析表明, 两基因在各器官中均表达, 但SPT在果实和雌蕊中表达量最高, 而HEC1在根和花蕾中的表达量最高。为检测两者的相互作用, 构建原核表达质粒pCold I-SPT和pGEX-HEC1, Pull-down试验表明两蛋白能够在体外相互作用。同时构建pGBKT7-SPT、pGADT7-HEC1及互换载体pGBKT7-HEC1和pGADT7-SPT酵母表达载体, 分别转化酵母Y2HGold和Y187感受态细胞后均未出现自激活和毒性现象, 融合后的二倍体酵母均能在SD/–Trp–Leu–Ade–His/X-α-gal/AbA板上长出蓝色菌斑, 表明SPT和HEC1能够相互作用激活下游的HIS3、AUR1-C、MEL1和ADE2报告基因, 酵母双杂交试验结果与Pull-down检测一致, 说明SPT和HEC1能够相互作用以调控雌蕊的发育。

关键词: 结球甘蓝, 雌蕊, SPT, HEC1, 相互作用, 酵母双杂交

Abstract:

To explore interaction of SPT and HECs in cabbage pistil development, we took stigma of self-incompatibility line E1 to obtain total RNA for first-strain cDNA synthesis, and cloned SPT gene fragment with the ORF of 1062 bp and HEC1 gene with 696bp by using primers according to SPT and HEC1 genes in Arabidopsis. Amino acid sequence analysis showed that SPT and HEC1we cloned encoded 353 and 231 amino acid residues, respectively; the predicted molecular weight of SPT protein was 37.67 kD, with pI of 6.83; the predicted molecular weight of HEC1 protein was 25.26 kD, with pI of 10.23. Relative expression of SPT was the highest in the fruit and pistil, that of HEC1 was the highest in the root and bud. Prokaryotic expression plasmids pCold I-SPT and pGEX-HEC1 were constructed and then transformed into E. coli Rosetta (DE3). The Pull-down assay showed that the two tagged expression products could interact with each other. To verify the interactions furtherly, we constructed yeast expression vectors pGBKT7-SPT, pGADT7-HEC1, and pGADT7-SPT, pGBKT7-HEC1 to transform into yeast Y2HGold and Y187 strains, respectively. Finally, diploid yeast showed positive results in SD/–Ade–Trp–Leu–His/X-α-gal/AbA synthetic dropout. It demonstrated that interaction between SPT and HEC1 can activate HIS3, AUR1-C,and ADE2 reporter genes downstream. Yeast two-hybrid experimental results as well as the pull-down assay showed that SPT and HEC1 can form heterodimer to regulate the development of pistil.

Key words: Brassica oleracea, Pistil, SPT, HEC1, Interaction, Yeast two-hybrid

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