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中国农学通报

中国农学通报 ›› 2012, Vol. 28 ›› Issue (15): 24-30.doi: 10.11924/j.issn.1000-6850.2011-3975

所属专题： 生物技术； 玉米

• 农学 农业基础科学 • 上一篇    下一篇

玉米淀粉分支酶SBEIIb基因片段酵母双杂交诱饵载体

崔喜艳 刘晶 刘忠野 韩琳   

  • 收稿日期:2011-12-29 修回日期:2012-02-28 出版日期:2012-05-25 发布日期:2012-05-25
  • 基金资助:

    吉林省教育厅“十一五”科学技术研究项目;吉林省博士后科研项目启动经费资助

Construction and Detection of Yeast Two-hybrid Bait Vector for SBEIIb Gene Fragment of Starch Branching Enzyme from Zea mays

  • Received:2011-12-29 Revised:2012-02-28 Online:2012-05-25 Published:2012-05-25

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摘要：

为探寻玉米淀粉分支酶(starch branching enzyme, SBEIIb)与其互作蛋白的作用位点，构建了酵母双杂交诱饵载体。根据CDD和NCBI对SBEIIb蛋白氨基酸序列的分析，确定玉米SBEIIb的功能域及非功能域，PCR扩增功能域及非功能域基因的目的片段，经回收纯化，用相同的限制性内切酶将其与载体pGBKT7进行双酶切后，分别将目的基因片段与载体pGBKT7进行连接。构建了酵母双杂交诱饵载体pGBKT7-SBEIIb-X(X=1,2,3,4,5,6)，转化酵母菌AH109的菌液，OD600值在0.8以上，证明诱饵载体pGBKT7-SBEIIb-X对酵母菌AH109没有毒性作用；转化诱饵载体后的酵母菌AH109，在SD/-Trp/X-α-Gal平板上长出白色菌落，在SD/-His/-Trp/X-α-Gal、SD/-Ade/-Trp/X-α-Gal平板上不能生长，排除了pGBKT7-SBEIIb-X具有自激活宿主菌AH109的作用。构建的酵母双杂交诱饵载体pGBKT7- SBEIIb-X(X=1,2,3,4,5,6)，其表达对酵母细胞无毒性及对报告基因无自激活作用，可为下一步分析SBEIIb与其互作蛋白的作用位点奠定基础。

关键词: 湖南, 湖南

Abstract:

The aim was to explore the effective situs of starch branching enzyme between its interactive protein in Zea mays and to construct bait vectors in yeast two-hybrid. The amino acid sequence of SBEIIb protein was analyzed by the CDD and NCBI, and the functional domain and non-functional domain were determined. The genes of the functional domain and non-functional domain were amplified by PCR, and purified and cloned into the pGBKT7 plasmid after digested with the same restriction endonuclease. The pGBKT7-SBEIIb-X (X=1, 2, 3, 4, 5, 6) of the yeast two-hybrid bait vector were successfully transformed into the yeast cells AH109, and OD600 value over 0.8. The result indicated that bait vectors were not toxic to AH109 cells. The yeast transformants grew white colonies in SD/-Trp/X-α-Gal plates, but couldn't in SD/-His/-Trp/X-α-Gal and SD/-Ade/-Trp/X-α-Gal plates, so except for self-activation of the bait protein. Vectors of pGBKT7-SBEIIb-X (X=1, 2, 3, 4, 5, 6) were constructed, and were not toxic to AH109 and could not activate the reporter genes, which would provide a foundation for further analyzing the effective situs of interactive protein with SBEIIb.