利用酵母双杂交系统鉴定甘蓝SCR与SRK胞外域片段间的相互作用

doi:10.3864/j.issn.0578-1752.2011.09.024

摘要/Abstract

摘要： 【目的】利用酵母双杂交系统鉴定甘蓝自交不亲和决定因子S位点富含半胱氨酸蛋白/S位点蛋白11（SCR/SP11）与S位点受体激酶（SRK）胞外域（eSRK）片段间可能的相互作用区域。【方法】以结球甘蓝B3为材料，利用分子克隆技术，分别构建以pGBKT7为载体的全长SCRB3、SCRB3-1、SCRB3-2的重组诱饵质粒；以pGADT7为载体的全长eSRKB3 、eSRKB3-1、eSRKB3-2的重组激活域（AD）质粒。用PEG/LiAc法将上述重组诱饵质粒转化感受态酵母菌Y2HGold，重组AD质粒转化感受态酵母菌Y187；Y2HGold[pGBKT7-SCRB3-s]、Y187[pGADT7-eSRKB3-s]两两融合培养，观察交配菌在SD/-Trp-Leu/x-α-gal/AbA(DDO/x/A)、SD/-Trp-Leu-Ade-His/x-α-gal/AbA(QDO/x/A)平板上的生长情况。【结果】酵母双杂交重组表达载体构建成功，且无毒性和自激活作用产生；9个试验组中只有SCRB3-1、SCRB3-2与eSRKB3-1、eSRKB3-2重组表达质粒转化子两两组合的4个融合株在QDO/x/A平板上出现蓝色克隆，激活了报告基因AUR1-C、MEL1、HIS3、ADE2。【结论】酵母双杂交系统适用于SCR与SRK蛋白相互作用的研究，初步确定了SCR与eSRK存在相互作用，SRK跨膜域的存在与否对其相互作用的研究没有影响。

关键词: 结球甘蓝 , 自交不亲和 , S位点受体激酶（SRK） , S位点富含半胱氨酸蛋白（SCR） , 酵母双杂交

Abstract: 【Objective】 Self-incompatibility of Brassica oleracea L. var capitata L. was determined by interaction between the female S-locus receptor kinase (SRK) and the male S-locus cysteine rich protein/S-locus protein 11 (SCR/SP11). In this study, a yeast two-hybrid system was adopted to investigate their potential interaction domains between SCR/SP11 and the extracellular fragment of SRK (eSRK).【Method】The full length SCRB3, SCRB3-1 and SCRB3-2 amplified from Brassica oleracea L. B3 were cloned into the pGBKT7 to construct the recombinant bait plasmids which were then transformed into yeast Y2HGold cells by PEG/LiAc method. The DNA fragments including eSRKB3, eSRKB3-1 and eSRKB3-2 were cloned into the pGADT7 to construct the recombinant activation domain (AD) plasmids which were transformed into yeast Y187 cells. Each transformant of Y2HGold [pGBKT7-SCRB3-s] was crossed with one of three transformants Y187 [pGADT7-eSRKB3-s]. Then the crossed yeast cells were screened consecutively on SD/-Trp-Leu/x-α-gal/AbA (DDO/x/A) and SD/-Trp-Leu-Ade-His/x-α-gal/AbA(QDO/x/A) nutritional media. 【Result】 The restriction enzyme analysis and DNA sequencing indicated that the above recombinant plasmids were correct. The yeast Y2HGold cells transformed with each of the three recombinant bait plasmids could grow on SD/-Trp nutritional media without autonomous activation effect on the reporter gene MEL1 and AUR1-C. The yeast Y187 cells transformed with each of the three recombinant AD plasmids could grow on SD/-Leu nutritional media without activation of the reporter gene MEL1. And these recombinant plasmids were no toxic to yeast cells. Four crossed yeast cell lines could grow on QDO/x/A nutritional media with transcription activation of the reporter genes AUR1-C, MEL1, HIS3, and ADE2.【Conclusion】The yeast two-hybrid system could be used to study the interaction domain between SCR and eSRK. The transmembrane domain of SRK is present or not which will have no effect on studying the interaction between SCR and SRK.

Key words: Brassica oleracea L. var capitata L. , self-incompatibility , S-locus receptor kinase (SRK) , S-locus cysteine-rich protein (SCR) , yeast two-hybrid

杨红1;朱利泉1;张贺翠1;杨永军1;薛丽琰1;杨昆1;余浩1;彭一波1;罗兵1;吴志刚1;黄丹1;高启国2;王小佳2. 利用酵母双杂交系统鉴定甘蓝SCR与SRK胞外域片段间的相互作用[J]. 中国农业科学, 2011, 44(9): 1953-1962.

YANG Hong; ZHU Li-quan; ZHANG He-cui;YANG Yong-jun; XUE Li-yan; YANG Kun; YU Hao; PENG Yi-bo; LUO Bing; WU Zhi-gang; HUANG Dan; GAO Qi-guo; WANG Xiao-jia. Study on the Interactions Between the Truncated Fragments of SCR and eSRK from Brassica oleracea L. by a Yeast Two-Hybrid System[J]. Scientia Agricultura Sinica, 2011, 44(9): 1953-1962.

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