论文部分内容阅读
【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。
【Objective】 Yeast two-hybrid system was used to identify the cysteine / S protein 11 (SCR / SP11) and S site receptor kinase (SRK) extracellular domain (eSRK) fragments may interact with the region. 【Method】 The recombinant bait plasmid with full-length SCRB3, SCRB3-1 and SCRB3-2 with pGBKT7 as the vector was constructed by using the Brassica oleracea B3 as a material. The full length eSRKB3 and eSRKB3- 1, a recombinant activation domain (eD) plasmid of eSRKB3-2. The recombined bait plasmids were transformed into competent yeast Y2HGold by PEG / LiAc method, and the recombinant AD plasmids were transformed into competent yeasts Y187; Y2HGold [pGBKT7-SCRB3-s] and Y187 [pGADT7-eSRKB3-s] The mating strains were cultured in SD / -Trp-Leu / x-α-gal / AbA (DDO / x / A), SD / -Trp- Leu- Ade-His / x- α-gal / AbA (QDO / x / Growth on the tablet. [Results] The yeast two-hybrid recombinant expression vector was constructed successfully and had no toxicity and self-activation. Only 9 SCRB3-1, SCRB3-2 and eSRKB3-1 recombinant eSRKB3-2 recombinant expression plasmids were used in combination Of the four fusion strains appeared on QDO / x / A plates blue clones, activating the reporter genes AUR1-C, MEL1, HIS3, ADE2. 【Conclusion】 Yeast two-hybrid system is suitable for the study of the interaction between SCR and SRK protein. The interaction between SCR and eSRK was preliminarily confirmed. The existence of SRK transmembrane domain had no effect on the interaction between SCR and SRK protein.