1ql1基因酵母双杂交诱饵载体的构建及自激活检测
onstruction of mC1ql1 Yeast Two-Hybrid Bait Vector and Detection of Its Self-activated Activity
目的：通过钓取C1ql1的相互作用蛋白研究未知功能的小分泌蛋白C1ql1的功能，本研究构建pGBKT7-C1ql1酵母双杂交诱饵载体并验证其自激活活性。方法：采用PCR技术扩增出小鼠C1ql1（mC1ql1）基因去除信号肽后的区段，连接到酵母双杂交诱饵载体pGBKT7中。采用醋酸锂转化法将阳性pGBKT7-mC1ql1质粒转化酵母Y2HGold菌株。利用不同营养缺陷型培养基进行自激活检测和毒性检测。结果：用PCR法、EcoR I /BamH I双酶切法和测序法鉴定重组pGBKT7-mC1ql1载体，结果表明构建的mC1ql1基因的诱饵载体构建成功，读码框正确。转化Y2HGold 酵母菌后，在SD/-Trp/X-α-gal固体培养基上未长出蓝色菌落，而在SD/-Trp/X-α-gal/AbA 固体培养基上未见有菌落生长。结论：诱饵载体对酵母菌株Y2HGold没有转录自激活活性和毒害作用。说明构建的诱饵载体可以用于酵母双杂交系统中，为下一步筛选mC1ql1互作蛋白奠定了基础。
Objective: To reveal the function of C1ql1 by screening its interaction protein, we constructed a bait vector of mC1ql1 gene and evaluated autoactivation in yeast two-hybrid system. Methods: The mC1ql1 fragment of coding region without signal peptide was amplified by polymerase chain reaction (PCR) and cloned into bait vector pGBKT7. The pGBKT7-mC1ql1 vector was transformed into the yeast strain Y2HGold by PEG/LiAc method and its self-activation was tested by the phenotype assay. Results: The bait vector pGBKT7-mC1ql1 was further confirmed by PCR, enzyme digestion and sequencing, results showed that the confirmed fragments were subcloned to bait vector pGBKT7. Blue plaque was not detected on SD/-Trp/X-α-gal solid medium．And no colony was grown on SD/-Trp/X-α-gal/AbA solid medium. Conclusion: The bait plasmid had no self-transcriptional activity and not toxicity to yeast strain Y2HGold. This bait vector constructed in this study could be used in yeast two hybrid system, which laid the foundation for screening interactional proteins of mC1ql1．
蔡冬青、肖銮娟、陈冉、廉滋珍、禹艳红、齐绪峰、武征、陈雷、秦俊文、薛樱子
分子生物学遗传学生物科学研究方法、生物科学研究技术
1ql1基因诱饵载体酵母双杂交自激活毒性检测
1ql1 geneBait vectorYeast two hybridSelf-activated transcriptional activateToxicity detection
蔡冬青,肖銮娟,陈冉,廉滋珍,禹艳红,齐绪峰,武征,陈雷,秦俊文,薛樱子.1ql1基因酵母双杂交诱饵载体的构建及自激活检测[EB/OL].(2013-05-20)[2025-08-11].http://www.paper.edu.cn/releasepaper/content/201305-304.点此复制
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