摘要
为了筛选巴西橡胶树蔗糖转运蛋白(HbSUT)的互作蛋白质,构建了以SOS恢复系统为原理的胞质酵母双杂交系统的6个HbSUT基因的诱饵载体,并进行了自激活检测。通过PCR技术从携带HbSUT基因的质粒中获得预期的基因编码序列,将目标片段定向插入诱饵载体pSOS的SalⅠ及NotⅠ酶切位点之间,酶切和测序结果表明,获得了序列和读框正确的6个HbSUT基因的诱饵载体pSOS-SUTs,进一步导入酵母温度敏感酵母cdc25H菌株中,检测其表达产物对酵母SOS恢复系统Ras信号通路的激活作用,除pSOS-SUT5外,其他5个诱饵载体对酵母菌株均无激活作用,因而适合于进行后续互作蛋白质筛选研究。
In order to screen the interacting proteins of sucrose transporters （SUTs） from Hevea brasiliensis,the bait vectors of the six sucrose transporters of cytoplasmic yeast two-hybrid system,which was based on Sos recruitment system （SRS）,were constructed and detected for their self activity. The coding sequences of SUTs were obtained by PCR from the plasmids containing respective Hevea SUTs,and the amplified products were cloned into the bait vector pSos at site Sal Ⅰ and Not Ⅰ. Restriction analysis and sequencing confirmed the sequence identity and the correct in frame fusion with the Sos gene of pSos. The resultant bait vectors of the 6 HbSUT genes were termed as pSos-SUTs. The pSos-SUTs were transformed into the temperature-sensitive strain cdc25H,and detected for their self activating effects. The results showed that except for pSos-SUT5,the other five bait vectors had no autonomous activation upon the yeast strain,which were suitable for the follow-up studies.
出处
《热带生物学报》
2010年第4期314-320,共7页
Journal of Tropical Biology
基金
国家自然科学基金(30760114)
农业部基本科研业务费(XJSYWFZX2008-16
XJSYWFZX2010-13)
公益性行业(农业)科研专项(nyhyzx07-033-6)
