应用酵母双杂交系统筛选虾夷扇贝FOXL2的相互作用蛋白

Screening of Interacting Proteins of FOXL2in Scallop Patinopecten yessoensis by Yeast Two-Hybrid System

FOXL2是脊椎动物的雌性决定基因,在多种无脊椎动物中也被认为是雌性决定与分化的关键基因,但其在无脊椎动物中的作用机制目前尚不清晰。本文利用Clontech酵母双杂交系统筛查虾夷扇贝FOXL2的相互作用蛋白。研究首先构建诱饵质粒pGBKT7-FOXL2,并检测诱饵蛋白的毒性及自激活活性,结果显示诱饵蛋白对酵母菌株无毒,也无自激活活性。利用该诱饵质粒,本研究从虾夷扇贝酵母双杂交cDNA文库筛选得到17个阳性克隆,测序验证得到12个读码框正确的阳性克隆,它们来自4种蛋白:COP9信号复合体亚基5、钠/钾ATP酶β3、哺乳动物室管膜相关蛋白1和胰脂肪酶相关蛋白2。研究进一步将诱饵质粒与4种蛋白的猎物质粒进行共转化验证,结果均为阳性,表明这4种蛋白与虾夷扇贝FOXL2都可能存在相互作用。本文为研究FOXL2在无脊椎动物性别决定与分化中的作用提供了参考。

FOXL2, a female sex-determining gene in vertebrates, is also considered a key gene for female determination and differentiation in a variety of invertebrates. But how it works in invertebrates remains unclear. In this study, the interacting proteins of FOXL2 of scallop Patinopecten yessoensis were screened using Clontech yeast two-hybrid system. Bait plasmid pGBKT7-FOXL2 was constructed, and the toxicity and self-activation ability of the bait protein was detected. Results showed that bait protein was non-toxic to yeast strain and had no self-activating activity. A total of 17 positive clones were screened from yeast two-hybrid cDNA library of P. yessoensis . Among them, 12 were confirmed to contain correct open reading frame. These proteins showed high sequence similarity with the following four proteins: COP9 signalosome complex subunit 5, sodium/potassium-transporting ATPase subunit beta-3, mammalian ependymin-related protein 1-like and pancreatic lipase-related protein 2-like. Co-transformation of the bait plasmid with prey plasmids of the 4 proteins confirmed that these 4 proteins could interact with scallop FOXL2. This study provides a basis for studying the role of FOXL2 in the sex determination and differentiation of invertebrates.