拟南芥NiNJA基因诱饵载体构建及酵母双杂互作筛选.doc

文档介绍：
拟南芥 NiNJA 基因诱饵载体构建及酵母双
杂互作筛选
訾亮,翟金玲,洪灏,马穗,黄惜**
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(海南大学生物科学技术研究所,海口 570228)
摘要:JAZ 蛋白是植物茉莉酸信号途经重要负调控因子,通过与 NiNJA 蛋白互作抑制茉莉
酸下游转录因子的转录活性,NiNJA 蛋白是衔接 JAZ 蛋白与下游基因之间重要的因子。为
了研究 NiNJA 调控的下游基因,本文首先通过 PCR 技术中扩增拟南芥全长 NiNJA 基因 cDNA
序列,构建 NiNJA 基因的诱饵载体 pGBKT7-NiNJA。然后将该载体转入酵母 Y2H Gold 感
受态,通过自转录激活验证实验发现诱饵载体 pGBKT7-NiNJA 在酵母双杂体系中没有发生
自转录激活。在此基础上,通过酵母双杂技术,从拟南芥“Mate & Plate™” Library 筛选与
NiNJA 互作的蛋白,最后进一步验证,获得若干个与 NiNJA 互作的蛋白,该结果为下一步
鉴定 NiNJA 的互作蛋白及茉莉酸的信号调控途径打下基础。
关键词:NiNJA 基因;pGBKT7 诱饵载体;酵母双杂;自转录激活验证
中图分类号:Q
Construction test of a bait vector of NiNJA gene in
Arabidopsis and yeast two-hybrid system interaction
screening
ZI Liang, ZHAI Jinling, HONG Hao, MA Sui, HUANG Xi
(Institute of Bio-Science and Technology,Hainan University, HaiKou 570228)
Abstract: JAZ proteins are important negative factors for jasmonic acid signaling pathways in
Arabidopsis thaliana, NiNJA interacted with JAZs and control the transcription activity of
JA-related transcription factors. NiNJA is a key factor that connected the JAZs and JA-related
transcription factors. In order to screen NiNJA interacted proteins, NiNJA full-length cDNA
sequence was PCR amplified inserted into pGBKT7 vector to construct a bait vector for yeast
two-hybrid system. Furthermore, the vector was transformed into Y2H Gold strain. The result of
autoactivation showed that pGBKT7-NiNJA vec