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CMO启动子盐诱导功能元件鉴定及转录因子分离
Identification of Salt-induced Cis-elements in CMO Promoter and Separation of Transcription Factors Interacting with the Cis-elements
【作者】 郭晋艳；
【导师】 李秋莉；
【作者基本信息】 辽宁师范大学 ， 植物学， 2011， 硕士
【摘要】 甜菜碱是植物体内的一种无毒渗透保护剂,它的积累使代谢中的许多重要酶类在渗透胁迫下能保持活性,保证植物正常生长。胆碱单加氧酶(CMO)是指导合成甜菜碱的关键酶。CMO基因的表达受其上游启动子及转录因子的调控,转录因子与启动子上的顺式作用元件相互作用,促进下游基因的表达。本文通过对CMO启动子盐诱导元件及与其结合的转录因子进行研究,分析CMO基因转录调控的机制。本论文通过凝胶迁移分析实验找到了CMO启动子区段中盐诱导功能元件,以盐诱导功能元件为诱饵,通过酵母单杂交实验分离得到了可能与之结合的转录因子,主要结果如下:1、用Genomatix软件对已知的辽宁碱蓬CMO启动子序列(-267～+128 bp)进行功能元件分析,发现在该序列上存在11个与非生物胁迫相关的顺式作用元件。从中选定了4个与盐诱导表达相关的元件:W-Box、Salt/drought responsive element、GT-1 cis-element和MYB元件用于凝胶迁移分析实验。2、以选定的4个元件为核心序列设计探针,与辽宁碱蓬叶片核蛋白结合,经凝胶阻滞分析,发现有2条探针具有阻滞信号,初步认为Salt/drought responsive elements和MYB元件是CMO启动子中盐诱导相关的功能元件。3、从获得的2个盐诱导功能元件中选取Salt/drought responsive element作为诱饵,将该元件的三串联体构建到酵母菌株中获得诱饵酵母菌株,同时构建辽宁碱蓬叶片cDNA文库,经酵母单杂交实验,筛选出阳性克隆,经测序及序列比对分析,获得了1个与Salt/drought responsive element相互作用的转录因子,该转录因子可能是PAPA-1- like family protein / zinc finger (HIT) family protein。
【Abstract】 Betaine is a non-toxic penetration of protective agents in plants. Its accumulation makes many important enzymes of metabolism keep activity under osmotic stress, and protect the normal growth of plants. Choline monooxygenase (CMO) is the key enzyme for betaine synthesis. CMO gene expression is regulated by upstream promoter and transcription factors. The interaction between transcription factors and promoter cis-acting elements promote the expression of genes downstream. In this paper, the salt-related cis-acting elements in CMO promoter and their binding transcription factors were studied, and transcriptional regulation mechanism of CMO gene was analyzed.This thesis finds salt-related cis-acting elements in CMO promoter by electrophoretic mobility shift assay(EMSA), and transcription factors binding to the cis-acting elements were isolated by yeast one-hybrid experiments. The main results are as follows:1. CMO promoter sequence (-267～+128 bp) were analyzed using Genomatix Online software. 11 abiotic stress-related cis-acting elements were found in this sequence. 4 cis-acting elements:MYB element, W-box, GT-box and Salt/drought responsive element, were selected for EMSA.2. 4 probes were designed using the selected cis-acting elements as the core sequence. The interaction between probes and nuclear proteins from Suaeda liaotungensis leaves was detected by EMSA, Blocking signals were found in 2 probes. It is concluded that MYB element and Salt/drought responsive element may be the salt-induced cis-acting elements in CMO promoter.3. Using the Salt/drought responsive element as bait, a transcription factor was isolated from the cDNA library of Suaeda liaotungensis by yeast one-hybrid experiments, It is a PAPA-1-like family protein / zinc finger (HIT) family protein.