辽宁碱蓬BADH启动子盐诱导功能元件鉴定及转录因子分离
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摘要
BADH（甜菜碱醛脱氢酶）是合成甜菜碱的关键酶，BADH基因受盐诱导表达。本实验室的前期工作发现辽宁碱蓬（Suaeda liaotungensis K.）BADH启动子-300～+62 bp区段具有启动子活性，是一个强的盐诱导表达启动子。本研究通过生物信息学分析找出BADH启动子中的盐诱导功能元件，通过凝胶阻滞分析对BADH启动子中的盐诱导功能元件进行鉴定，通过酵母单杂交实验，分离与BADH启动子中的盐诱导功能元件相互作用的转录因子。本研究旨在寻找该区段启动子中盐诱导功能元件及与其相互作用的转录因子，为盐诱导启动子功能元件及转录因子研究提供可靠的参考和依据。具体工作及结果如下：
1、利用Genomatix软件，对BADH启动子-300～+62 bp区段进行生物信息学分析，得到约200个具有不同功能的元件，从中挑选7个可能的抗盐功能元件：PB1（GT-1元件）、PB2（MYB元件）、PB3（NAC元件）、PB4（WRKY元件）、PB5（MYB元件）、PB6（MYB元件）、PB7（G-box/MYC/ABA元件）。
2、分别以这7个元件为核心序列设计探针，与辽宁碱蓬叶片核蛋白结合，进行凝胶阻滞分析，发现有5条探针具有阻滞信号，初步认为PB1（GT-1元件）、PB2（MYB元件）、PB3（NAC元件）、PB6（MYB元件）、PB7（G-box/MYC/ABA元件）是BADH启动子中的盐诱导功能元件。
3、从所获得的5个盐诱导功能元件中选取PB3（NAC元件）作为诱饵，将该元件的4串联体构建到酵母菌株中，合成辽宁碱蓬叶片cDNA，经酵母单杂交实验，筛选出阳性克隆，经测序获得了6条序列，序列比对分析发现，其中两条序列是干旱及冷诱导相关基因，可能是与PB3相互作用的转录因子。
Betaine aldehyde dehydrogenase (BADH) is the key enzyme for betaine synthesis, itsexpression is induced by salt. Our preliminary work found that BADH promoter fragment(-300bp to+62bp) from Suaeda liaotungensis K. possesses all the essential cis-acting elementsand is sufficient for NaCl induction. The purpose of this study is to find salt-inducedcis-acting elements in BADH promoter by bioinformatics analysis, to identify salt-inducedcis-acting elements by EMSA, to isolate the transcription factors interacting with thesalt-induced cis-acting elements by yeast one-hybrid system. This study will provide areliable reference and research basis for the study of salt induction promoter elements andtranscription factors. Specific work and results are as follows:
1. BADH promoter fragment (-300bp to+62bp) was analyzed using Genomatix software, andabout 200 different elements were obtained. From these elements, we selected sevenpossible salt-induced cis-acting elements. They are named as PB1 (GT-1), PB2 (MYB),PB3 (NAC), PB4 (WRKY), PB5 (MYB ), PB6 (MYB), PB7 (G-box/MYC/ABA).
2. 7 probes were designed using the selected cis-acting elements as the core sequence. Theinteraction between probes and nuclear proteins from Suaeda liaotungensis leaves wasdetected by EMSA, Blocking signals were found in 7 probes. It is concluded that PB1(GT-1 ), PB2 (MYB), PB3 (NAC), PB6 (MYB), PB7 (G-box/MYC/ABA) may be thesalt-induced cis-acting elements of BADH promoter.
3. Using PB3 (NAC) as bait, 2 transcription factors were isolated from the cDNA library ofSuaeda liaotungensis by yeast one-hybrid experiments. They are drought/cold induciblegenes. They may be the transcription factors interacting with PB3.
1、利用Genomatix软件，对BADH启动子-300～+62 bp区段进行生物信息学分析，得到约200个具有不同功能的元件，从中挑选7个可能的抗盐功能元件：PB1（GT-1元件）、PB2（MYB元件）、PB3（NAC元件）、PB4（WRKY元件）、PB5（MYB元件）、PB6（MYB元件）、PB7（G-box/MYC/ABA元件）。
2、分别以这7个元件为核心序列设计探针，与辽宁碱蓬叶片核蛋白结合，进行凝胶阻滞分析，发现有5条探针具有阻滞信号，初步认为PB1（GT-1元件）、PB2（MYB元件）、PB3（NAC元件）、PB6（MYB元件）、PB7（G-box/MYC/ABA元件）是BADH启动子中的盐诱导功能元件。
3、从所获得的5个盐诱导功能元件中选取PB3（NAC元件）作为诱饵，将该元件的4串联体构建到酵母菌株中，合成辽宁碱蓬叶片cDNA，经酵母单杂交实验，筛选出阳性克隆，经测序获得了6条序列，序列比对分析发现，其中两条序列是干旱及冷诱导相关基因，可能是与PB3相互作用的转录因子。
Betaine aldehyde dehydrogenase (BADH) is the key enzyme for betaine synthesis, itsexpression is induced by salt. Our preliminary work found that BADH promoter fragment(-300bp to+62bp) from Suaeda liaotungensis K. possesses all the essential cis-acting elementsand is sufficient for NaCl induction. The purpose of this study is to find salt-inducedcis-acting elements in BADH promoter by bioinformatics analysis, to identify salt-inducedcis-acting elements by EMSA, to isolate the transcription factors interacting with thesalt-induced cis-acting elements by yeast one-hybrid system. This study will provide areliable reference and research basis for the study of salt induction promoter elements andtranscription factors. Specific work and results are as follows:
1. BADH promoter fragment (-300bp to+62bp) was analyzed using Genomatix software, andabout 200 different elements were obtained. From these elements, we selected sevenpossible salt-induced cis-acting elements. They are named as PB1 (GT-1), PB2 (MYB),PB3 (NAC), PB4 (WRKY), PB5 (MYB ), PB6 (MYB), PB7 (G-box/MYC/ABA).
2. 7 probes were designed using the selected cis-acting elements as the core sequence. Theinteraction between probes and nuclear proteins from Suaeda liaotungensis leaves wasdetected by EMSA, Blocking signals were found in 7 probes. It is concluded that PB1(GT-1 ), PB2 (MYB), PB3 (NAC), PB6 (MYB), PB7 (G-box/MYC/ABA) may be thesalt-induced cis-acting elements of BADH promoter.
3. Using PB3 (NAC) as bait, 2 transcription factors were isolated from the cDNA library ofSuaeda liaotungensis by yeast one-hybrid experiments. They are drought/cold induciblegenes. They may be the transcription factors interacting with PB3.
引文
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