水稻OsRINGzf基因的功能研究
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摘要
水稻是世界上最主要的粮食作物之一,生产消耗水量非常大,这与近年来全球范围内日益严重的水资源短缺形成了尖锐的矛盾,因此发展抗旱的栽培稻被认为是节约水资源的有效手段。根据抗旱基因作用方式的不同,将抗旱基因分成两类:一类是功能基因,其编码产物对水稻抗旱性直接起保护作用;一类是调节基因,其编码产物在信号转导和基因表达过程中起调节功能基因的作用。本研究的目的基因OsRINGzf是基于遗传定位、生物信息学和表达谱分析的分析方法获得的,在水稻第4染色体QTL区间,预测为E3连接酶。
OsRINGzf基因全长cDNA序列长度为1197bp,其编码框ORF长度为564bp,编码一个187个氨基酸的蛋白质。该序列C端有一个保守的RING(really interestingnew gene)结构域,N端有三个转膜(TM)区。预测为RING-H2(C3H2C3)型E3连接酶。E3连接酶可以识别特异蛋白,并通过26S蛋白酶体途径降解。本研究的主要内容:
1.建立超表达载体、抑制表达载体、GFP融合表达载体和启动子:GFP载体,转化水稻和拟南芥,获得T0代植株。其中,超表达载体转化的水稻阳性植株已收获T1代种子。对水稻转基因种子进行发芽实验,结果显示转基因材料的相对发芽率和平均株高都比对照中花11低。
2.通过TILLING、测序等分析方法,对OsRINGzf基因在野生稻、早稻核心种质、珍汕97B和IRAT109中的序列进行对比分析。与IRAT109中该基因相比,野生稻中OsRINGzf基因序列变化可分为5种类型,其中S1061和100916中OsRINGzf基因因为一个碱基的插入突变,致使RING结构域发生变化,可能导致该基因丧失E3连接酶活性;旱稻核心种质中,凝胶电泳图显示有3中不同的序列类型,通过测序分析发现,在ORF内变化的有两种类型,其中在ORF变化相同的类型,彼此间在内含子序列上有变化。
3.通过RT-PCR分析在正常生长情况下和旱胁迫处理的情况下,OsRINGzf基因的表达差异。在IRAT109的苗期、分蘖盛期和孕穗期,分别进行干旱胁迫处理,结果发现:在苗期和分蘖盛期,OsRINGzf基因的表达都呈现上升趋势,在苗期的表达变化最为明显。在孕穗期,OsRINGzf基因的表达开始呈上升趋势,但是在4-6天下降,表达几乎为0,在胁迫7天的时候表达又上升,且表达量达到了最大。复水后,三个时期中的OsRINGzf基因表达都呈下降趋势,且低于对照时期。在植株正常生长情况下,转基因植株中OsRINGzf基因的表达量显著高于对照中花11。对植株进行20%PEG 6000模拟干旱处理144小时后,结果显示,在转基因植株中,OsRINGzf基因的表达量呈下降趋势,复水之后OsRINGzf基因的达量并无太大的变化。部分转基因株系在复水之后长势好于中花11对照,显示了较好的复原抗旱性。
4.构建原核表达载体PET-32a+,通过IPTG诱导,从大肠杆菌中提取OsRINGzf基因的表达蛋白,通过SDS-PAGE电泳,分离获得了OsRINGzf基因的蛋白,优化建立了该基因的表达和纯化体系。
Rice (Oryza sativa L) is one of the most main grain crops in the world. It needs the very large consumption of water, which has formed a sharp contradiction with more and more severe shortage of global water resources in recent years. Therefore, development of drought resistance of cultivation rice is considered as the effective measure of water resources conservation. According to their mode of action, drought resistance genes can be divided into two groups: one is functional gene, which encoding products can directly protect drought resistance of rice; one is regulatory gene, which encoding products can regulate functional gene in gene expression and signal transduction pathways. Based on the heredity localization, bioinformatics and expression spectrum analysis, we got OsRINGzf gene located on chromosome IV in rice, which was speculated to be an E3 ligase.
The full-length cDNA of OsRINGzf is 1197 bp and the opening reading frame (ORF) is 564 bp, which codes a 187 amino acids protein. The C terminus of the sequence contains a conserved RING (really interesting new gene) domain and the N terminus has three transmembranes (TM) domains. It is speculated to be E3 ligase of RING-H2 (C3H2C3) type. The E3 ligase can recognize specific protein and degenerate it by the 26S proteasome pathway. The main contents of this study are here:
1. Construct over-expression vector, Inhibitor expression vector, GFP fusion expression vector and Promoter: GFP fusion, transformed into rice and Arabidopsis, got TO generation plants. Now, we got the seeds of T1 generation transgenic positive plants through transformed over expression vector into rice. Seeds germination test of transgenic plants, the results suggest that the average height and germination rate of the transgenic plants are lower than those of the zhonghua 11 control.
2. Different sequences analysis of OsRINGzf in wild rice, upland rice core collection, Zhenshan 97B, and IRAT109 by TILLING, sequencing and other analytical methods. Compared to IRAT109, five types are classified by different sequences of the OsRINGzf gene in wild rice. Because of the OsRINGzf gene in S1061 and 100916 plant have a base insert mutation, which changes RING domain, maybe cause the gene E3 ubiqiutin ligase activity abolished; Gel electrophoresis indicated three different sequences type in upland rice core collection. Through the sequencing analysis, the ORF domain including two different types, and in the same ORF type, the difference occurred within intron.
3. Analysis of the different expression of OsRINGzf gene by RT-PCR under the condition of normal and drought stress treatment. Treated with IRAT109 drought stress at seedling, vigorous division and booting stage. The results suggest that, at seedling and vigorous division stage, OsRINGzf gene expressions show an upward trend and the most obvious change occur at seedling stage. At booting stage, OsRINGzf gene expression shows an upward trend. But at 4-6th days, the expression almost disappeared. At the seventh day, the increase change is largest. After rehydration, OsRINGzf gene expressions show a downward trend at three stages and lower than control. Compared to zhonghua11 control, OsRINGzf gene expressions are markedly increased under normal situation in transgenic plants. Treated with transgenic plants 20% PEG 6000 144 hours, the results suggest that OsRINGzf gene expressions show a downward trend in transgenic plants and no obvious change after rehydration. Some trangenic plants are better than zhonghua 11 control at rehydration, showed the better recovery of drought resistance.
4. Construct prokaryotic expression vector PET-32a+ of OsRINGzf and extracted the expression protein through IPTG induction. Through SDS-PAGE electrophoresis, isolate protein OsRINGzf gene, establish and optimize the gene expression and purification system.
OsRINGzf基因全长cDNA序列长度为1197bp,其编码框ORF长度为564bp,编码一个187个氨基酸的蛋白质。该序列C端有一个保守的RING(really interestingnew gene)结构域,N端有三个转膜(TM)区。预测为RING-H2(C3H2C3)型E3连接酶。E3连接酶可以识别特异蛋白,并通过26S蛋白酶体途径降解。本研究的主要内容:
1.建立超表达载体、抑制表达载体、GFP融合表达载体和启动子:GFP载体,转化水稻和拟南芥,获得T0代植株。其中,超表达载体转化的水稻阳性植株已收获T1代种子。对水稻转基因种子进行发芽实验,结果显示转基因材料的相对发芽率和平均株高都比对照中花11低。
2.通过TILLING、测序等分析方法,对OsRINGzf基因在野生稻、早稻核心种质、珍汕97B和IRAT109中的序列进行对比分析。与IRAT109中该基因相比,野生稻中OsRINGzf基因序列变化可分为5种类型,其中S1061和100916中OsRINGzf基因因为一个碱基的插入突变,致使RING结构域发生变化,可能导致该基因丧失E3连接酶活性;旱稻核心种质中,凝胶电泳图显示有3中不同的序列类型,通过测序分析发现,在ORF内变化的有两种类型,其中在ORF变化相同的类型,彼此间在内含子序列上有变化。
3.通过RT-PCR分析在正常生长情况下和旱胁迫处理的情况下,OsRINGzf基因的表达差异。在IRAT109的苗期、分蘖盛期和孕穗期,分别进行干旱胁迫处理,结果发现:在苗期和分蘖盛期,OsRINGzf基因的表达都呈现上升趋势,在苗期的表达变化最为明显。在孕穗期,OsRINGzf基因的表达开始呈上升趋势,但是在4-6天下降,表达几乎为0,在胁迫7天的时候表达又上升,且表达量达到了最大。复水后,三个时期中的OsRINGzf基因表达都呈下降趋势,且低于对照时期。在植株正常生长情况下,转基因植株中OsRINGzf基因的表达量显著高于对照中花11。对植株进行20%PEG 6000模拟干旱处理144小时后,结果显示,在转基因植株中,OsRINGzf基因的表达量呈下降趋势,复水之后OsRINGzf基因的达量并无太大的变化。部分转基因株系在复水之后长势好于中花11对照,显示了较好的复原抗旱性。
4.构建原核表达载体PET-32a+,通过IPTG诱导,从大肠杆菌中提取OsRINGzf基因的表达蛋白,通过SDS-PAGE电泳,分离获得了OsRINGzf基因的蛋白,优化建立了该基因的表达和纯化体系。
Rice (Oryza sativa L) is one of the most main grain crops in the world. It needs the very large consumption of water, which has formed a sharp contradiction with more and more severe shortage of global water resources in recent years. Therefore, development of drought resistance of cultivation rice is considered as the effective measure of water resources conservation. According to their mode of action, drought resistance genes can be divided into two groups: one is functional gene, which encoding products can directly protect drought resistance of rice; one is regulatory gene, which encoding products can regulate functional gene in gene expression and signal transduction pathways. Based on the heredity localization, bioinformatics and expression spectrum analysis, we got OsRINGzf gene located on chromosome IV in rice, which was speculated to be an E3 ligase.
The full-length cDNA of OsRINGzf is 1197 bp and the opening reading frame (ORF) is 564 bp, which codes a 187 amino acids protein. The C terminus of the sequence contains a conserved RING (really interesting new gene) domain and the N terminus has three transmembranes (TM) domains. It is speculated to be E3 ligase of RING-H2 (C3H2C3) type. The E3 ligase can recognize specific protein and degenerate it by the 26S proteasome pathway. The main contents of this study are here:
1. Construct over-expression vector, Inhibitor expression vector, GFP fusion expression vector and Promoter: GFP fusion, transformed into rice and Arabidopsis, got TO generation plants. Now, we got the seeds of T1 generation transgenic positive plants through transformed over expression vector into rice. Seeds germination test of transgenic plants, the results suggest that the average height and germination rate of the transgenic plants are lower than those of the zhonghua 11 control.
2. Different sequences analysis of OsRINGzf in wild rice, upland rice core collection, Zhenshan 97B, and IRAT109 by TILLING, sequencing and other analytical methods. Compared to IRAT109, five types are classified by different sequences of the OsRINGzf gene in wild rice. Because of the OsRINGzf gene in S1061 and 100916 plant have a base insert mutation, which changes RING domain, maybe cause the gene E3 ubiqiutin ligase activity abolished; Gel electrophoresis indicated three different sequences type in upland rice core collection. Through the sequencing analysis, the ORF domain including two different types, and in the same ORF type, the difference occurred within intron.
3. Analysis of the different expression of OsRINGzf gene by RT-PCR under the condition of normal and drought stress treatment. Treated with IRAT109 drought stress at seedling, vigorous division and booting stage. The results suggest that, at seedling and vigorous division stage, OsRINGzf gene expressions show an upward trend and the most obvious change occur at seedling stage. At booting stage, OsRINGzf gene expression shows an upward trend. But at 4-6th days, the expression almost disappeared. At the seventh day, the increase change is largest. After rehydration, OsRINGzf gene expressions show a downward trend at three stages and lower than control. Compared to zhonghua11 control, OsRINGzf gene expressions are markedly increased under normal situation in transgenic plants. Treated with transgenic plants 20% PEG 6000 144 hours, the results suggest that OsRINGzf gene expressions show a downward trend in transgenic plants and no obvious change after rehydration. Some trangenic plants are better than zhonghua 11 control at rehydration, showed the better recovery of drought resistance.
4. Construct prokaryotic expression vector PET-32a+ of OsRINGzf and extracted the expression protein through IPTG induction. Through SDS-PAGE electrophoresis, isolate protein OsRINGzf gene, establish and optimize the gene expression and purification system.
引文
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