中国华东葡萄抗白粉病新基因原核表达与抗体制备
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摘要
葡萄白粉病是由葡萄白粉病菌[ Uncinula necator (Schw.) Burr.]引起的一种世界性的真菌病害。中国是葡萄属植物最重要的原产地之一,中国野生葡萄蕴藏丰富的抗病种质资源。本实验室研究人员通过mRNA差异显示技术(mRNA differential display reverse transcription-PCR , DDRT-PCR)及cDNA末端快速扩增技术(Rapid amplification of cDNA ends, RACE),从高抗白粉病的华东葡萄白河-35-1(Vitis pseudoreticulata)中克隆分离得到一条抗白粉病相关的新基因GLOXrg的cDNA全长序列。
本研究利用生物信息学的常用工具及软件初步分析预测了该基因编码蛋白的功能及理化性质,将该新基因构建原核融合表达载体,运用体外表达技术获得其体外表达产物融合蛋白,并对其进行了纯化研究,为该基因体外功能的进一步验证提供依据。同时将获得的融合蛋白作抗原制备多克隆抗体,为目的基因产物在植株体内的定位研究、表达量的时空变化研究以及转基因植物的筛选和检测奠定基础。取得的主要结果如下:
1新基因GLOXrg编码蛋白的物理化学性质与功能预测结果显示,其可能是一个定位于细胞膜上的催化醛类的氧化而产生H2O2的乙二醛氧化酶,但其编码蛋白的分子量56 kDa比已知真菌上乙二醛氧化酶分子量68kDa小。通过对H2O2抗病作用的分析,GLOXrg基因可能在葡萄抗白粉病的过程中起着重要的作用。
2根据已知中国野生葡萄抗白粉病相关新基因GLOXrg基因序列,设计一对特异PCR引物,通过PCR方法,以华东葡萄白河-35-1(V. pseudoreticulata )5’RACE cDNA第一链为模板,扩增出一条约1572 bp的DNA条带,经克隆测序,结果显示所获片段含有实际大小为1572 bp的完整阅读框,与已知中国野生葡萄GLOXrg基因序列完全一致。将目的基因分别构建重组原核表达载体pGEX-GLOXrg,经双酶切鉴定,目的基因与表达载体均正确连接,重组表达载体构建成功。
3重组原核表达载体pGEX- GLOXrg转入大肠杆菌E.coli BL21(coden plus)中,经诱导培养后,重组菌特异性表达了在SDS-PAGE上显示大小为83 kDa的融合蛋白,说明GLOXrg基因全长在E.coli BL21(coden plus)中得到表达。通过诱导剂IPTG不同浓度诱导对照、不同诱导培养时间对照以及不同诱导培养温度对照,确定了最佳表达条件(37℃,0.1 mM IPTG诱导表达5 h)。
4以包涵体形式表达的GST-GLOXrg融合蛋白能够通过变性溶解、梯度透析方法复性,复性蛋白经GST亲和介质进行富集,SDS-PAGE结果显示获得了很好的纯化蛋白,
Powdery mildew, caused by Uncinula necator (Schw.) Burr., is the most ubiquitously damaging fungal disease of grapevines worldwide. China is one of the major centers of origin of Vitis species with the potential as sources of disease resistance. In our lab, by mRNA DDRT-PCR (mRNA differential display reverse transcription-PCR ) and RACE (Rapid amplification of cDNA ends ) , the novel GLOXrg gene of Chinese wild Vitis was cloned from Vitis pseudoreticulata Baihe-35-1, which has a high resistance to Uncinula necator.
In our research, the physical and chemical character and the function of GLOXrg were predicted by the usual tools and softwares of bioinformatics. GLOXrg was cloned as fusion with GST, and expressed, and the fusion protein was purified, which provide the excellent tools for detecting further its function of resistance to Uncinula necator. Fusion protein GST-GLOXrg was isolated and used to raise the polyclonal anti-(GST-GLOXrg) in rabbits. These offer powful base for the research of the specific features of GLOXrg spatial and temporal expression and the detecting of transgenic grapevine plants. The research results are as follows:
1. The prediction results of physical and chemical character and function of the induced protein of GLOXrg shows that GLOXrg was probably glyoxal oxidase.which catalyzes the oxidation of a number of aldehyde and a-hydroxy carbonyl compounds, reducing O2 to H2O2 in the process.But molecular weight of the induced protein of GLOXrg was smaller than knowed glyoxal oxidase of fungi. By the analysis of the disease-resistant H2O2, GLOXrg probably play a crucial role in the resistance to Uncinula necator.
2. Based on the cDNA full sequence of GLOXrg gene of Vitis pseudoreticulata, we designed a pair of specific primers, and obtained DNA band of about 1572 bp length by PCR from the first strain of 5’RACE cDNA of Vitis pseudoreticulata Baihe-35-1. The sequencing result shows that the factual length of the open reading frames is 1572 bp, and the sequence is
本研究利用生物信息学的常用工具及软件初步分析预测了该基因编码蛋白的功能及理化性质,将该新基因构建原核融合表达载体,运用体外表达技术获得其体外表达产物融合蛋白,并对其进行了纯化研究,为该基因体外功能的进一步验证提供依据。同时将获得的融合蛋白作抗原制备多克隆抗体,为目的基因产物在植株体内的定位研究、表达量的时空变化研究以及转基因植物的筛选和检测奠定基础。取得的主要结果如下:
1新基因GLOXrg编码蛋白的物理化学性质与功能预测结果显示,其可能是一个定位于细胞膜上的催化醛类的氧化而产生H2O2的乙二醛氧化酶,但其编码蛋白的分子量56 kDa比已知真菌上乙二醛氧化酶分子量68kDa小。通过对H2O2抗病作用的分析,GLOXrg基因可能在葡萄抗白粉病的过程中起着重要的作用。
2根据已知中国野生葡萄抗白粉病相关新基因GLOXrg基因序列,设计一对特异PCR引物,通过PCR方法,以华东葡萄白河-35-1(V. pseudoreticulata )5’RACE cDNA第一链为模板,扩增出一条约1572 bp的DNA条带,经克隆测序,结果显示所获片段含有实际大小为1572 bp的完整阅读框,与已知中国野生葡萄GLOXrg基因序列完全一致。将目的基因分别构建重组原核表达载体pGEX-GLOXrg,经双酶切鉴定,目的基因与表达载体均正确连接,重组表达载体构建成功。
3重组原核表达载体pGEX- GLOXrg转入大肠杆菌E.coli BL21(coden plus)中,经诱导培养后,重组菌特异性表达了在SDS-PAGE上显示大小为83 kDa的融合蛋白,说明GLOXrg基因全长在E.coli BL21(coden plus)中得到表达。通过诱导剂IPTG不同浓度诱导对照、不同诱导培养时间对照以及不同诱导培养温度对照,确定了最佳表达条件(37℃,0.1 mM IPTG诱导表达5 h)。
4以包涵体形式表达的GST-GLOXrg融合蛋白能够通过变性溶解、梯度透析方法复性,复性蛋白经GST亲和介质进行富集,SDS-PAGE结果显示获得了很好的纯化蛋白,
Powdery mildew, caused by Uncinula necator (Schw.) Burr., is the most ubiquitously damaging fungal disease of grapevines worldwide. China is one of the major centers of origin of Vitis species with the potential as sources of disease resistance. In our lab, by mRNA DDRT-PCR (mRNA differential display reverse transcription-PCR ) and RACE (Rapid amplification of cDNA ends ) , the novel GLOXrg gene of Chinese wild Vitis was cloned from Vitis pseudoreticulata Baihe-35-1, which has a high resistance to Uncinula necator.
In our research, the physical and chemical character and the function of GLOXrg were predicted by the usual tools and softwares of bioinformatics. GLOXrg was cloned as fusion with GST, and expressed, and the fusion protein was purified, which provide the excellent tools for detecting further its function of resistance to Uncinula necator. Fusion protein GST-GLOXrg was isolated and used to raise the polyclonal anti-(GST-GLOXrg) in rabbits. These offer powful base for the research of the specific features of GLOXrg spatial and temporal expression and the detecting of transgenic grapevine plants. The research results are as follows:
1. The prediction results of physical and chemical character and function of the induced protein of GLOXrg shows that GLOXrg was probably glyoxal oxidase.which catalyzes the oxidation of a number of aldehyde and a-hydroxy carbonyl compounds, reducing O2 to H2O2 in the process.But molecular weight of the induced protein of GLOXrg was smaller than knowed glyoxal oxidase of fungi. By the analysis of the disease-resistant H2O2, GLOXrg probably play a crucial role in the resistance to Uncinula necator.
2. Based on the cDNA full sequence of GLOXrg gene of Vitis pseudoreticulata, we designed a pair of specific primers, and obtained DNA band of about 1572 bp length by PCR from the first strain of 5’RACE cDNA of Vitis pseudoreticulata Baihe-35-1. The sequencing result shows that the factual length of the open reading frames is 1572 bp, and the sequence is
引文
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