小麦与叶锈菌互作体系中TaHIR2基因克隆及其特性分析
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摘要
过敏性反应(Hypersensitive Response,HR)是植物抗病机制中最常见的一种抗病表现形式。过敏性诱导反应蛋白是参与植物HR过程的一类蛋白。目前在小麦上尚未见TaHIR2的报道。为明确TaHIR2及其所编码蛋白在小麦中的存在状况及其与小麦抗叶锈病性的关系,本研究以受叶锈菌侵染的小麦抗叶锈病近等基因系TcLr15为材料,利用PCR技术克隆获得小麦过敏性诱导反应蛋白2(TaHIR2)的cDNA及基因组DNA序列;利用实时定量PCR(qRT-PCR)技术对小麦与叶锈菌亲和及非亲和互作过程中TaHIR2的表达模式进行了分析;通过构建原核表达载体对该基因进行原核表达,产生的蛋白用于制备抗体,利用Western杂交技术对小麦中该基因所编码蛋白的表达情况进行检测;构建了含有TaHIR2基因的重组质粒,通过基因枪介导法分别对小麦品种TcLr15和Thatcher的成熟胚性愈伤组织进行了转化。主要内容包括以下几个方面:
1.小麦TaHIR2基因克隆及序列分析。以小麦抗叶锈病近等基因系TcLr15和非亲和叶锈菌株05-19-43②为材料,以接菌24 h、48 h和72 h小麦叶片总RNA的等量混合物为模板,通过RT-PCR技术获得小麦TaHIR2的cDNA序列。同时以TcLr15的基因组DNA为模板,通过PCR技术获得TaHIR2的DNA序列。
2.小麦TaHIR2时空表达模式分析。以接种非亲和叶锈菌05-19-43②和亲和叶锈菌05-5-137③的小麦TcLr15叶片为材料,应用qRT-PCR技术检测接种不同毒力叶锈菌后小麦叶片中TaHIR2的时间表达模式。结果表明TaHIR2受叶锈菌诱导18 h时表达量均上升,在接种36 h时达到最大,然后下降。但在非亲和组合中,24 h和36 h的表达量要明显高于亲和组合。以小麦TcLr15的幼根、幼茎、幼叶和种子为材料,应用qRT-PCR技术检测TaHIR2在这几种组织中的表达量差异。结果表明TaHIR2在小麦幼叶中的表达量最大。
3. TaHIR2蛋白在叶锈菌侵染不同时间点的小麦叶片中表达模式分析。将TaHIR2基因的编码区插入到表达载体pET-30(+)中,获得原核表达载体TaHIR2-pET-30a,重组质粒转化大肠杆菌表达菌株BL21(DE3)。对阳性菌株的蛋白表达条件进行优化,确定IPTG的最佳诱导浓度为0.3 mM,最佳诱导时间为7 h。表达的融合蛋白分子量为37 kDa左右,该蛋白存在于包涵体中。用Ni~(2+)-His亲和层析柱对蛋白进行纯化后,经SDS-PAGE电泳检测呈现单一条带。纯化的蛋白用于免疫兔子,制备抗体,当效价达到1:76800时,利用Western杂交检测抗体质量,并对小麦中TaHIR2蛋白的表达情况进行检测。杂交结果表明,在小麦中确实存在TaHIR2蛋白。对叶锈菌侵染不同时间点的小麦叶片中TaHIR2蛋白的表达模式进行分析。结果表明在非亲和组合中,TaHIR2蛋白的表达量在检测的时间进程中平稳增长,60 h时达到最大,而在亲和组合中,蛋白的表达量在96 h时达到最大。
4. TaHIR2基因植物高效表达载体构建及再生小麦幼苗获得。将TaHIR2基因的编码区定向插入到载体pAHC-25中,获得重组质粒TaHIR2-pAHC-25,利用基因枪介导法将该重组载体转化小麦品种TcLr15和Thatcher的成熟胚性愈伤组织,经分化筛选后获得再生的小麦幼苗。
本研究在克隆获得小麦TaHIR2 cDNA及基因组DNA序列基础上,利用实时定量、Western杂交及转基因等技术对TaHIR2基因进行系统研究,研究结果一方面丰富了小麦的基因资源,另一方面对于揭示该基因在小麦中的功能,同时为阐明HR的发生机制提供新的实验支持。
Hypersensitive response (HR) is one of the most common resistant styles in plant. Hypersensitive induced response protein is a protein superfamily involved in plant HR. TaHIR2 had not been reported in wheat till now. In order to understand the existence of TaHIR2 in wheat and the relationship between TaHIR2 with wheat resistant to leaf rust pathogen, wheat near isogenic line TcLr15 inoculated with different virulent leaf rust pathogen was used as the initial materials, TaHIR2 full length cDNA and genomic DNA sequences were amplified by PCR. Quantitative real-time PCR (qRT-PCR) was used to detect the expression profiles of TaHIR2 in the compatible and incompatible interactions between wheat and leaf rust pathogen. Prokaryotic expression vector with TaHIR2 gene was successfully constructed, and transformed to E. coli competent cells. The fusion protein produced by E. coli expression system was used to immune rabbit and produce antibody. The expression profiles of TaHIR protein in wheat leaves inoculated with different virulent leaf rust strains were detected by Western blotting. The recombinant plasmids TaHIR2-pAHC-25 were constructed and transformed into embryogenic callus of wheat cv. TcLr15 and Thatcher by particle bombardment respectively. Main contents were as followed:
1. Cloning and sequence analysis of wheat TaHIR2. Wheat near isogenic line TcLr15 and avirulent strain 05-19-43②were used as the initial material. Total RNA of wheat leaves at 24, 48 and 72 hours post inoculation (hpi) were mixed and used to synthesize first strand cDNA, and TaHIR2 cDNA and DNA were cloned by PCR using the cDNA and genomic DNA of TcLr15 as templates, respectively.
2. Temporal and spatial expression profile of wheat TaHIR2. Wheat cv. TcLr15 inoculated with avirulent strain 05-19-43②and virulent strain 05-5-137③were used as the initial materials, temporal expression profile of TaHIR2 in wheat leaves at 0, 6, 12, 18, 24, 36, 48, 60, 72, 96, 120 hpi were detected by qRT-PCR. The results showed that TaHIR2 transcripts were up-regulated when wheat was attacked by leaf rust pathogen at 18 hpi, and reached the peak at 36 hpi both in the compatible and incompatible combination, then declined. But more transcripts were accumulated in incompatible interaction than compatible interaction at 24 and 36 hpi. Furthermore, four different tissues, including young roots, young stems, young leaves and mature seeds, from wheat cv. TcLr15 were used as the initial materials. The accumulation of TaHIR2 transcripts in the 4 tissues was detected by qRT-PCR, respectively. The results showed more TaHIR2 transcripts existed in young leaves.
3. Expression profile of TaHIR2 protein in the wheat leaves inoculated with leaf rust at different time points. The prokaryotic expression vectors TaHIR2-pET-30a were successfully constructed after inserting open reading frame of TaHIR2 into pGEM-T easy vector, and then transformed into E.coli BL21 (DE3). After optimization for the producing conditions of target protein, the optimal IPTG concentration was 0.3 mM, the optimal inducing time was 7 h. The molecular weight of the fusion protein was about 37 kDa. The fusion protein expressed in inclusion body. A single band was detected by SDS-PAGE after the fusion protein was purified by Ni~(2+)-His affinity columns. The purified protein was used to immune rabbit to produce polylclonal antibody. When potence of the antibody reached to 1:76800, the antibody was isolated and used to detect the specificity by Western blotting. The result showed that the protein corresponding to TaHIR2 was existed in wheat. The expression profiles of TaHIR2 protein were detected by Western blotting, and the results showed the accumulation of TaHIR2 increased steadily from 0 to 96 hpi in the incompatible combination, the peak occurred at 60 hpi, while the accumulation of TaHIR2 reached the peak at 96 hpi in the compatible combination.
4. Construction of the plant expression vectors with TaHIR2 and obtaining of the regenerate wheat seedlings. The transgene vector TaHIR2-pAHC-25 was successfully constructed after inserting TaHIR2 open reading frame into plant expression vector pAHC-25, then transformed into the embryogenic callus of the wheat cv. TcLr15 and Thatcher by particle bombardment, the regenerate wheat seedlings were obtained after series of selective cultivation.
Based on cloning of TaHIR2 cDNA and DNA sequences, series of analyses including qRT-PCR, western blotting and particle bombardment were carried out in this research. The results not only increase the understanding of wheat gene resources, but also make clear the functions of TaHIR2 in wheat, and supply new experimental supports for HR mechanism.
1.小麦TaHIR2基因克隆及序列分析。以小麦抗叶锈病近等基因系TcLr15和非亲和叶锈菌株05-19-43②为材料,以接菌24 h、48 h和72 h小麦叶片总RNA的等量混合物为模板,通过RT-PCR技术获得小麦TaHIR2的cDNA序列。同时以TcLr15的基因组DNA为模板,通过PCR技术获得TaHIR2的DNA序列。
2.小麦TaHIR2时空表达模式分析。以接种非亲和叶锈菌05-19-43②和亲和叶锈菌05-5-137③的小麦TcLr15叶片为材料,应用qRT-PCR技术检测接种不同毒力叶锈菌后小麦叶片中TaHIR2的时间表达模式。结果表明TaHIR2受叶锈菌诱导18 h时表达量均上升,在接种36 h时达到最大,然后下降。但在非亲和组合中,24 h和36 h的表达量要明显高于亲和组合。以小麦TcLr15的幼根、幼茎、幼叶和种子为材料,应用qRT-PCR技术检测TaHIR2在这几种组织中的表达量差异。结果表明TaHIR2在小麦幼叶中的表达量最大。
3. TaHIR2蛋白在叶锈菌侵染不同时间点的小麦叶片中表达模式分析。将TaHIR2基因的编码区插入到表达载体pET-30(+)中,获得原核表达载体TaHIR2-pET-30a,重组质粒转化大肠杆菌表达菌株BL21(DE3)。对阳性菌株的蛋白表达条件进行优化,确定IPTG的最佳诱导浓度为0.3 mM,最佳诱导时间为7 h。表达的融合蛋白分子量为37 kDa左右,该蛋白存在于包涵体中。用Ni~(2+)-His亲和层析柱对蛋白进行纯化后,经SDS-PAGE电泳检测呈现单一条带。纯化的蛋白用于免疫兔子,制备抗体,当效价达到1:76800时,利用Western杂交检测抗体质量,并对小麦中TaHIR2蛋白的表达情况进行检测。杂交结果表明,在小麦中确实存在TaHIR2蛋白。对叶锈菌侵染不同时间点的小麦叶片中TaHIR2蛋白的表达模式进行分析。结果表明在非亲和组合中,TaHIR2蛋白的表达量在检测的时间进程中平稳增长,60 h时达到最大,而在亲和组合中,蛋白的表达量在96 h时达到最大。
4. TaHIR2基因植物高效表达载体构建及再生小麦幼苗获得。将TaHIR2基因的编码区定向插入到载体pAHC-25中,获得重组质粒TaHIR2-pAHC-25,利用基因枪介导法将该重组载体转化小麦品种TcLr15和Thatcher的成熟胚性愈伤组织,经分化筛选后获得再生的小麦幼苗。
本研究在克隆获得小麦TaHIR2 cDNA及基因组DNA序列基础上,利用实时定量、Western杂交及转基因等技术对TaHIR2基因进行系统研究,研究结果一方面丰富了小麦的基因资源,另一方面对于揭示该基因在小麦中的功能,同时为阐明HR的发生机制提供新的实验支持。
Hypersensitive response (HR) is one of the most common resistant styles in plant. Hypersensitive induced response protein is a protein superfamily involved in plant HR. TaHIR2 had not been reported in wheat till now. In order to understand the existence of TaHIR2 in wheat and the relationship between TaHIR2 with wheat resistant to leaf rust pathogen, wheat near isogenic line TcLr15 inoculated with different virulent leaf rust pathogen was used as the initial materials, TaHIR2 full length cDNA and genomic DNA sequences were amplified by PCR. Quantitative real-time PCR (qRT-PCR) was used to detect the expression profiles of TaHIR2 in the compatible and incompatible interactions between wheat and leaf rust pathogen. Prokaryotic expression vector with TaHIR2 gene was successfully constructed, and transformed to E. coli competent cells. The fusion protein produced by E. coli expression system was used to immune rabbit and produce antibody. The expression profiles of TaHIR protein in wheat leaves inoculated with different virulent leaf rust strains were detected by Western blotting. The recombinant plasmids TaHIR2-pAHC-25 were constructed and transformed into embryogenic callus of wheat cv. TcLr15 and Thatcher by particle bombardment respectively. Main contents were as followed:
1. Cloning and sequence analysis of wheat TaHIR2. Wheat near isogenic line TcLr15 and avirulent strain 05-19-43②were used as the initial material. Total RNA of wheat leaves at 24, 48 and 72 hours post inoculation (hpi) were mixed and used to synthesize first strand cDNA, and TaHIR2 cDNA and DNA were cloned by PCR using the cDNA and genomic DNA of TcLr15 as templates, respectively.
2. Temporal and spatial expression profile of wheat TaHIR2. Wheat cv. TcLr15 inoculated with avirulent strain 05-19-43②and virulent strain 05-5-137③were used as the initial materials, temporal expression profile of TaHIR2 in wheat leaves at 0, 6, 12, 18, 24, 36, 48, 60, 72, 96, 120 hpi were detected by qRT-PCR. The results showed that TaHIR2 transcripts were up-regulated when wheat was attacked by leaf rust pathogen at 18 hpi, and reached the peak at 36 hpi both in the compatible and incompatible combination, then declined. But more transcripts were accumulated in incompatible interaction than compatible interaction at 24 and 36 hpi. Furthermore, four different tissues, including young roots, young stems, young leaves and mature seeds, from wheat cv. TcLr15 were used as the initial materials. The accumulation of TaHIR2 transcripts in the 4 tissues was detected by qRT-PCR, respectively. The results showed more TaHIR2 transcripts existed in young leaves.
3. Expression profile of TaHIR2 protein in the wheat leaves inoculated with leaf rust at different time points. The prokaryotic expression vectors TaHIR2-pET-30a were successfully constructed after inserting open reading frame of TaHIR2 into pGEM-T easy vector, and then transformed into E.coli BL21 (DE3). After optimization for the producing conditions of target protein, the optimal IPTG concentration was 0.3 mM, the optimal inducing time was 7 h. The molecular weight of the fusion protein was about 37 kDa. The fusion protein expressed in inclusion body. A single band was detected by SDS-PAGE after the fusion protein was purified by Ni~(2+)-His affinity columns. The purified protein was used to immune rabbit to produce polylclonal antibody. When potence of the antibody reached to 1:76800, the antibody was isolated and used to detect the specificity by Western blotting. The result showed that the protein corresponding to TaHIR2 was existed in wheat. The expression profiles of TaHIR2 protein were detected by Western blotting, and the results showed the accumulation of TaHIR2 increased steadily from 0 to 96 hpi in the incompatible combination, the peak occurred at 60 hpi, while the accumulation of TaHIR2 reached the peak at 96 hpi in the compatible combination.
4. Construction of the plant expression vectors with TaHIR2 and obtaining of the regenerate wheat seedlings. The transgene vector TaHIR2-pAHC-25 was successfully constructed after inserting TaHIR2 open reading frame into plant expression vector pAHC-25, then transformed into the embryogenic callus of the wheat cv. TcLr15 and Thatcher by particle bombardment, the regenerate wheat seedlings were obtained after series of selective cultivation.
Based on cloning of TaHIR2 cDNA and DNA sequences, series of analyses including qRT-PCR, western blotting and particle bombardment were carried out in this research. The results not only increase the understanding of wheat gene resources, but also make clear the functions of TaHIR2 in wheat, and supply new experimental supports for HR mechanism.
引文
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