黄萎病菌诱导下海岛棉全长cDNA文库构建与抗病相关基因克隆
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摘要
黄萎病（Verticillium wilt）是影响世界棉花生产的主要病害之一。育种实践证明，培育抗病品种是控制棉花黄萎病的经济有效途径。研究证明海岛棉Pima90-53对黄萎病具有高度抗性；对其抗黄萎病相关基因进行克隆研究不仅有利于阐明品种抗病机制，而且对棉花抗黄萎病的分子育种具有重要价值。本研究以海岛棉Pima90-53为材料，构建其在黄萎病菌诱导下的根系cDNA文库，并依据文库克隆棉花抗黄萎病相关基因。主要结果如下：
1.构建了黄萎病菌诱导下的海岛棉Pima90-53根系的全长cDNA文库
采用SMART技术成功构建了黄萎病菌诱导下的Pima90-53根系全长cDNA文库，该文库基础库容量为1.28×106PFU，重组率94%，扩增后文库滴度大于1010PFU·mL-1，插入片段平均长度1.1kb。经随机测序共获得45条cDNA序列，利用Blastx程序进行同源分析，4个contigs与假定抗病相关基因同源性较高；3个contigs没有注释，可能为新基因。
2.利用文库筛选和RACE技术相结合，克隆了一个新的GbWRKY1基因
利用构建的cDNA文库，结合RACE技术，获得了一个新WRKY基因（GbWRKY1）（GenBank No. JF831361）。GbWRKY1基因cDNA全长1971bp，包括5′端非编码区271bp，3′端非编码区230bp，含有1个可能的polyA加尾信号：AATAAT；基因开放阅读框为1470bp，编码一个由489个氨基酸构成的多肽。
生物信息学分析发现GbWRKY1包括2高度保守的WRKY基本结构域和锌指结构，属于WRKY家族第Ⅰ类，与VvWRKY2、 AtWRKY4和AtWRKY3蛋白的同源性分别为62%、61%和59%；GbWRKY1属可溶性蛋白，无信号肽和跨膜结构域，包含N-糖基化位点、蛋白激酶C磷酸化位点、N端肉豆蔻酰化位点、酪蛋白激酶II磷酸化位点、依赖cAMP和cGMP蛋白激酶磷酸化位点等。
GbWRKY1基因含有3个内含子，具有有保守的GT-AG剪切位点，分别为599bp、81bp、325bp。克隆得到GbWRKY1的1762bp启动子，包含植物病原诱导性启动子调控元件（RAV1AAT、Box-W1、ARE、W-box、CGTCA-motif、ERE、 TGACG-motif）与非生物胁迫应答元件（HSE、 TC-rich repeats、WRKY71OS、WBOXNTERF3、MYCATERD1）。
构建了基因融合表达载体pCamE-GbWRKY1-GFP，基因枪转化洋葱内表皮细胞，结果显示转化pCamE-GbWRKY1-GFP表皮细胞的细胞核出现绿色荧光，GbWRKY1蛋白定位于细胞核。
3. Northern杂交分析了GbWRKY1在黄萎病菌接种后的表达模式，揭示了基因与棉花抗黄萎病的联系；实时定量PCR分析了GbWRKY1在不同激素处理下的表达模式，为解析基因作用机制提供了依据；构建了GbWRKY1超表达与RNAi载体，获得了转基因T3拟南芥
Northern杂交分析GbWRKY1表达模式发现，基因在Pima90-53的根、茎和叶组织均有表达；在受黄萎病菌、SA、MeJA和ACC的诱导后呈现不同的表达模式。在黄萎病菌诱导后，GbWRKY1基因在接种后出现持续高表达，在8h出现表达高峰，并持续高表达至接菌后12h。SA诱导后，其表达量在12h降到最低，24h基本恢复正常水平；MeJA诱导后，GbWRKY1表达量上升，在12h达到高峰，24h出现下降；ACC诱导12h，GbWRKY1表达呈现明显上升，并持续到24h。
构建了GbWRKY1基因超表达和反义载体pCamE-GbWRKY1、pCamE-iGbWRKY1，通过农杆菌介导法转化拟南芥，经潮霉素筛选和PCR检测，获得了GbWRKY1过表达转基因T3拟南芥。
Verticillium wilt is one of the most serious diseases which influence the cottonproduction in the world. Breeding for resistant cultivars has been proved the mosteffeetive way to control this disease. Previous researches indicated that，Pima90–53possessing resistance to Verticillium wilt genes. Cloning the resistance genes or therelated genes is important to understand disease resistance mechanism and molecularbreeding. In this study, a cDNA expression library of Pima90-53(G. barbadense)was constructed after inoculation with defoliating strain of Verticillium dahliae.cDNAlibrary screening and RACE technique were used, structure and expression of thetarget sequences were analysed in order to obtain disease-resistant related gene.Theresults are as follows:
1. Construction and characterization of full-length cDNA expression libraryfrom Pima90-53root induced by Verticillium dahliae
The SMART technology was used to construct a full-length cDNA Library ofPima90-53root tissue after inoculation with defoliating strain of Verticillium dahliae.The capacity of the primary cDNA library was1.28×106PFU, the titer of theamplified cDNA library was more than1010PFU·mL-1and the recombination ratiowas94%. The average insert size of the cDNA library was about1.1kb. Fourty-fivesequences were obtained. Blastx was used to homologue searching for thesesequences. Four ESTs sequences showed homologue to putative genes related todisease resistance, and three EST failed to show significant homology to any proteinsin the public databases, suggesting that they represent novel sequences.
2. A novel GbWRKY1gene was isolated by cDNA library screening and RACEtechnique
GbWRKY1gene was isolated by cDNA library screening and RACE (GenBankaccession No. JF831361). The1971bp GbWRKY1cDNA sequence contained a271bp5′-UTR, a230bp3′-UTR and a polyA polyadenylation signal，as well as an openread frame of1470bp encoding a489amino acids. The deduced GbWRKY1proteincontains two characteristic WRKY domains and two zinc finger motifs. It couldclearly be assigned to GroupⅠ. Alignment of the putative GbWRKY1protein withrelated sequences indicated that it had the highest sequence homology with theVvWRKY2(amino acid identity62%), AtWRKY4(61%) and AtWRKY3(59%). The putative GbWRKY1protein contained N-glycosylation site, Protein kinase Cphosphorylation site, N-myristoylation site, and so on. It was soluble protein and nosignal peptide and transmembrane domains.
In GbWRKY1, there were three introns which contained conservative GT-AGcleavage site with size of599bp,81bp and325bp, respectively. A1762bp fragmentof the GbWRKY1promoter was obtained using PCR from Pima90-53genome. Weidentified pathogen/elicitor-related elements including RAV1AAT, Box-W1, ARE,W-box, CGTCA-motif, ERE and TGACG, abiotic stress responsive element includingHSE, TC-rich repeats, WRKY71OS, WBOXNTERF3and MYCATERD1. It suggeststhat GbWRKY1may play a role in the response to environmental stress.
We constructed an expression vector containing the GbWRKY1-GFP fusiongene driven by the CaMV35S promoter. Afterwards, the35S::GbWRKY1-GFPvector and the control35S::GFP vector were directly introduced into onion epidermiscells by particle bombardment. Those transiently expressed GbWRKY1-GFP fusionproteins were found exclusively in the nucleus, whereas GFP occurred in both thenucleus and cytoplasm. Therefore, these results indicated that the GbWRKY1proteinis indeed localized to the nucleus.
3. Understanding expression patterns of GbWRKY1by inoculation andhormone treatment, the plant expression vector pCamE-GbWRKY1wasconstructed and successfully transferred into Arabidopsis thaliana
Quantitative real-time PCR showed that GbWRKY1expressed in all tissues ofroot, stem and leaf. The GbWRKY1gene expression pattern was different afterinoculation and hormone treatment. GbWRKY1was induced and reached to a peak at8h after inoculation with Verticillium dahliae and maintained at high level within12h. After SA treatment, the expression levels declined at8h, dropped to the nadir at12h, and then increased normal lever at24h. MeJA treatment induced the GbWRKY1expression quickly accumulated and reached maximum level at12h, the expressionlevels declined at24h. For ACC treatment, GbWRKY1expression increasedobviously at12h, and maintained at high level within24h.
The plant overexpression and antisense vector pCamE-GbWRKY1andpCamE-iGbWRKY1were constructed and then transferred into Arabidopsis thalianavia Agrobacterium-madiated method. Seven transgenic plants were verified byeffective selection for hygromycin B resistance and PCR.
1.构建了黄萎病菌诱导下的海岛棉Pima90-53根系的全长cDNA文库
采用SMART技术成功构建了黄萎病菌诱导下的Pima90-53根系全长cDNA文库，该文库基础库容量为1.28×106PFU，重组率94%，扩增后文库滴度大于1010PFU·mL-1，插入片段平均长度1.1kb。经随机测序共获得45条cDNA序列，利用Blastx程序进行同源分析，4个contigs与假定抗病相关基因同源性较高；3个contigs没有注释，可能为新基因。
2.利用文库筛选和RACE技术相结合，克隆了一个新的GbWRKY1基因
利用构建的cDNA文库，结合RACE技术，获得了一个新WRKY基因（GbWRKY1）（GenBank No. JF831361）。GbWRKY1基因cDNA全长1971bp，包括5′端非编码区271bp，3′端非编码区230bp，含有1个可能的polyA加尾信号：AATAAT；基因开放阅读框为1470bp，编码一个由489个氨基酸构成的多肽。
生物信息学分析发现GbWRKY1包括2高度保守的WRKY基本结构域和锌指结构，属于WRKY家族第Ⅰ类，与VvWRKY2、 AtWRKY4和AtWRKY3蛋白的同源性分别为62%、61%和59%；GbWRKY1属可溶性蛋白，无信号肽和跨膜结构域，包含N-糖基化位点、蛋白激酶C磷酸化位点、N端肉豆蔻酰化位点、酪蛋白激酶II磷酸化位点、依赖cAMP和cGMP蛋白激酶磷酸化位点等。
GbWRKY1基因含有3个内含子，具有有保守的GT-AG剪切位点，分别为599bp、81bp、325bp。克隆得到GbWRKY1的1762bp启动子，包含植物病原诱导性启动子调控元件（RAV1AAT、Box-W1、ARE、W-box、CGTCA-motif、ERE、 TGACG-motif）与非生物胁迫应答元件（HSE、 TC-rich repeats、WRKY71OS、WBOXNTERF3、MYCATERD1）。
构建了基因融合表达载体pCamE-GbWRKY1-GFP，基因枪转化洋葱内表皮细胞，结果显示转化pCamE-GbWRKY1-GFP表皮细胞的细胞核出现绿色荧光，GbWRKY1蛋白定位于细胞核。
3. Northern杂交分析了GbWRKY1在黄萎病菌接种后的表达模式，揭示了基因与棉花抗黄萎病的联系；实时定量PCR分析了GbWRKY1在不同激素处理下的表达模式，为解析基因作用机制提供了依据；构建了GbWRKY1超表达与RNAi载体，获得了转基因T3拟南芥
Northern杂交分析GbWRKY1表达模式发现，基因在Pima90-53的根、茎和叶组织均有表达；在受黄萎病菌、SA、MeJA和ACC的诱导后呈现不同的表达模式。在黄萎病菌诱导后，GbWRKY1基因在接种后出现持续高表达，在8h出现表达高峰，并持续高表达至接菌后12h。SA诱导后，其表达量在12h降到最低，24h基本恢复正常水平；MeJA诱导后，GbWRKY1表达量上升，在12h达到高峰，24h出现下降；ACC诱导12h，GbWRKY1表达呈现明显上升，并持续到24h。
构建了GbWRKY1基因超表达和反义载体pCamE-GbWRKY1、pCamE-iGbWRKY1，通过农杆菌介导法转化拟南芥，经潮霉素筛选和PCR检测，获得了GbWRKY1过表达转基因T3拟南芥。
Verticillium wilt is one of the most serious diseases which influence the cottonproduction in the world. Breeding for resistant cultivars has been proved the mosteffeetive way to control this disease. Previous researches indicated that，Pima90–53possessing resistance to Verticillium wilt genes. Cloning the resistance genes or therelated genes is important to understand disease resistance mechanism and molecularbreeding. In this study, a cDNA expression library of Pima90-53(G. barbadense)was constructed after inoculation with defoliating strain of Verticillium dahliae.cDNAlibrary screening and RACE technique were used, structure and expression of thetarget sequences were analysed in order to obtain disease-resistant related gene.Theresults are as follows:
1. Construction and characterization of full-length cDNA expression libraryfrom Pima90-53root induced by Verticillium dahliae
The SMART technology was used to construct a full-length cDNA Library ofPima90-53root tissue after inoculation with defoliating strain of Verticillium dahliae.The capacity of the primary cDNA library was1.28×106PFU, the titer of theamplified cDNA library was more than1010PFU·mL-1and the recombination ratiowas94%. The average insert size of the cDNA library was about1.1kb. Fourty-fivesequences were obtained. Blastx was used to homologue searching for thesesequences. Four ESTs sequences showed homologue to putative genes related todisease resistance, and three EST failed to show significant homology to any proteinsin the public databases, suggesting that they represent novel sequences.
2. A novel GbWRKY1gene was isolated by cDNA library screening and RACEtechnique
GbWRKY1gene was isolated by cDNA library screening and RACE (GenBankaccession No. JF831361). The1971bp GbWRKY1cDNA sequence contained a271bp5′-UTR, a230bp3′-UTR and a polyA polyadenylation signal，as well as an openread frame of1470bp encoding a489amino acids. The deduced GbWRKY1proteincontains two characteristic WRKY domains and two zinc finger motifs. It couldclearly be assigned to GroupⅠ. Alignment of the putative GbWRKY1protein withrelated sequences indicated that it had the highest sequence homology with theVvWRKY2(amino acid identity62%), AtWRKY4(61%) and AtWRKY3(59%). The putative GbWRKY1protein contained N-glycosylation site, Protein kinase Cphosphorylation site, N-myristoylation site, and so on. It was soluble protein and nosignal peptide and transmembrane domains.
In GbWRKY1, there were three introns which contained conservative GT-AGcleavage site with size of599bp,81bp and325bp, respectively. A1762bp fragmentof the GbWRKY1promoter was obtained using PCR from Pima90-53genome. Weidentified pathogen/elicitor-related elements including RAV1AAT, Box-W1, ARE,W-box, CGTCA-motif, ERE and TGACG, abiotic stress responsive element includingHSE, TC-rich repeats, WRKY71OS, WBOXNTERF3and MYCATERD1. It suggeststhat GbWRKY1may play a role in the response to environmental stress.
We constructed an expression vector containing the GbWRKY1-GFP fusiongene driven by the CaMV35S promoter. Afterwards, the35S::GbWRKY1-GFPvector and the control35S::GFP vector were directly introduced into onion epidermiscells by particle bombardment. Those transiently expressed GbWRKY1-GFP fusionproteins were found exclusively in the nucleus, whereas GFP occurred in both thenucleus and cytoplasm. Therefore, these results indicated that the GbWRKY1proteinis indeed localized to the nucleus.
3. Understanding expression patterns of GbWRKY1by inoculation andhormone treatment, the plant expression vector pCamE-GbWRKY1wasconstructed and successfully transferred into Arabidopsis thaliana
Quantitative real-time PCR showed that GbWRKY1expressed in all tissues ofroot, stem and leaf. The GbWRKY1gene expression pattern was different afterinoculation and hormone treatment. GbWRKY1was induced and reached to a peak at8h after inoculation with Verticillium dahliae and maintained at high level within12h. After SA treatment, the expression levels declined at8h, dropped to the nadir at12h, and then increased normal lever at24h. MeJA treatment induced the GbWRKY1expression quickly accumulated and reached maximum level at12h, the expressionlevels declined at24h. For ACC treatment, GbWRKY1expression increasedobviously at12h, and maintained at high level within24h.
The plant overexpression and antisense vector pCamE-GbWRKY1andpCamE-iGbWRKY1were constructed and then transferred into Arabidopsis thalianavia Agrobacterium-madiated method. Seven transgenic plants were verified byeffective selection for hygromycin B resistance and PCR.
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