海岛棉枯萎病抗性相关基因的克隆及功能验证
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摘要
新疆是中国唯一的海岛棉种植基地。海岛棉比起陆地棉有着纤维长度、比强度、细度等方面优异的特性，是纺高支纱必不可缺少的原料，在国民经济中具有举足轻重的地位。然而海岛棉枯萎病的蔓延严重制约着其生产，影响了棉农种植海岛棉的积极性，使新疆海岛棉种植面积及产量都大幅度下降。棉花抗病新品种的选育和种植是解决这个难题的有效途径之一。培育抗病棉花新品种的关键是要研究海岛棉抗枯萎病的机理机抗病相关基因的作用，从而在分子水平上对海岛棉加以改良，获得具有高抗特性的棉花新品种。
本研究首先以定位到的与海岛棉枯萎病抗性基因紧密连锁的分子标记为基础，通过对功能标记位点的克隆，获得抗性相关基因的全序列；利用RT-PCR研究该基因的表达特性，并通过转化烟草进一步揭示该基因在枯萎病抗性中的作用，结果如下：
1.根据GenBank数据库上公布的抗病基因序列，设计出107对RGA引物，利用高抗枯萎病的海岛棉品系“06-146”、高感枯萎病的海岛棉品种新海14号及以杂交F_2群体中随机选取的180个单株为实验材料，利用RGA引物在亲本间筛选出来的12对引物在抗、感基因池中进行筛选，初步确定Gbrga14是与海岛棉枯萎病紧密连锁的分子标记，该分子标记与海岛棉枯萎病抗性的交换值为15.5%，与海岛棉抗枯萎病基因的遗传距离为15.92cM。查找连锁标记的来源发现，根据拟南芥RPP5基因设计的引物最多，有4对（占33.33%）。这12对具有多态性的RGA标记主要由3类组成，其中TIR-NBS-LRR类6对，占50%；NBS-LRR类5对，占41.67%； NBS类1对，占8.33%。
2.通过对Gbrga14在两亲本间扩增出来的位点进行回收测序，以该测序片段为探针，通过同源克隆的方法，分别克隆到两条抗性相关基因，命名为RGBCH和SGBCH。BLAST比对分析显示，RGBCH、SGBCH基因具有较高的基因多态性。生物信息学分析表明，α-螺旋均是两个蛋白二级结构的主要部分。ProtFun预测RGBCH蛋白的主要功能是参与能量代谢和生物合成辅助因子；SGBCH蛋白的主要功能是参与蛋白质翻译过程中的能量转换。
3.选取5份不同的海岛棉及陆地棉材料，经枯萎病诱导后，取不同的组织器官分别进行荧光定量PCR检测。利用特异性引物GBCH–Q对材料的荧光定量PCR分析可知：品系“06-146”目的基因在不同组织器官的表达量在4d达到峰值；新海14号目的基因在6d时出现一个最高值，随后降低到最初水平；中棉所35号、军棉1号和TM-1在诱导前和诱导后表达量均很微弱或者比较一致，基本维持在低水平表达；利用引物StVe-Q对材料的荧光定量PCR分析可知：品系“06-146”、新海14号、中棉所35号、军棉1号和TM-1的目的基因在病菌诱导条件下，没有随着诱导时间的变化而呈现表达变化状态。说明已克隆的RGBCH和SGBCH只在海岛棉材料中表达，是与枯萎病抗性相关的两个基因。
4.将RGBCH、SGBCH基因重组到pCAMBIA1301植物表达载体上，构建了海岛棉pCAMBIA1301-RGBCH、pCAMBIA1301-SGBCH的植物表达载体，同时利用农杆菌介导的方法将RGBCH、SGBCH基因整合到烟草基因组中，并获得T1代种子。对T1代转基因及非转基因烟草采用菌液浇根法（每种材料种植30株），转RGBCH基因植株的存活率为78%，叶片呈显黄化状态，，而转SGBCH基因大部分的叶片干枯直至死亡，植株的存活率为24%，非转基因植株几乎全部死亡。研究表明转RGBCH基因的转入提高了烟草的抗枯萎病病菌的能力。
Cotton is the main cash crops,which has a pivotal position in the national economy.Xinjiangcotton district,as the main raw cotton production base,which has an important role in the wholecountry.Fusarium Wilt is one of the main diseases of serious harm to the cotton production,breeding and planting new disease-resistant varieties of cotton is the most cost-effective only wayto solve this problem.Using traditional breeding methods to cultivate the new varieties needs toolong cycle,and it is unable to meet the urgent need of the production,so cloning of resistance geneand studying of the gene expression have great significance in breeding for disease resistance.
In this study,resistance to Fusarium wilt resistance to sea-island cotton tightly linkedmolecular markers sites,obtaining the sequence of resistance related genes;And then using theprocess of Fusarium Wilt pathogen infection of cotton resulting in the onset,in order to clear theabove gene expression level,further to explain the role of this gene in resistance to Fusarium Wilt.The results are as follows:
1. Resistance gene sequence published in the GenBank database,designed107pairs the RGAprimers,the use of high resistance to Fusarium wilt Island Cotton “06-146”highly susceptible toFusarium wilt Island Cotton Xinhai14andmiscellaneous home F2groups randomly select180plant materials,use of RGA primer material between parent screening out12pairs of primersmatter screening anti,sense gene pool, preliminary to determine Gbrga14island cotton wilt diseaseclosely linked to themolecular markers,molecular markers and the Sea Island cotton wilt diseaseresistance exchange values were15.5%, and Sea Island cotton resistance to Fusarium wilt genegenetic distance to15.92cm.Finding the source of the linked markers found,according to theprimers of the Arabidopsis thaliana the RPP5gene design,4(33.33%). This12pairs ofpolymorphic RGA markers mainly composed of three classes, which TIR-NBS-LRR class of sixpairs, accounting for50%; NBS-LRR class of five pairs, accounting for41.67%; NBS class1pair,accounting for8.33%.
2. The through on for recycling Gbrga14loci amplification between the two parentalsequencing,the sequencing fragment as a probe,by means of silico cloning method,obtaining twodisease-resistant gene, named into RGBCH and SGBCH,BLAST alignment analysis showed thatRGBCH、SGBCH possess a high genetic polymorphism.Bioinformatics analysis showed that theα-helix is the main part of the secondary structure of the two proteins.ProtFun forecasted the mainfunction of the RGBCH protein is participating energy metabolism and biosynthesizingcofactors;The main function of the SGBCH protein is participating energy conversion in theprocess of protein translation.
3. Induced by Fusarium oxysporum five different materials in different tissues and fluorescence quantitative PCR using two primers,respectively,the results show, the use of thespecific primers GBCH-Q material quantitative PCR analysis shows:“06-146”strainsthe targetgene expression in different tissues4d peak;Xinhai14target genes in6d there is a maximumvalue, and then decreased to the original level;Zhongmiansuo35,Junmian1cotton and TM-1inthe induction ofbefore and after the induction of expression of the volume is very weak orrelatively consistent,remaining at a low level of expression, quantitative PCR analysis of thematerial shows through the use of primers StVe-Q:lines “06-146”,Xinhai14,Zhongmiansuo35,Junmian1cotton and TM-1target gene in the bacteria-induced conditions,there is no change withthe induction time showing expression status.Description the cloned RGBCH、SGBCH onlyexpressed in Sea Island cotton material,two genes associated with resistance to Fusarium wilt.
4. RGBCH and SGBCH recombinating into pCAMBIA1301plant expression vector,constructing plant expression vector pCAMBIA1301-RGBCH and pCAMBIA1301-SGBCH,RGBCH and SGBCH gene integrated into the tobacco genome using of agrobacterium-mediatedmethod,obtaining T1generation seeds.T1transgenic and non-transgenic tobacco broth poured rootmethod(Each material of30planting),Turn RGBCH gene most of the leaves were yellowing, plantsurvival rate was78%,while the survival rate of to turn SGBCH transgenic plants to24%,most ofthe leaves dry death,non-transgenic plants almost all died.These results suggest that the genetransferred RGBCH tobacco resistance to Fusarium wilt bacteria.
本研究首先以定位到的与海岛棉枯萎病抗性基因紧密连锁的分子标记为基础，通过对功能标记位点的克隆，获得抗性相关基因的全序列；利用RT-PCR研究该基因的表达特性，并通过转化烟草进一步揭示该基因在枯萎病抗性中的作用，结果如下：
1.根据GenBank数据库上公布的抗病基因序列，设计出107对RGA引物，利用高抗枯萎病的海岛棉品系“06-146”、高感枯萎病的海岛棉品种新海14号及以杂交F_2群体中随机选取的180个单株为实验材料，利用RGA引物在亲本间筛选出来的12对引物在抗、感基因池中进行筛选，初步确定Gbrga14是与海岛棉枯萎病紧密连锁的分子标记，该分子标记与海岛棉枯萎病抗性的交换值为15.5%，与海岛棉抗枯萎病基因的遗传距离为15.92cM。查找连锁标记的来源发现，根据拟南芥RPP5基因设计的引物最多，有4对（占33.33%）。这12对具有多态性的RGA标记主要由3类组成，其中TIR-NBS-LRR类6对，占50%；NBS-LRR类5对，占41.67%； NBS类1对，占8.33%。
2.通过对Gbrga14在两亲本间扩增出来的位点进行回收测序，以该测序片段为探针，通过同源克隆的方法，分别克隆到两条抗性相关基因，命名为RGBCH和SGBCH。BLAST比对分析显示，RGBCH、SGBCH基因具有较高的基因多态性。生物信息学分析表明，α-螺旋均是两个蛋白二级结构的主要部分。ProtFun预测RGBCH蛋白的主要功能是参与能量代谢和生物合成辅助因子；SGBCH蛋白的主要功能是参与蛋白质翻译过程中的能量转换。
3.选取5份不同的海岛棉及陆地棉材料，经枯萎病诱导后，取不同的组织器官分别进行荧光定量PCR检测。利用特异性引物GBCH–Q对材料的荧光定量PCR分析可知：品系“06-146”目的基因在不同组织器官的表达量在4d达到峰值；新海14号目的基因在6d时出现一个最高值，随后降低到最初水平；中棉所35号、军棉1号和TM-1在诱导前和诱导后表达量均很微弱或者比较一致，基本维持在低水平表达；利用引物StVe-Q对材料的荧光定量PCR分析可知：品系“06-146”、新海14号、中棉所35号、军棉1号和TM-1的目的基因在病菌诱导条件下，没有随着诱导时间的变化而呈现表达变化状态。说明已克隆的RGBCH和SGBCH只在海岛棉材料中表达，是与枯萎病抗性相关的两个基因。
4.将RGBCH、SGBCH基因重组到pCAMBIA1301植物表达载体上，构建了海岛棉pCAMBIA1301-RGBCH、pCAMBIA1301-SGBCH的植物表达载体，同时利用农杆菌介导的方法将RGBCH、SGBCH基因整合到烟草基因组中，并获得T1代种子。对T1代转基因及非转基因烟草采用菌液浇根法（每种材料种植30株），转RGBCH基因植株的存活率为78%，叶片呈显黄化状态，，而转SGBCH基因大部分的叶片干枯直至死亡，植株的存活率为24%，非转基因植株几乎全部死亡。研究表明转RGBCH基因的转入提高了烟草的抗枯萎病病菌的能力。
Cotton is the main cash crops,which has a pivotal position in the national economy.Xinjiangcotton district,as the main raw cotton production base,which has an important role in the wholecountry.Fusarium Wilt is one of the main diseases of serious harm to the cotton production,breeding and planting new disease-resistant varieties of cotton is the most cost-effective only wayto solve this problem.Using traditional breeding methods to cultivate the new varieties needs toolong cycle,and it is unable to meet the urgent need of the production,so cloning of resistance geneand studying of the gene expression have great significance in breeding for disease resistance.
In this study,resistance to Fusarium wilt resistance to sea-island cotton tightly linkedmolecular markers sites,obtaining the sequence of resistance related genes;And then using theprocess of Fusarium Wilt pathogen infection of cotton resulting in the onset,in order to clear theabove gene expression level,further to explain the role of this gene in resistance to Fusarium Wilt.The results are as follows:
1. Resistance gene sequence published in the GenBank database,designed107pairs the RGAprimers,the use of high resistance to Fusarium wilt Island Cotton “06-146”highly susceptible toFusarium wilt Island Cotton Xinhai14andmiscellaneous home F2groups randomly select180plant materials,use of RGA primer material between parent screening out12pairs of primersmatter screening anti,sense gene pool, preliminary to determine Gbrga14island cotton wilt diseaseclosely linked to themolecular markers,molecular markers and the Sea Island cotton wilt diseaseresistance exchange values were15.5%, and Sea Island cotton resistance to Fusarium wilt genegenetic distance to15.92cm.Finding the source of the linked markers found,according to theprimers of the Arabidopsis thaliana the RPP5gene design,4(33.33%). This12pairs ofpolymorphic RGA markers mainly composed of three classes, which TIR-NBS-LRR class of sixpairs, accounting for50%; NBS-LRR class of five pairs, accounting for41.67%; NBS class1pair,accounting for8.33%.
2. The through on for recycling Gbrga14loci amplification between the two parentalsequencing,the sequencing fragment as a probe,by means of silico cloning method,obtaining twodisease-resistant gene, named into RGBCH and SGBCH,BLAST alignment analysis showed thatRGBCH、SGBCH possess a high genetic polymorphism.Bioinformatics analysis showed that theα-helix is the main part of the secondary structure of the two proteins.ProtFun forecasted the mainfunction of the RGBCH protein is participating energy metabolism and biosynthesizingcofactors;The main function of the SGBCH protein is participating energy conversion in theprocess of protein translation.
3. Induced by Fusarium oxysporum five different materials in different tissues and fluorescence quantitative PCR using two primers,respectively,the results show, the use of thespecific primers GBCH-Q material quantitative PCR analysis shows:“06-146”strainsthe targetgene expression in different tissues4d peak;Xinhai14target genes in6d there is a maximumvalue, and then decreased to the original level;Zhongmiansuo35,Junmian1cotton and TM-1inthe induction ofbefore and after the induction of expression of the volume is very weak orrelatively consistent,remaining at a low level of expression, quantitative PCR analysis of thematerial shows through the use of primers StVe-Q:lines “06-146”,Xinhai14,Zhongmiansuo35,Junmian1cotton and TM-1target gene in the bacteria-induced conditions,there is no change withthe induction time showing expression status.Description the cloned RGBCH、SGBCH onlyexpressed in Sea Island cotton material,two genes associated with resistance to Fusarium wilt.
4. RGBCH and SGBCH recombinating into pCAMBIA1301plant expression vector,constructing plant expression vector pCAMBIA1301-RGBCH and pCAMBIA1301-SGBCH,RGBCH and SGBCH gene integrated into the tobacco genome using of agrobacterium-mediatedmethod,obtaining T1generation seeds.T1transgenic and non-transgenic tobacco broth poured rootmethod(Each material of30planting),Turn RGBCH gene most of the leaves were yellowing, plantsurvival rate was78%,while the survival rate of to turn SGBCH transgenic plants to24%,most ofthe leaves dry death,non-transgenic plants almost all died.These results suggest that the genetransferred RGBCH tobacco resistance to Fusarium wilt bacteria.
引文
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