辣椒疫霉菌4个果胶甲基酯酶基因的克隆和pcpme6的真核表达研究
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摘要
辣椒疫病在世界范围内广泛发生的土传病害,严重影响世界各国的农业生产,造成巨大的经济损失,病原是辣椒疫霉菌(Phytophthora capsici)。辣椒疫霉菌是一种土传卵菌,以卵孢子或厚垣孢子在土壤病残体中越冬,可以存活数月甚至更长时间,在适宜条件下引起青椒茎腐、根腐和枯萎;寄主范围广,除侵染辣椒外,还可以侵染西葫芦、黄瓜、番茄等20多种作物。
目前,对于辣椒疫病的防治主要采用化学防治,而培育抗病品种效果不稳定,原因之一是辣椒疫霉具有较强的变异能力。因此,探索辣椒疫霉菌重要的致病因子,研究抗病性生理生化机制,结合抗病性遗传,抗病基因的QTL定位(quantitative trait loci)与基因工程,进行抗病性鉴定和抗病育种方面研究成为病害防治的前景。其中细胞壁降解酶在病害致病关键因子之一,包括角质酶(cutinase),果胶酶(pectinase),纤维素酶(cellulase),半纤维素酶(hemicellulase),蛋白酶(protease)等。而果胶酶又包括多聚半乳糖醛酸酶(PG),果胶甲基酯酶(PME),果胶裂解酶(PL)。果胶甲基酯酶(PME)是许多植物病原菌分泌的一种果胶酶,能降解植物细胞壁,许多病原真菌,卵菌,细菌在侵染过程都能分泌果胶甲基酯酶。本文以辣椒疫霉菌为研究对象,克隆辣椒疫霉果胶甲基酯酶基因及表达研究。
以辣椒疫霉强致病和果胶甲基酯酶活性高的菌株为实验材料构建的DNA文库,借助Pool-PCR技术分离了4个果胶甲基酯酶基因。对基因结构分析发现4个pme基因具有很高的同源性,并具有不同数目的糖基化位点。所编码的蛋白具果胶甲基酯酶的保守序列和蛋白活性位点。本研究发现,辣椒疫霉pme基因与卵菌pme基因具高度保守序列,也证明了卵菌在自然界中的分类地位。并且对其中一个基因构建质粒进行体外诱导表达,制备了多克隆抗体,借助蛋白印记杂交验证了重组蛋白的免疫活性,其具体研究结果如下:
1、Pool-PCR法筛选基因文库,分离了4个果胶甲基酯酶基因
从GenBank中下载若干pme基因,同源比对设计多对引物( P230+ AP650,P230+ P920,P230+ P795,P445+ P920,P445+AP650, P620 + P920 ,P650+P920,P445+ P795),然后进行反复筛选,分离了3个全长和1个非全长基因。利用引物P230+AP650筛选出pcpme6,P650+P920筛选出pcpme7和pcpme8,其中P445+P920和P445+P795均能扩增出pcpme9。将所克隆的4个基因经过blast,显示全部为果胶甲基酯酶基因。经过DNAman软件处理分析了这4个基因的基本结构,最大开放阅读框长度差别不大,均在1100bp左右,编码345-348个氨基酸,预测氨基酸分子量为37-38kDa。利用http://www.expasy.org和http://www.cbs.dfu.dk/service/signalP对这四个基因的信号肽和N糖基化位点进行预测,信号肽长度16-20个氨基酸不等,N-端糖基化位点4-7个不等,4个基因的理论PI值为5-9。
2、RACE技术扩增pcpme9的3’端
将辣椒疫霉菌置于三角瓶进行振荡培养7天后,收集菌丝。然后液氮研磨,利用Trizol法提取总RNA,经反转录酶作用,合成cDNA,作为模板,以通用引物(USP)和基因特异引物(GSP)进行第一轮PCR;以第一轮产物做模板,用巢式特异引物(NGSP)和巢式通用引物(NUSP)进行第二轮PCR,目的片段回收,连接,转化大肠杆菌DH-5α后,送样测序。将测序片段拼接后,对其进行了初步分析,果胶甲基酯酶基因pcpme9序列全长为1041 bp,最大开放阅读框ORF为1038bp(1-1038),ORF编码一个346个氨基酸的蛋白质,预测编码产物大小约为38kDa,并有7个N糖基化位点,信号肽含16个氨基酸。
3、pcpme6真核表达和Western blot
根据已经克隆的pcpme6基因序列,设计特异性引物,PCR扩增其成熟肽片断。重组至酵母分泌型表达载体pPIC9K,构建重组表达载体pPIC9K/pcpme6,转化至大肠杆菌JM109中,筛选得到阳性克隆。重组质粒经stuⅠ线性化后转化毕赤酵母GS115感受态细胞,经G418筛选和PCR扩增转化子基因组筛选,得到数株基因工程酵母菌。SDS-PAGE分析发现,重组蛋白进行了特异表达,分子量大小为50KDa左右。通过将酵母分泌的PME蛋白免疫家兔,制备特异性抗体。Western blot分析结果进一步表明,转基因酵母成功表达,重组抗原有良好的免疫活性,多克隆抗体具有较高特异性和灵敏度,重组蛋白与制备的抗体特异性结合。
Pepper blight is a worldwide disease, which impacted on agriculture almost in every country, and caused enormous loss on economy. The disease was caused by phytophthora capsici , a soilborn Oomycete, which can survive for several months even longer time in the soil as oospores or chlamydospore. In the optimum condition, the pathogen can not only impact pepper, and pumpkin, cucumber, tomato, and other plant can be its host as well.
At present, the pepper blight was controlled mainly by chemical methods and breeding resisitant cultivars. However, strong variability in pathogenicity of P. capsici caused the instability of resistant breeds. It is very important to research infection mechanism, resistance biochemistry mechanism, resistance genetics, and quantitative trait loci and gene clone, as well, for resistance identification and application in breeding for disease resistance. Cell wall degrading-enzyme plays very important roll in the relation of pathogenesis action by peper blight in peper, including cutinase, pectinase, cellulose, hemicellulase, protease and so on. And pectinase can be classified as polygalacturonase (PG), Pectinmelythesterase (PME), and pectinlyase(PL) . PME is a key roll in infection process of P. capsici. , through degrading plant cell wall , and is ubiquitously secrated in fungi, bactera and Oomycets. The research is primary on pme gene cloning and expression.
The paper expatiated selecting pme genes through a DNA genetic library constructed basing on a strong infection strain of P. capsici, and eukaryotic expression and western blot of pcpme6 .Four pme genes were isolated by screening genomic DNA library with PCR method. Then pcpme6 recombinant protein was obtained through eukaryotic expression, and identified by western blot with multiclone antibody. The process and results are as follows:
1 Selecting DNA libraty whith PCR, 4 pme obtained
A serious of primers were designed according to data including nucleotide sequences of fungi,Oomycets, bacteria, and plants from Genbank ,and the resultant comprised 8 pairs, P230+ AP650,P230+ P920,P230+ P795,P445+ P920,P445+AP650, P620 + P920 ,P650+P920,P445+ P795. Repeating PCR to screen the DNA library with different primer pair respectively, we got three full length genes and one fragment. We got pcpme6 with P230+AP650,and pcpme7and pcpme8 with P650+P920, with P445+P920 and P445+P795 having the same gene pcpme9. Blast result of the 4 gene on NCBI, showed they were all pectin melythesterase genes.
Analysis of sequences indicated that there were highly conserved sequence in the four pme genes and various N-glycosylation sites. The pme genes of P. capsici showed high homogeneity compared with pme genes of other Oomycetes and consequently defined the station of Oomycete in the nature.They had the same basic structure through analysis using DNAman soft ware, the biggest open reading frame was around 1100bp in length and encoded a deduced amino acid sequence of 345-348 residues, and the molecular weight was about 37-38kd. The signal peptide cleavage site was predicted by SignalP programe (http://www.expasy.org), showing their signal peptide was around 16-20 residues .The N-glycosylation sites were predicted by scan prosite (http://www.cbs.dfu.dk/service/signalP) , which also dipcted difference among the four gene, they had 4-7 N-glycosylation sites. And the deduced isoelectric point was between 5-9, through analyse online programe (http://us.expasy.org).
2 Amplification of the 3′end sequence of pcpme9 by 3′race
Collect mycelium of the phytophthora capsici strain after cultivation in liquid shake medium in conical flask for seven days. Then grind the mycelium in liquid nitrogen and extract total RNA using Trizol. Use reverse transcriptase to synthesis cDNA as model for the first-round PCR in which the primer were USP and GSP. Then process the second-round PCR whose model was product of the first-round PCR ,and primers NUSP and NGSP. The target DNA fragment recovered from agarose gel and cloned into E.coli DH-5α,and then sequenced in biology company. Recombine the full length gene, and analyze using DNAman soft ware.The result showed that its full length was 1041bp, and the biggest opening reading frame contained 1038bp, encoding 346 residues. The deduced molecular weight was about 38kd. The signal peptide 16 residues and N-glycosylation sites number was seven, according to the online programe (http://us.expasy.org).
3 Eukaryotic expression and western blot of pcpme6
Gene-specific primers were designed and synthesized to amplify the pcpme6 mature peptide sequence according to known sequence. The amplified fragment was inserted into pPIC9K. Recombinant expression plasmid pPIC9K/pcpme6 was constructed. The plasmid was transferred into E. coli JM109 to obtain positive clones and to ampify culture. The plasmid pPIC9K/ pcpme6 was transformed to Pichia pastoris GS115 competent cell after linearized with restriction enzyme stu I. After screening on plates with density gradient of G418, transformants’DNA was extrcted and PCR was performed for identification . The expressed protein was detected about 50KDa by SDS-PAGE .The protein secreated from recombinant strains was purified through SDS-PAGE to prepare polyclonal antibody by immunizing rabbits. Western blot showed that expressed protein could specially react with this antibody, which dipcited that the recombinant successfully expressed, accompanied with prepared antigen’s relative immunocompetence, and the sensitivity and specificity of polyclonal antibody were high, as well.
目前,对于辣椒疫病的防治主要采用化学防治,而培育抗病品种效果不稳定,原因之一是辣椒疫霉具有较强的变异能力。因此,探索辣椒疫霉菌重要的致病因子,研究抗病性生理生化机制,结合抗病性遗传,抗病基因的QTL定位(quantitative trait loci)与基因工程,进行抗病性鉴定和抗病育种方面研究成为病害防治的前景。其中细胞壁降解酶在病害致病关键因子之一,包括角质酶(cutinase),果胶酶(pectinase),纤维素酶(cellulase),半纤维素酶(hemicellulase),蛋白酶(protease)等。而果胶酶又包括多聚半乳糖醛酸酶(PG),果胶甲基酯酶(PME),果胶裂解酶(PL)。果胶甲基酯酶(PME)是许多植物病原菌分泌的一种果胶酶,能降解植物细胞壁,许多病原真菌,卵菌,细菌在侵染过程都能分泌果胶甲基酯酶。本文以辣椒疫霉菌为研究对象,克隆辣椒疫霉果胶甲基酯酶基因及表达研究。
以辣椒疫霉强致病和果胶甲基酯酶活性高的菌株为实验材料构建的DNA文库,借助Pool-PCR技术分离了4个果胶甲基酯酶基因。对基因结构分析发现4个pme基因具有很高的同源性,并具有不同数目的糖基化位点。所编码的蛋白具果胶甲基酯酶的保守序列和蛋白活性位点。本研究发现,辣椒疫霉pme基因与卵菌pme基因具高度保守序列,也证明了卵菌在自然界中的分类地位。并且对其中一个基因构建质粒进行体外诱导表达,制备了多克隆抗体,借助蛋白印记杂交验证了重组蛋白的免疫活性,其具体研究结果如下:
1、Pool-PCR法筛选基因文库,分离了4个果胶甲基酯酶基因
从GenBank中下载若干pme基因,同源比对设计多对引物( P230+ AP650,P230+ P920,P230+ P795,P445+ P920,P445+AP650, P620 + P920 ,P650+P920,P445+ P795),然后进行反复筛选,分离了3个全长和1个非全长基因。利用引物P230+AP650筛选出pcpme6,P650+P920筛选出pcpme7和pcpme8,其中P445+P920和P445+P795均能扩增出pcpme9。将所克隆的4个基因经过blast,显示全部为果胶甲基酯酶基因。经过DNAman软件处理分析了这4个基因的基本结构,最大开放阅读框长度差别不大,均在1100bp左右,编码345-348个氨基酸,预测氨基酸分子量为37-38kDa。利用http://www.expasy.org和http://www.cbs.dfu.dk/service/signalP对这四个基因的信号肽和N糖基化位点进行预测,信号肽长度16-20个氨基酸不等,N-端糖基化位点4-7个不等,4个基因的理论PI值为5-9。
2、RACE技术扩增pcpme9的3’端
将辣椒疫霉菌置于三角瓶进行振荡培养7天后,收集菌丝。然后液氮研磨,利用Trizol法提取总RNA,经反转录酶作用,合成cDNA,作为模板,以通用引物(USP)和基因特异引物(GSP)进行第一轮PCR;以第一轮产物做模板,用巢式特异引物(NGSP)和巢式通用引物(NUSP)进行第二轮PCR,目的片段回收,连接,转化大肠杆菌DH-5α后,送样测序。将测序片段拼接后,对其进行了初步分析,果胶甲基酯酶基因pcpme9序列全长为1041 bp,最大开放阅读框ORF为1038bp(1-1038),ORF编码一个346个氨基酸的蛋白质,预测编码产物大小约为38kDa,并有7个N糖基化位点,信号肽含16个氨基酸。
3、pcpme6真核表达和Western blot
根据已经克隆的pcpme6基因序列,设计特异性引物,PCR扩增其成熟肽片断。重组至酵母分泌型表达载体pPIC9K,构建重组表达载体pPIC9K/pcpme6,转化至大肠杆菌JM109中,筛选得到阳性克隆。重组质粒经stuⅠ线性化后转化毕赤酵母GS115感受态细胞,经G418筛选和PCR扩增转化子基因组筛选,得到数株基因工程酵母菌。SDS-PAGE分析发现,重组蛋白进行了特异表达,分子量大小为50KDa左右。通过将酵母分泌的PME蛋白免疫家兔,制备特异性抗体。Western blot分析结果进一步表明,转基因酵母成功表达,重组抗原有良好的免疫活性,多克隆抗体具有较高特异性和灵敏度,重组蛋白与制备的抗体特异性结合。
Pepper blight is a worldwide disease, which impacted on agriculture almost in every country, and caused enormous loss on economy. The disease was caused by phytophthora capsici , a soilborn Oomycete, which can survive for several months even longer time in the soil as oospores or chlamydospore. In the optimum condition, the pathogen can not only impact pepper, and pumpkin, cucumber, tomato, and other plant can be its host as well.
At present, the pepper blight was controlled mainly by chemical methods and breeding resisitant cultivars. However, strong variability in pathogenicity of P. capsici caused the instability of resistant breeds. It is very important to research infection mechanism, resistance biochemistry mechanism, resistance genetics, and quantitative trait loci and gene clone, as well, for resistance identification and application in breeding for disease resistance. Cell wall degrading-enzyme plays very important roll in the relation of pathogenesis action by peper blight in peper, including cutinase, pectinase, cellulose, hemicellulase, protease and so on. And pectinase can be classified as polygalacturonase (PG), Pectinmelythesterase (PME), and pectinlyase(PL) . PME is a key roll in infection process of P. capsici. , through degrading plant cell wall , and is ubiquitously secrated in fungi, bactera and Oomycets. The research is primary on pme gene cloning and expression.
The paper expatiated selecting pme genes through a DNA genetic library constructed basing on a strong infection strain of P. capsici, and eukaryotic expression and western blot of pcpme6 .Four pme genes were isolated by screening genomic DNA library with PCR method. Then pcpme6 recombinant protein was obtained through eukaryotic expression, and identified by western blot with multiclone antibody. The process and results are as follows:
1 Selecting DNA libraty whith PCR, 4 pme obtained
A serious of primers were designed according to data including nucleotide sequences of fungi,Oomycets, bacteria, and plants from Genbank ,and the resultant comprised 8 pairs, P230+ AP650,P230+ P920,P230+ P795,P445+ P920,P445+AP650, P620 + P920 ,P650+P920,P445+ P795. Repeating PCR to screen the DNA library with different primer pair respectively, we got three full length genes and one fragment. We got pcpme6 with P230+AP650,and pcpme7and pcpme8 with P650+P920, with P445+P920 and P445+P795 having the same gene pcpme9. Blast result of the 4 gene on NCBI, showed they were all pectin melythesterase genes.
Analysis of sequences indicated that there were highly conserved sequence in the four pme genes and various N-glycosylation sites. The pme genes of P. capsici showed high homogeneity compared with pme genes of other Oomycetes and consequently defined the station of Oomycete in the nature.They had the same basic structure through analysis using DNAman soft ware, the biggest open reading frame was around 1100bp in length and encoded a deduced amino acid sequence of 345-348 residues, and the molecular weight was about 37-38kd. The signal peptide cleavage site was predicted by SignalP programe (http://www.expasy.org), showing their signal peptide was around 16-20 residues .The N-glycosylation sites were predicted by scan prosite (http://www.cbs.dfu.dk/service/signalP) , which also dipcted difference among the four gene, they had 4-7 N-glycosylation sites. And the deduced isoelectric point was between 5-9, through analyse online programe (http://us.expasy.org).
2 Amplification of the 3′end sequence of pcpme9 by 3′race
Collect mycelium of the phytophthora capsici strain after cultivation in liquid shake medium in conical flask for seven days. Then grind the mycelium in liquid nitrogen and extract total RNA using Trizol. Use reverse transcriptase to synthesis cDNA as model for the first-round PCR in which the primer were USP and GSP. Then process the second-round PCR whose model was product of the first-round PCR ,and primers NUSP and NGSP. The target DNA fragment recovered from agarose gel and cloned into E.coli DH-5α,and then sequenced in biology company. Recombine the full length gene, and analyze using DNAman soft ware.The result showed that its full length was 1041bp, and the biggest opening reading frame contained 1038bp, encoding 346 residues. The deduced molecular weight was about 38kd. The signal peptide 16 residues and N-glycosylation sites number was seven, according to the online programe (http://us.expasy.org).
3 Eukaryotic expression and western blot of pcpme6
Gene-specific primers were designed and synthesized to amplify the pcpme6 mature peptide sequence according to known sequence. The amplified fragment was inserted into pPIC9K. Recombinant expression plasmid pPIC9K/pcpme6 was constructed. The plasmid was transferred into E. coli JM109 to obtain positive clones and to ampify culture. The plasmid pPIC9K/ pcpme6 was transformed to Pichia pastoris GS115 competent cell after linearized with restriction enzyme stu I. After screening on plates with density gradient of G418, transformants’DNA was extrcted and PCR was performed for identification . The expressed protein was detected about 50KDa by SDS-PAGE .The protein secreated from recombinant strains was purified through SDS-PAGE to prepare polyclonal antibody by immunizing rabbits. Western blot showed that expressed protein could specially react with this antibody, which dipcited that the recombinant successfully expressed, accompanied with prepared antigen’s relative immunocompetence, and the sensitivity and specificity of polyclonal antibody were high, as well.
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