pthA-nls及其ScFv基因转化柑橘获得抗溃疡病新种质的研究
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摘要
柑橘溃疡病(citrus bacterial canker disease)是由地毯黄单胞杆菌柑橘致病变种(Xanthomonas axonopodis pv.citri)引起的一种世界性检疫性病害,对柑橘产量及品质影响极大,严重危害着柑橘产业的健康发展。然而,到目前为止,尚无一种能根除柑橘溃疡病菌的最佳方法,柑橘品种资源中亦未发现完全抗病的品种,长期的常规育种,也没有获得抗柑橘溃疡病的优良杂种,柑橘抗溃疡病育种没有取得实质性进展。病原基因pthA是柑橘溃疡病亚洲型(A型)致病所必需的因子,其中3个核定位信号区是PthA的重要功能区,在寄主细胞内起识别定位的作用。本研究以柑橘溃疡病致病基因pthA为出发点,利用病原基因诱导植物获得抗性原理,将致病基因pthA的核定位信号NLSs构建正义和反义基因,导入柑橘感病品种甜橙中,使其过量表达失去原有功能的蛋白,或表达蛋白失去原有的时空性,或表达蛋白激活植物本身的防御系统,从而干扰病原菌的正常的生理代谢;另一方面,利用单克隆抗体和植物抗体技术,制备抗致病因子PthA核定位信号肽NLS单克隆抗体,分离出抗体基因的轻链和重链,并构建单链抗体基因ScFv,将其导入柑橘感病品种,通过在柑橘体内表达抗体蛋白,结合致病因子PthA蛋白的功能位点,阻断致病蛋白向细胞核的运输途径,从而达到抗病的目的。通过对转基因甜橙的抗性筛选和外源基因的表达分析,对进一步研究柑橘溃疡病的致病机理具有重要的理论意义。同时,选择高感病的主栽品种甜橙进行试验,获得的抗病新种质,从根本上控制柑橘溃疡病的危害,对柑橘产业意义重大。本研究取得研究结果如下:
1、从湖南省各柑橘产区感病果园分离纯化柑橘溃疡病菌株50份,并根据致病基因pthA和柑橘溃疡病菌独有的保守蛋白基因差异序列,设计筛选出特异性引物对,通过优化PCR反应条件,利用PCR技术从质粒和基因组两方面对柑橘溃疡病进行鉴定,并利用离体接种对获得的菌样作最终鉴定,为后续试验提供了试验材料。
2、利用long-PCR和net-PCR方法,得到pthA完整的编码区并将其克隆到植物表达载体pBI121中。序列分析结果表明,克隆得到的pthA′与Genbank中的pthA(U28802)序列有99.8%的同源性。pthA′基因的克隆为进一步研究其功能奠定了基础。
3、构建了pthA-nls正义基因和pthA-a-nls反义基因植物表达载体,利用农杆菌介导法转化甜橙实生苗,经PCR检测,成功获得了9株转pthA-nls和12株转pthA-a-nls基因甜橙转基因苗,目前转基因苗长势良好。对9株转pthA-nls基因甜橙中的生长势好的3株进行Southern杂交分析,证明pthA-nls基因已成功整合到甜橙基因组中,通过RT-PCR对这3株转基因植株进行表达分析,检测到了外源目的基因的表达。以溃疡病菌液接种转pthA-nls基因和pthA-a-nls及对照甜橙植株离体叶片,接种试验结果表明,转pthA-nls基因植株叶片对溃疡病表现出一定的抗性,而转pthA-a-nls基因植株叶片对溃疡病表现与对照一样敏感。
4、采用PCR法扩增出pthA基因末端NLSs序列,并将其克隆到原核表达载体PET32a(+)的EcoRⅠ和XhoⅠ位点。重组质粒转化大肠杆菌BL21(DE3)并诱导表达重组蛋白,经Ni~(2+)-NTA纯化柱纯化得到纯化的重组蛋白,免疫Balb/c小白鼠,制备了相应的抗血清。Western blotting、ELISA鉴定结果表明,获得的抗血清可特异地结合重组蛋白亦可识别溃疡病菌PthA天然蛋白。利用抗血清与溃疡病菌混合接种离体冰糖橙叶片,发现抗血清对溃疡病的发生具有一定的拮抗作用,能推迟溃疡病菌的致病过程,且病斑比对照小,但未能达到抗病的程度。
5、制备了抗PthA-NLS重组多肽的3株单克隆抗体(McAb),McAb能够特异性、高亲和力地与PthA-NLS多肽结合。这3株McAb可被进一步用于柑橘溃疡病的鉴定检测,并为下一步制备单链抗体的研究提供杂交瘤细胞株。
6、通过RT-PCR成功地扩增出重链可变区基因和轻链可变区基因,采用重叠延伸PCR将两个可变区基因串联,获得的单链抗体基因大小约750 bp,将其克隆到T载体和原核表达载体pET32a(+),通过酶切和PCR鉴定均获得了约750 bp大小的条带。测序结果显示,重链可变区基因有360 bp,轻链可变区基因有342 bp,中间连接肽基因45 bp,经过IPTG诱导ScFv原核表达,获得了大小为44.5 KD的ScFv重组蛋白。
7、将ScFv基因构建到植物表达载体pBI121中,利用农杆菌介导法转化甜橙实生苗,经PCR检测,成功获得了12株转ScFv基因甜橙,利用RT-PCR分析外源基因ScFv在转基因植株中的表达,初步表明外源基因ScFv已成功转入到甜橙基因组中,并能在甜橙体内正常表达。采用离体接种对部分转ScFv基因植株进行抗性筛选,结果表明,转基因植株对溃疡病表现出一定的抗性,株系之间抗性存在着差异,其中D9可能是很有希望的抗性材料。
Citrus canker disease caused by Xanthomonas axonopodis pv.citri.(Xac.),affects almost all of the citrus species and cultivars in the world causing severe damages to the citrus industry in most citrus producing areas worldwide,and China is one of the most severely damaged countries.All cultivars of citrus are susceptible to canker and highly susceptible when being artificially inoculated,implying that no function genes resistant to canker resistance have been identified in citrus,pthA is essential for the pathogen to cause hyperplastic canker symptoms on citrus.The carboxyl terminal portion of pthA encodes three nuclear localization signals(NLSs) that are critical for PthA function and for localization to the host cell nucleus.NLSs were found in all members of Xanthomonas avr/pth gene family.The avr/pth gene encodes signals affecting plant cell programs including "programmed death",as avr/pthA gene products can elicit resistance,it can be used to enhance bacterial disease resistance in plants.In this research,the COOH terminal of pthA,encoding three nuclear localizing signals,was cloned into a plant expression vector system and transferred into sweet orange via Agrobacterium-mediated transformation.Meanwhile,antibody technique and genetic transformation can overcome problems associated with conventional breeding and provide new approaches for disease resistance.A number of antibody genes have been introduced into plant cells,and the transgenic plants expressed the transgenes.These recombinant antibody molecules can be effective in insect or disease resistance.In this study,monoclonal antibody of the anti-recombinant PthA-NLS was prepared and the related ScFv gene was transferred into sweet orange, in order to confer resistance in citrus to canker disease.The main results were as follows:
1.50 Xanthomonas axonopodis pv.citri isolates were obtained from diseased materials of several citrus cultivars collected from Hengyang,Chenzhou and other areas of Hunan province.The isolated samples was identified by PCR assay using two pairs of specific primers,i.e.JYF5/JYR5 designed from published sequence encoding a conserved hypothetical protein gene in genome of the bacterium and P8/P9 designed from published sequence of pthA(U28802).The accurate and rapid molecular diagnosis technique was established and all the isolates were verified by PCR.The obtained pathogen isolates offered important materials for the subsequent studies.
2.pthA was amplified by long-PCR and net-PCR from the plasmid of Xanthomonas axonopodis pv.citri and cloned into pBI121 plant expression vector.The recombinant plasmid named pBI-pthA was identified by restriction digesting and sequencing.The result of sequence analysis showed that pthA' had 99.8%similarity with that of pthA in Genbank.
3.The COOH terminal of pthA,encoding three nuclear localizing signals,was amplified by PCR from the plasmid of Xanthomonas axonopodis pv.citri.,then the sense and antisense sequences were inserted in plant expression vectors,the sense recombinant plasmid named pBI-nls and the antisense one named pBI-a-nls.After identified by restriction digesting and sequencing,the pthA-nls and the pthA-α-nls genes were transferred into sweet orange respectively via Agrobacterium mediated transformation.Successful integration were confirmed by PCR and southern blotting, 9 transgenic pthA-nls gene and 12 transgenic pthA-α-nls gene sweet orange plants were abtained.Disease resistance assays in vitro indicated that the transgenic pthA-nls gene clones showed significantly less lesion development than the controls.And the transgenicα-nls gene sweet orange plants were susceptivity to the disease.
4.NLSs of pthA was amplified by PCR and cloned into PET32a(+) vector.The recombinant plasmid named PET-NLS was identified by restriction digesting and sequencing.The recombinant fusion protein was expressed in E.coli BL21(DE3) and analyzed by 12%SDS-PAGE.A 48 KD of recombinant fusion protein was purified with Ni~(2+)-NTA resin.The antiserum was obtained by immunizing Balb/c mouse with the purified recombinant protein then identified by western blotting and ELISA.The result demonstrated that the antiserum could specifically bind to the recombinant protein and the PthA from Xanthomonas axonopodis pv.citri.,as well as to partially inhibit the disease development.
5.Three cell lines producing monoclonal antibody,which can specifically recognize and strongly bound to pthA-NLS,were acquired.The immunoglobulin subtype of McAb was identified as mouse IgG1.
6.The variable region genes were amplified by RT-PCR from the RNA of hybridoma cell line 3D10H2,then strung together by SOE-PCR(splicing by overlap extension) and the ScFv gene was constructed.After cloned into pGEM-T and pET32a(+) vector,the recombinant ScFv gene was identified by endonuclease digestion,PCR and sequencing.The sequencing result showed that the ScFv gene consists of 747 bp,including 360 bp heavy chain variable region gene,342 bp light chain variable region gene and 45 bp linker gene.The recombinant fusion ScFv protein was expressed in E.coli BL21(DE3) with IPTG induction and analysised by SDS-PAGE,a 44.5 KDa of recombinant fusion protein was obtained.
7.ScFv gene was cloned into pBI121 plant expression vector,named p35S. Agrobacterium-mediated transformation was carried out to introduce p35S into sweet orange.12 kanamycin resistant plants were obtained.PCR analysis showed the integration of the ScFv gene in sweet orange genome.The expression of the transgene ScFv was detected by using RT-PCR,ScFv were expression successful in the 4 transgenic plants.
1、从湖南省各柑橘产区感病果园分离纯化柑橘溃疡病菌株50份,并根据致病基因pthA和柑橘溃疡病菌独有的保守蛋白基因差异序列,设计筛选出特异性引物对,通过优化PCR反应条件,利用PCR技术从质粒和基因组两方面对柑橘溃疡病进行鉴定,并利用离体接种对获得的菌样作最终鉴定,为后续试验提供了试验材料。
2、利用long-PCR和net-PCR方法,得到pthA完整的编码区并将其克隆到植物表达载体pBI121中。序列分析结果表明,克隆得到的pthA′与Genbank中的pthA(U28802)序列有99.8%的同源性。pthA′基因的克隆为进一步研究其功能奠定了基础。
3、构建了pthA-nls正义基因和pthA-a-nls反义基因植物表达载体,利用农杆菌介导法转化甜橙实生苗,经PCR检测,成功获得了9株转pthA-nls和12株转pthA-a-nls基因甜橙转基因苗,目前转基因苗长势良好。对9株转pthA-nls基因甜橙中的生长势好的3株进行Southern杂交分析,证明pthA-nls基因已成功整合到甜橙基因组中,通过RT-PCR对这3株转基因植株进行表达分析,检测到了外源目的基因的表达。以溃疡病菌液接种转pthA-nls基因和pthA-a-nls及对照甜橙植株离体叶片,接种试验结果表明,转pthA-nls基因植株叶片对溃疡病表现出一定的抗性,而转pthA-a-nls基因植株叶片对溃疡病表现与对照一样敏感。
4、采用PCR法扩增出pthA基因末端NLSs序列,并将其克隆到原核表达载体PET32a(+)的EcoRⅠ和XhoⅠ位点。重组质粒转化大肠杆菌BL21(DE3)并诱导表达重组蛋白,经Ni~(2+)-NTA纯化柱纯化得到纯化的重组蛋白,免疫Balb/c小白鼠,制备了相应的抗血清。Western blotting、ELISA鉴定结果表明,获得的抗血清可特异地结合重组蛋白亦可识别溃疡病菌PthA天然蛋白。利用抗血清与溃疡病菌混合接种离体冰糖橙叶片,发现抗血清对溃疡病的发生具有一定的拮抗作用,能推迟溃疡病菌的致病过程,且病斑比对照小,但未能达到抗病的程度。
5、制备了抗PthA-NLS重组多肽的3株单克隆抗体(McAb),McAb能够特异性、高亲和力地与PthA-NLS多肽结合。这3株McAb可被进一步用于柑橘溃疡病的鉴定检测,并为下一步制备单链抗体的研究提供杂交瘤细胞株。
6、通过RT-PCR成功地扩增出重链可变区基因和轻链可变区基因,采用重叠延伸PCR将两个可变区基因串联,获得的单链抗体基因大小约750 bp,将其克隆到T载体和原核表达载体pET32a(+),通过酶切和PCR鉴定均获得了约750 bp大小的条带。测序结果显示,重链可变区基因有360 bp,轻链可变区基因有342 bp,中间连接肽基因45 bp,经过IPTG诱导ScFv原核表达,获得了大小为44.5 KD的ScFv重组蛋白。
7、将ScFv基因构建到植物表达载体pBI121中,利用农杆菌介导法转化甜橙实生苗,经PCR检测,成功获得了12株转ScFv基因甜橙,利用RT-PCR分析外源基因ScFv在转基因植株中的表达,初步表明外源基因ScFv已成功转入到甜橙基因组中,并能在甜橙体内正常表达。采用离体接种对部分转ScFv基因植株进行抗性筛选,结果表明,转基因植株对溃疡病表现出一定的抗性,株系之间抗性存在着差异,其中D9可能是很有希望的抗性材料。
Citrus canker disease caused by Xanthomonas axonopodis pv.citri.(Xac.),affects almost all of the citrus species and cultivars in the world causing severe damages to the citrus industry in most citrus producing areas worldwide,and China is one of the most severely damaged countries.All cultivars of citrus are susceptible to canker and highly susceptible when being artificially inoculated,implying that no function genes resistant to canker resistance have been identified in citrus,pthA is essential for the pathogen to cause hyperplastic canker symptoms on citrus.The carboxyl terminal portion of pthA encodes three nuclear localization signals(NLSs) that are critical for PthA function and for localization to the host cell nucleus.NLSs were found in all members of Xanthomonas avr/pth gene family.The avr/pth gene encodes signals affecting plant cell programs including "programmed death",as avr/pthA gene products can elicit resistance,it can be used to enhance bacterial disease resistance in plants.In this research,the COOH terminal of pthA,encoding three nuclear localizing signals,was cloned into a plant expression vector system and transferred into sweet orange via Agrobacterium-mediated transformation.Meanwhile,antibody technique and genetic transformation can overcome problems associated with conventional breeding and provide new approaches for disease resistance.A number of antibody genes have been introduced into plant cells,and the transgenic plants expressed the transgenes.These recombinant antibody molecules can be effective in insect or disease resistance.In this study,monoclonal antibody of the anti-recombinant PthA-NLS was prepared and the related ScFv gene was transferred into sweet orange, in order to confer resistance in citrus to canker disease.The main results were as follows:
1.50 Xanthomonas axonopodis pv.citri isolates were obtained from diseased materials of several citrus cultivars collected from Hengyang,Chenzhou and other areas of Hunan province.The isolated samples was identified by PCR assay using two pairs of specific primers,i.e.JYF5/JYR5 designed from published sequence encoding a conserved hypothetical protein gene in genome of the bacterium and P8/P9 designed from published sequence of pthA(U28802).The accurate and rapid molecular diagnosis technique was established and all the isolates were verified by PCR.The obtained pathogen isolates offered important materials for the subsequent studies.
2.pthA was amplified by long-PCR and net-PCR from the plasmid of Xanthomonas axonopodis pv.citri and cloned into pBI121 plant expression vector.The recombinant plasmid named pBI-pthA was identified by restriction digesting and sequencing.The result of sequence analysis showed that pthA' had 99.8%similarity with that of pthA in Genbank.
3.The COOH terminal of pthA,encoding three nuclear localizing signals,was amplified by PCR from the plasmid of Xanthomonas axonopodis pv.citri.,then the sense and antisense sequences were inserted in plant expression vectors,the sense recombinant plasmid named pBI-nls and the antisense one named pBI-a-nls.After identified by restriction digesting and sequencing,the pthA-nls and the pthA-α-nls genes were transferred into sweet orange respectively via Agrobacterium mediated transformation.Successful integration were confirmed by PCR and southern blotting, 9 transgenic pthA-nls gene and 12 transgenic pthA-α-nls gene sweet orange plants were abtained.Disease resistance assays in vitro indicated that the transgenic pthA-nls gene clones showed significantly less lesion development than the controls.And the transgenicα-nls gene sweet orange plants were susceptivity to the disease.
4.NLSs of pthA was amplified by PCR and cloned into PET32a(+) vector.The recombinant plasmid named PET-NLS was identified by restriction digesting and sequencing.The recombinant fusion protein was expressed in E.coli BL21(DE3) and analyzed by 12%SDS-PAGE.A 48 KD of recombinant fusion protein was purified with Ni~(2+)-NTA resin.The antiserum was obtained by immunizing Balb/c mouse with the purified recombinant protein then identified by western blotting and ELISA.The result demonstrated that the antiserum could specifically bind to the recombinant protein and the PthA from Xanthomonas axonopodis pv.citri.,as well as to partially inhibit the disease development.
5.Three cell lines producing monoclonal antibody,which can specifically recognize and strongly bound to pthA-NLS,were acquired.The immunoglobulin subtype of McAb was identified as mouse IgG1.
6.The variable region genes were amplified by RT-PCR from the RNA of hybridoma cell line 3D10H2,then strung together by SOE-PCR(splicing by overlap extension) and the ScFv gene was constructed.After cloned into pGEM-T and pET32a(+) vector,the recombinant ScFv gene was identified by endonuclease digestion,PCR and sequencing.The sequencing result showed that the ScFv gene consists of 747 bp,including 360 bp heavy chain variable region gene,342 bp light chain variable region gene and 45 bp linker gene.The recombinant fusion ScFv protein was expressed in E.coli BL21(DE3) with IPTG induction and analysised by SDS-PAGE,a 44.5 KDa of recombinant fusion protein was obtained.
7.ScFv gene was cloned into pBI121 plant expression vector,named p35S. Agrobacterium-mediated transformation was carried out to introduce p35S into sweet orange.12 kanamycin resistant plants were obtained.PCR analysis showed the integration of the ScFv gene in sweet orange genome.The expression of the transgene ScFv was detected by using RT-PCR,ScFv were expression successful in the 4 transgenic plants.
引文
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