绿色木霉LTR-2 42kDa几丁质酶基因的克隆及转基因番茄的构建
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摘要
番茄作为一种主要的经济作物和遗传学上的模式生物,具有重要的研究价值,在植物基因工程研究上被广泛利用。通过插入外源抗性基因,构建转基因抗病番茄,是降低农药使用量和培育新优良作物品种的有效途径。几丁质酶(Chitinase)是一类细胞壁降解酶,普遍存在于各种动物、植物和微生物中。利用几丁质酶编码基因构建的转基因植物能有效地抵抗多种病原菌。然而不同来源的几丁质酶抗病效果差异很大,因此发现一种合适的几丁质酶编码基因对于构建转基因抗病植物具有重要意义。木霉作为一种重要的生防真菌,在其重寄生过程中产生的几丁质酶能够降解多种病原微生物细胞壁。研究证实,在木霉产生的多种几丁质酶中,42kDa几丁质酶被认为是降解病原菌细胞壁效果最好的一种。因此将木霉中的42kDa几丁质酶编码基因整合到植物染色体中,获得抗性植株是目前构建转基因植物研究的重要内容之一。
绿色木霉LTR-2(Trichoderma virede)由本实验室从蔬菜植物根系土壤中筛选获得,其制剂已经获得国家发明专利授权,并已经在农业部登记注册为新农药,对多种病原菌具有较好的拮抗作用,其作用机制之一就是产生多种细胞壁降解酶,尤其是42kDa几丁质酶。根据GenBank上已有的42kDa几丁质酶编码基因序列设计适宜的引物,通过PCR技术,从绿色木霉LTR-2基因组DNA中扩增到一段序列,测序结果表明,该编码基因片段大小为1508bp,其中包括一个1459bp的开放阅读框,起始密码子位于45bp,终止密码子位于1501bp,共编码氨基酸424个。通过GenBank进行序列比对,发现该序列同已发表的其他Trichoderma Viride 42kDa几丁质酶氨基酸序列具有99%的同源性,因此确定该序列为42kDa几丁质酶编码基因,该序列已在GenBank上登记(GenbankID:EF635427)。
将42kDa几丁质酶基因同质粒pCAMBIA1300中的CaMV35S启动子和35S-polyA终止子连接后,插入植物表达载体pCAMBIA1302多克隆位点中,构建成植物表达载体pCHI1302-42,利用冻融法将载体pCHI1302-42导入根癌农杆菌LBA4404中。利用农杆菌介导的叶盘转化法将42kDa基因导入番茄,组织培养后获得了番茄的再生植株。最后采用PCR及Southern杂交对再生转化番茄进行检测,证明42kDa几丁质酶基因已经插入番茄的染色体基因组中。
As an important economic crop and a biological model in genetics,tomato is of considerable value in science research and is widely studied in plant genetic engineering.Construction of transgenic tomato by inserting foreign resistant genes cloned from other organisms to its genome is an effective method in developing new disease-resistant species to reduce the chemical pesticide application.Chitinase is one of the fungal cell wall degrading enzymes which exist in animals,plant,and microorganisms.Transgenic plants expressing chitinase encoding gene can resist many kinds of pathogens efficiently.However,chitinases coming from different organisms have quite variable effects in resisting pathogens and/or diseases.Thus,to choose the most effective chitinase gene is one of the most important steps in constructing pathogen-resistant transgenic plant.Trichoderma is an important biocontrol fungus which produces various chitinases to degrade the cell wall of the fungal pathogens in the process of hyperparasitization.Among all the chitinases produced by Trichoderma, 42kDa chitinase is considered to be the most effective one in degrading the cell wall of pathogens.
Trichoderma viride strain LTR-2 isolated from the rhizosphere of vegetable field is effective in controlling various plant pathogens,and its preparation has been patented in China Bureau for Patent and registered as a new bio-pesticide in China Ministry of Agriculture.One of the important mechanisms of LTR-2 is the production of chitinases. A fragment from Trichoderma viride LTR-2 was successfully cloned by the method of PCR amplification with a pair of primers designed for 42kDa chitinase encoding sequence.The PCR product is 1508 bp and contains an open reading frame of 1459 nucleotides starting with the initiation codon ATG at position 45 and ending with the termination codon TAA at position 1501,and the number of deduction amino acid is about 424.DNA sequencing analysis shows that the sequence has a homology of 99%to those Trichoderma 42kDa chitinase encoding genes published in GenBank,and thus the PCR fragment was verified as the 42kDa chitinase encoding gene and was published in Genebank(GenbankID:EF635427).
The fragment was ligated with CaMV35S promoter and 35S-polyA terminator,and then was inserted into the vector of the pCAMBIA1302.Then,the vector of pCHI1302-42 was introduced into Agrobacterium tumefaciens LBA4404 by freezing-thawing method.Leaf disc method was adopted to transfer 42kDa chitinase gene into tomato.Finally,the insertion of the target gene in tomato chromosomal DNA was verified by PCR and Southern blotting methods.
绿色木霉LTR-2(Trichoderma virede)由本实验室从蔬菜植物根系土壤中筛选获得,其制剂已经获得国家发明专利授权,并已经在农业部登记注册为新农药,对多种病原菌具有较好的拮抗作用,其作用机制之一就是产生多种细胞壁降解酶,尤其是42kDa几丁质酶。根据GenBank上已有的42kDa几丁质酶编码基因序列设计适宜的引物,通过PCR技术,从绿色木霉LTR-2基因组DNA中扩增到一段序列,测序结果表明,该编码基因片段大小为1508bp,其中包括一个1459bp的开放阅读框,起始密码子位于45bp,终止密码子位于1501bp,共编码氨基酸424个。通过GenBank进行序列比对,发现该序列同已发表的其他Trichoderma Viride 42kDa几丁质酶氨基酸序列具有99%的同源性,因此确定该序列为42kDa几丁质酶编码基因,该序列已在GenBank上登记(GenbankID:EF635427)。
将42kDa几丁质酶基因同质粒pCAMBIA1300中的CaMV35S启动子和35S-polyA终止子连接后,插入植物表达载体pCAMBIA1302多克隆位点中,构建成植物表达载体pCHI1302-42,利用冻融法将载体pCHI1302-42导入根癌农杆菌LBA4404中。利用农杆菌介导的叶盘转化法将42kDa基因导入番茄,组织培养后获得了番茄的再生植株。最后采用PCR及Southern杂交对再生转化番茄进行检测,证明42kDa几丁质酶基因已经插入番茄的染色体基因组中。
As an important economic crop and a biological model in genetics,tomato is of considerable value in science research and is widely studied in plant genetic engineering.Construction of transgenic tomato by inserting foreign resistant genes cloned from other organisms to its genome is an effective method in developing new disease-resistant species to reduce the chemical pesticide application.Chitinase is one of the fungal cell wall degrading enzymes which exist in animals,plant,and microorganisms.Transgenic plants expressing chitinase encoding gene can resist many kinds of pathogens efficiently.However,chitinases coming from different organisms have quite variable effects in resisting pathogens and/or diseases.Thus,to choose the most effective chitinase gene is one of the most important steps in constructing pathogen-resistant transgenic plant.Trichoderma is an important biocontrol fungus which produces various chitinases to degrade the cell wall of the fungal pathogens in the process of hyperparasitization.Among all the chitinases produced by Trichoderma, 42kDa chitinase is considered to be the most effective one in degrading the cell wall of pathogens.
Trichoderma viride strain LTR-2 isolated from the rhizosphere of vegetable field is effective in controlling various plant pathogens,and its preparation has been patented in China Bureau for Patent and registered as a new bio-pesticide in China Ministry of Agriculture.One of the important mechanisms of LTR-2 is the production of chitinases. A fragment from Trichoderma viride LTR-2 was successfully cloned by the method of PCR amplification with a pair of primers designed for 42kDa chitinase encoding sequence.The PCR product is 1508 bp and contains an open reading frame of 1459 nucleotides starting with the initiation codon ATG at position 45 and ending with the termination codon TAA at position 1501,and the number of deduction amino acid is about 424.DNA sequencing analysis shows that the sequence has a homology of 99%to those Trichoderma 42kDa chitinase encoding genes published in GenBank,and thus the PCR fragment was verified as the 42kDa chitinase encoding gene and was published in Genebank(GenbankID:EF635427).
The fragment was ligated with CaMV35S promoter and 35S-polyA terminator,and then was inserted into the vector of the pCAMBIA1302.Then,the vector of pCHI1302-42 was introduced into Agrobacterium tumefaciens LBA4404 by freezing-thawing method.Leaf disc method was adopted to transfer 42kDa chitinase gene into tomato.Finally,the insertion of the target gene in tomato chromosomal DNA was verified by PCR and Southern blotting methods.
引文
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