橡胶炭疽病菌（Colletotrichum gloeosporioides）致病相关突变体表型分析及插入位点定位
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摘要
橡胶树炭疽病(Rubber plant anthracnose)主要是由胶孢炭疽菌[Colletotrichum gloeosporioides (Penz.)Sacc]侵染造成的,是橡胶树重要的叶部病害之一。构建炭疽菌ATMT (Agrobacterium tumefaciens-Mediated Transformation)转化体库,鉴定相关突变体,利用分子生物学技术分离和鉴定橡胶树胶胞炭疽菌的致病相关基因,是了解其致病机理及互作机制的重要手段。不仅在植物病理学上有重要意义,而且也为生产上进一步探索新的防病途径提供线索,为选育持久抗病品种、制定橡胶炭疽病菌持续管理策略等提供理论依据。
本研究利用启动子捕获技术通过农杆菌介导的遗传转化体系构建橡胶炭疽病菌RC-178的突变体库,使转化子数目增加到1685个。通过致病力的测定,从该突变体库中筛选到15个致病力明显减弱或丧失的突变体。对这15个突变体进行了生物学特性的观察分析,发现2个颜色异常,6个菌落生长缓慢,8个产孢量显著降低,2个分生孢子形态异常,2个附着胞形态异常,3个不能形成附着胞,在洋葱表皮上侵染钉形成率显著降低。
对这15个突变体的基因组DNA进行PCR扩增,结果全部都扩增到潮霉素磷酸转移酶(hph)基因序列,而野生型基因组DNA扩增不到,表明突变体的表型变化是由于外源DNA插入所致。采用TAIL-PCR的方法对这15个突变体基因组DNA进行扩增,得到特异扩增片段,并对这些DNA序列进行克隆和测序,获得3条序列信息,将这些序列在NCBI网站上进行Blast搜索,结果如下：
突变体T-0900-1的T-DNA插入位点右边界克隆到1275bp片段,其中950bp-1143bp的134bp与脉孢菌(Neurospora)的一个保守假设蛋白同源性最高,同时966bp-1143bp的113bp与稻瘟菌(Magnaporthe grisea)的70-15保守假设蛋白(MGG_03680)也具有很高同源性。T-0003-3的T-DNA插入位点右边界克隆到688bp片段,其中1bp-240bp、240bp-301bp的序列与耐热子囊菌(Neosartorya fischeri)、棒状曲霉(Aspergillus fumigatus)真核翻译起始因子3同源性很高。T-1103-2左边界克隆到712bp片段BLAST分析未找到与其同源性较高的基因序列,推测其被破坏的基因是目前在真菌中生物功能尚未确定的基因。
Rubber plant anthracnose, one of the most serious rubber foliar diseases, mainly caused by the infection of Colletotrichum gloeosporioides (Penz.) Sacc.The Construction of anthracnose ATMT (Agrobacterium tumefaciens-Mediated Transformation) transformants library, identification of the relevant mutant, using molecular biology techniques to isolate and identificate the disease related genes of Colletotrichum gloeosporioides (Penz.) Sacc, is the way to understand its pathogenesis and mechanism of interaction important tool.This is not only important in plant pathology, but also significant for the production of disease control and further explore new ways to provide clues for the breeding of durable resistant varieties, at the same time, it provide theoretical basis for the developing sustainable management strategies Colletotrichum gloeosporioides (Penz.).
In the study, we mutated Colletotrichum gloeosporioides RC-178 with T-DNA insertion Agrobacterium tumefaciens-mediated transformation (ATMT), using the promoter trapping vector pCAHPH which has been constructed by our lab. We increased the number of the T-DNA inserted mutants to 1,685.15 transformants which reduced greatly in pathogenicitv were selected for detection. Observation of the biological properties of these mutants and found that two color abnormalities, three colonies grow slowly, eight sporulation was significantly reduced, two conidia morphology abnormalities, two attached to abnormal cell morphology,3 could not form appressoria, in the onion skin infection nail on the formation rate decreased significantly.
These 15 mutants were tested with PCR for their insertional sequence, the results showed that all the genomes of these mutants contained the sequence of hygromycin phosphotransferase (hph) gene, while the wild type genomic DNA amplification of less than, that means mutant phenotype is as the result of exogenous DNA insertion. TAIL-PCR method using these 15 mutant genomic DNA were amplified by specific fragment, and these DNA sequences were cloned and sequenced to obtain three sequences, these sequences in the NCBI Blast search on the site.The results are as follows:
A 1275bp fragment was cloned from Mutant T-0900-1 to sites of T-DNA insertion of the right sector, in which there are 134bp (950bp-1143bp)and Neurospora as a conservative assumption that the highest protein homology, while the 113bp (966bp-1143bp) and the Magnaporthe grisea in the 70-15 conserved hypothetical protein (MGG_03680) also has high homology. T-0003-3 of T-DNA insertion sites cloned 688bp fragment of the right sector, Flanking sequence 1bp-240bp and 240bp-301bp of sequence and Neosartorya fischeri, Aspergillus fumigatus eukaryotic translation initiation factor 3 are highly homologous.712bp fragment of T-1103-2 was cloned into the left border is not found with BLAST analysis of homologous gene sequences, we speculate that the gene is damaged in the fungi in the biological function of genes has not been determined.
本研究利用启动子捕获技术通过农杆菌介导的遗传转化体系构建橡胶炭疽病菌RC-178的突变体库,使转化子数目增加到1685个。通过致病力的测定,从该突变体库中筛选到15个致病力明显减弱或丧失的突变体。对这15个突变体进行了生物学特性的观察分析,发现2个颜色异常,6个菌落生长缓慢,8个产孢量显著降低,2个分生孢子形态异常,2个附着胞形态异常,3个不能形成附着胞,在洋葱表皮上侵染钉形成率显著降低。
对这15个突变体的基因组DNA进行PCR扩增,结果全部都扩增到潮霉素磷酸转移酶(hph)基因序列,而野生型基因组DNA扩增不到,表明突变体的表型变化是由于外源DNA插入所致。采用TAIL-PCR的方法对这15个突变体基因组DNA进行扩增,得到特异扩增片段,并对这些DNA序列进行克隆和测序,获得3条序列信息,将这些序列在NCBI网站上进行Blast搜索,结果如下：
突变体T-0900-1的T-DNA插入位点右边界克隆到1275bp片段,其中950bp-1143bp的134bp与脉孢菌(Neurospora)的一个保守假设蛋白同源性最高,同时966bp-1143bp的113bp与稻瘟菌(Magnaporthe grisea)的70-15保守假设蛋白(MGG_03680)也具有很高同源性。T-0003-3的T-DNA插入位点右边界克隆到688bp片段,其中1bp-240bp、240bp-301bp的序列与耐热子囊菌(Neosartorya fischeri)、棒状曲霉(Aspergillus fumigatus)真核翻译起始因子3同源性很高。T-1103-2左边界克隆到712bp片段BLAST分析未找到与其同源性较高的基因序列,推测其被破坏的基因是目前在真菌中生物功能尚未确定的基因。
Rubber plant anthracnose, one of the most serious rubber foliar diseases, mainly caused by the infection of Colletotrichum gloeosporioides (Penz.) Sacc.The Construction of anthracnose ATMT (Agrobacterium tumefaciens-Mediated Transformation) transformants library, identification of the relevant mutant, using molecular biology techniques to isolate and identificate the disease related genes of Colletotrichum gloeosporioides (Penz.) Sacc, is the way to understand its pathogenesis and mechanism of interaction important tool.This is not only important in plant pathology, but also significant for the production of disease control and further explore new ways to provide clues for the breeding of durable resistant varieties, at the same time, it provide theoretical basis for the developing sustainable management strategies Colletotrichum gloeosporioides (Penz.).
In the study, we mutated Colletotrichum gloeosporioides RC-178 with T-DNA insertion Agrobacterium tumefaciens-mediated transformation (ATMT), using the promoter trapping vector pCAHPH which has been constructed by our lab. We increased the number of the T-DNA inserted mutants to 1,685.15 transformants which reduced greatly in pathogenicitv were selected for detection. Observation of the biological properties of these mutants and found that two color abnormalities, three colonies grow slowly, eight sporulation was significantly reduced, two conidia morphology abnormalities, two attached to abnormal cell morphology,3 could not form appressoria, in the onion skin infection nail on the formation rate decreased significantly.
These 15 mutants were tested with PCR for their insertional sequence, the results showed that all the genomes of these mutants contained the sequence of hygromycin phosphotransferase (hph) gene, while the wild type genomic DNA amplification of less than, that means mutant phenotype is as the result of exogenous DNA insertion. TAIL-PCR method using these 15 mutant genomic DNA were amplified by specific fragment, and these DNA sequences were cloned and sequenced to obtain three sequences, these sequences in the NCBI Blast search on the site.The results are as follows:
A 1275bp fragment was cloned from Mutant T-0900-1 to sites of T-DNA insertion of the right sector, in which there are 134bp (950bp-1143bp)and Neurospora as a conservative assumption that the highest protein homology, while the 113bp (966bp-1143bp) and the Magnaporthe grisea in the 70-15 conserved hypothetical protein (MGG_03680) also has high homology. T-0003-3 of T-DNA insertion sites cloned 688bp fragment of the right sector, Flanking sequence 1bp-240bp and 240bp-301bp of sequence and Neosartorya fischeri, Aspergillus fumigatus eukaryotic translation initiation factor 3 are highly homologous.712bp fragment of T-1103-2 was cloned into the left border is not found with BLAST analysis of homologous gene sequences, we speculate that the gene is damaged in the fungi in the biological function of genes has not been determined.
引文
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