棉花黄萎病菌T-DNA插入突变体表型特征及侧翼序列分析
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摘要
棉花黄萎病(Verticllium wilt)是由大丽轮枝菌(Verticillium dahliae kleb)引起的土传维管束病害,每年在世界范围内给棉花生产造成重大损失。多年来,国内外学者对大丽轮枝菌的致病机理进行了大量研究,但有关其致病的分子机制研究却非常滞后。本研究以我国落叶型棉花黄萎病菌VD991为野生型菌株,通过农杆菌介导的遗传转化,获得了大量大丽轮枝菌的T-DNA插入突变体,并进行了转化条件优化、突变体表型鉴定和插入位点侧翼序列的分析等研究,建立了大丽轮枝菌T-DNA插入突变和致病相关基因筛选研究的技术平台,为开展黄萎病菌致病的分子机制研究奠定了基础。
在Mullins建立的尖孢镰刀菌(Fusarium oxysporum)转化方法基础上进一步优化了介导黄萎病菌T-DNA插入突变的条件,获得插入突变体2628个。经过分子验证和连续继代培养,证明这些突变体的抗性遗传稳定。部分突变体的表型鉴定结果表明：(1)VD991在正常恒温培养时并不产生微菌核,突变后部分突变体由菌丝型变成了菌核型,在培养5-7天开始产生大量微菌核,这部分突变体约占7.3%左右；(2)无论在MM还是PDA固体培养基上,突变体的生长速度普遍小于野生型菌株。在摇瓶培养条件下,大部分突变体产孢量的高峰在培养后第5-6天；菌丝型突变体的产孢能力和野生型接近或略低,而大部分菌核型突变体的产孢能力都高于野生型VD991,最高的达到VD991的4.9倍；(3)胞外酶检测结果表明：在平板培养时,VD991可以产生胞外蛋白酶、淀粉酶、纤维素酶和果胶酶,并能在以几丁质为惟一碳源的查比克培养基上生长。筛选出不能在MM培养基上正常生长的营养缺陷型突变体8个,不能利用脱脂奶粉的突变体3个,不能利用可溶性淀粉的突变体5个,果胶酶分泌降低的突变体7个；(4)利用棉花幼苗孢子悬浮液茎部穿刺和灌根接种方法,对部分突变体的致病性进行了鉴定,筛选出致病力减弱的突变体6个。
采用hiTAIL-PCR的方法,获得了30个突变体T-DNA插入位点的右侧序列,与公布的大丽轮枝菌VDLs.17和黑白轮枝菌VaMs.102基因组序列比对结果表明：(1)侧翼序列与大丽轮枝菌VDLS.17DNA序列的一致性,均在95-100%之间,与黑白轮枝菌VaMs.102的一致性较低,为87-94%,说明VDLs.17基因组可以用作VD991功能基因研究的参考序列；(2)VD991和VDLs.17序列之间,除存在大量的单碱基差异和缺口外,有少数基因序列间表现出片段的插入和重新排序；(3)有7个突变体T-DNA插在基因编码区,13个突变体插入在基因编码序列上游500bp以内,这可能直接影响了下游基因的表达；(4)根据插入位置和表型分析,5个突变体T-DNA插入位点下游基因可以作为功能基因研究的候选基因；(5)突变体H20侧翼序列在大丽轮枝菌VDLs.17中未找到匹配序列,而在黑白轮枝菌VaMs.102基因组中存在一致性88%的同源序列,这在进化上是如何形成的值得进一步研究；(6)将30个突变体的T-DNA插入位置进行对比,发现它们插入位点不同,并广泛分布在所有8个染色体上,进一步证明了T-DNA插入事件的随机性。
Verticillium dahliae is the causal agent of the soil-borne vascular wilt disease, which results in severe yield and quality losses in cotton worldwide every year. Over the years, many studies were done by the domestic and foreign scholars on about the pathogenic mechanism of Verticillium dahliae, but little achievements were obtained. In this study, using the defoliated Verticillium wilt strain VD991as a wild type strain, a large number of mutants were constructed by the method of Agrobacterium tumefaciens mediaed transformation (ATMT). Subsequently, the optimizing of transformation protocol, the identification of mutants phenotype and their pathogenicity, and the flanking sequences of T-DNA insertional sites were studied. Accordingly, the technical platform on studying the T-DNA insertional mutation and the cloning of the pathogenicity related genes were built in our laboratory. This was a foundation for the study of pathogenic molecular mechanisms in Verticillium dahliae.
Based on the transformation method of Fusarium oxysporum described by Mullins, the parameters of T-DNA insertional mutation for Verticillium dahliae were optimized.2628T-DNA insertional mutants were obtained and the stability of resistance heredity was testified by series culture and molecular verification. Some of the mutants were selected for the phenotype identification, the results indicated that:(1) VD991did not produce microsclerotia when cultivating at normal temperature on PDA. Some mutants, accounted for7.3%, changed from the original mycelial type into a sclerotium type, and producing large amount of microsclerotia at5-7d post cultivation.(2) The growth rate of mutants was slightly slower than that of wild-type strains on either MM or PDA medium. In shake-flask cultivation, the sporulation peak of most mutants was at5-6d post-inoculation. The sporulation capacity of mycelial type mutants was close to or slightly lower than that of wild-type, however, the sporulation capacity of sclerotium type mutants were higher than that of VD991, even up to4.9times.(3) The determination results of extracellular enzyme showed that VD991could produce extracellular protease, amylase, cellulase and pectinase, and it could grow on Czapek medium appending chitin as only carbon sources. Eight auxotrophic mutants that could not grow well on MM medium, three mutants that could not use skimmed milk powder, five mutants that could not make use of soluble starch and seven mutants with pectinase secretion reduced were selected.(4) Using the inoculation methods of root-dipping and stem puncturing with spore suspensions on cotton seedlings, the virulence of some mutants were identified. Six mutants that virulence reduced comparing with VD991were screened.
The right flanking sequences of T-DNA insertional sites of30mutants were obtained by using the method of hiTAIL-PCR, and the results BLAST to the database of Verticillium dahliae VDLs.17and Verticillium albo-atrum VaMs.102genomes indicated that:(1) The flanking sequences showed95-100%identity over its full length to the corresponding site of VDLs.17, and87-94%identity to that of VaMs.102. Therefore, the genome sequence of VDLs.17could be used as a conference sequence for the functional genome research.(2) Between the sequences of VD991and VDLs.17, with the exception of the existence of a large number of single base differences and gaps, there are a small number of gene sequences showed fragments insertion and rearrangement;(3) There were7mutants with T-DNA inserted in coding region, thirteen mutants with insertional sites located in the range of500bp upstream of start codon may have direct impact on the expression of its downstream genes.(4) The downstream genes of T-DNA insertional sites of5mutants could serve as candidate genes for functional genetic research.(5) The matching sequence to H20flanking sequence could not be found in Verticillium dahliae VDLs.17, but it existed in Verticillium albo-atrum VaMs.102and shared homologous of88%. Further study should be done on how it happened on evolution.(6) Comparing the T-DNA insertional sites of30mutants showed that they were different from each other, and widely distributed in all8chromosomes. This demonstrated that the T-DNA insertional events happened randomly.
在Mullins建立的尖孢镰刀菌(Fusarium oxysporum)转化方法基础上进一步优化了介导黄萎病菌T-DNA插入突变的条件,获得插入突变体2628个。经过分子验证和连续继代培养,证明这些突变体的抗性遗传稳定。部分突变体的表型鉴定结果表明：(1)VD991在正常恒温培养时并不产生微菌核,突变后部分突变体由菌丝型变成了菌核型,在培养5-7天开始产生大量微菌核,这部分突变体约占7.3%左右；(2)无论在MM还是PDA固体培养基上,突变体的生长速度普遍小于野生型菌株。在摇瓶培养条件下,大部分突变体产孢量的高峰在培养后第5-6天；菌丝型突变体的产孢能力和野生型接近或略低,而大部分菌核型突变体的产孢能力都高于野生型VD991,最高的达到VD991的4.9倍；(3)胞外酶检测结果表明：在平板培养时,VD991可以产生胞外蛋白酶、淀粉酶、纤维素酶和果胶酶,并能在以几丁质为惟一碳源的查比克培养基上生长。筛选出不能在MM培养基上正常生长的营养缺陷型突变体8个,不能利用脱脂奶粉的突变体3个,不能利用可溶性淀粉的突变体5个,果胶酶分泌降低的突变体7个；(4)利用棉花幼苗孢子悬浮液茎部穿刺和灌根接种方法,对部分突变体的致病性进行了鉴定,筛选出致病力减弱的突变体6个。
采用hiTAIL-PCR的方法,获得了30个突变体T-DNA插入位点的右侧序列,与公布的大丽轮枝菌VDLs.17和黑白轮枝菌VaMs.102基因组序列比对结果表明：(1)侧翼序列与大丽轮枝菌VDLS.17DNA序列的一致性,均在95-100%之间,与黑白轮枝菌VaMs.102的一致性较低,为87-94%,说明VDLs.17基因组可以用作VD991功能基因研究的参考序列；(2)VD991和VDLs.17序列之间,除存在大量的单碱基差异和缺口外,有少数基因序列间表现出片段的插入和重新排序；(3)有7个突变体T-DNA插在基因编码区,13个突变体插入在基因编码序列上游500bp以内,这可能直接影响了下游基因的表达；(4)根据插入位置和表型分析,5个突变体T-DNA插入位点下游基因可以作为功能基因研究的候选基因；(5)突变体H20侧翼序列在大丽轮枝菌VDLs.17中未找到匹配序列,而在黑白轮枝菌VaMs.102基因组中存在一致性88%的同源序列,这在进化上是如何形成的值得进一步研究；(6)将30个突变体的T-DNA插入位置进行对比,发现它们插入位点不同,并广泛分布在所有8个染色体上,进一步证明了T-DNA插入事件的随机性。
Verticillium dahliae is the causal agent of the soil-borne vascular wilt disease, which results in severe yield and quality losses in cotton worldwide every year. Over the years, many studies were done by the domestic and foreign scholars on about the pathogenic mechanism of Verticillium dahliae, but little achievements were obtained. In this study, using the defoliated Verticillium wilt strain VD991as a wild type strain, a large number of mutants were constructed by the method of Agrobacterium tumefaciens mediaed transformation (ATMT). Subsequently, the optimizing of transformation protocol, the identification of mutants phenotype and their pathogenicity, and the flanking sequences of T-DNA insertional sites were studied. Accordingly, the technical platform on studying the T-DNA insertional mutation and the cloning of the pathogenicity related genes were built in our laboratory. This was a foundation for the study of pathogenic molecular mechanisms in Verticillium dahliae.
Based on the transformation method of Fusarium oxysporum described by Mullins, the parameters of T-DNA insertional mutation for Verticillium dahliae were optimized.2628T-DNA insertional mutants were obtained and the stability of resistance heredity was testified by series culture and molecular verification. Some of the mutants were selected for the phenotype identification, the results indicated that:(1) VD991did not produce microsclerotia when cultivating at normal temperature on PDA. Some mutants, accounted for7.3%, changed from the original mycelial type into a sclerotium type, and producing large amount of microsclerotia at5-7d post cultivation.(2) The growth rate of mutants was slightly slower than that of wild-type strains on either MM or PDA medium. In shake-flask cultivation, the sporulation peak of most mutants was at5-6d post-inoculation. The sporulation capacity of mycelial type mutants was close to or slightly lower than that of wild-type, however, the sporulation capacity of sclerotium type mutants were higher than that of VD991, even up to4.9times.(3) The determination results of extracellular enzyme showed that VD991could produce extracellular protease, amylase, cellulase and pectinase, and it could grow on Czapek medium appending chitin as only carbon sources. Eight auxotrophic mutants that could not grow well on MM medium, three mutants that could not use skimmed milk powder, five mutants that could not make use of soluble starch and seven mutants with pectinase secretion reduced were selected.(4) Using the inoculation methods of root-dipping and stem puncturing with spore suspensions on cotton seedlings, the virulence of some mutants were identified. Six mutants that virulence reduced comparing with VD991were screened.
The right flanking sequences of T-DNA insertional sites of30mutants were obtained by using the method of hiTAIL-PCR, and the results BLAST to the database of Verticillium dahliae VDLs.17and Verticillium albo-atrum VaMs.102genomes indicated that:(1) The flanking sequences showed95-100%identity over its full length to the corresponding site of VDLs.17, and87-94%identity to that of VaMs.102. Therefore, the genome sequence of VDLs.17could be used as a conference sequence for the functional genome research.(2) Between the sequences of VD991and VDLs.17, with the exception of the existence of a large number of single base differences and gaps, there are a small number of gene sequences showed fragments insertion and rearrangement;(3) There were7mutants with T-DNA inserted in coding region, thirteen mutants with insertional sites located in the range of500bp upstream of start codon may have direct impact on the expression of its downstream genes.(4) The downstream genes of T-DNA insertional sites of5mutants could serve as candidate genes for functional genetic research.(5) The matching sequence to H20flanking sequence could not be found in Verticillium dahliae VDLs.17, but it existed in Verticillium albo-atrum VaMs.102and shared homologous of88%. Further study should be done on how it happened on evolution.(6) Comparing the T-DNA insertional sites of30mutants showed that they were different from each other, and widely distributed in all8chromosomes. This demonstrated that the T-DNA insertional events happened randomly.
引文
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