一粒系小麦Pina基因与小麦生防细菌荧光假单胞菌研究
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摘要
小麦适应性强,分布广,用途多,是世界上最重要的粮食作物,其总面积、总产量及总贸易额均居粮食作物第一位,是全世界1/3以上人口的主要食粮。在我国,小麦的地位仅次于水稻。小麦的品质和抗病性是小麦研究的重点领域。本实验主要研究了影响一粒系小麦籽粒硬度的Pina基因和拮抗小麦真菌病害的生防细菌荧光假单胞菌,主要研究结果如下：
1.以一粒系小麦为载体,采用PCR方法分离克隆一粒系小麦籽粒硬度蛋白Puroindoline a基因。得到了56份一粒系小麦影响籽粒硬度的Pina基因序列,并进行了单核苷酸多态性(SNP)分析和单倍型分析。在获得的1595 bp核苷酸序列中,有1270个保守位点,21个SNP位点和16个插入缺失(Indels)。分析得到14种单倍型,其发生频率由1到18不等。在野生一粒、栽培一粒和乌拉尔图小麦中,分别有9、8和2种单倍型。系统发育分析发现来自不同染色体组来源(Am and Au)的Pina能通过开放阅读框分析清楚地区分开来,这些研究为进一步研究一粒系小麦Pina基因功能和利用打好了基础。
2.针对小麦真菌病害(全蚀病、立枯病、赤霉病)的生物防治细菌筛选鉴定试验,以分离自加拿大渥太华地区5个试验点土样的约300株荧光假单胞菌为研究对象,运用分子生物学技术,系统的研究荧光假单胞菌的遗传多样性。
3.针对其中一土样分离得到的143株假单胞菌株中可分为两种表型群体FormⅠ和Ⅱ。运用电子显微镜和BOX-PCR、ERIC-PCR和新的Miniprimer-PCR技术以及16S rRNA和gacA基因序列测序,系统研究分离菌株不同表型和基因型的特性。基因组指纹图谱BOX-和ERIC-PCR的结果相近而不完全相同。与预期结果一致,新的图谱分析方法Miniprimer-PCR与BOX-和ERIC-PCR的相似性则较低,因其是基于16S rRNA序列设计的引物。16S rRNA基因测序及系统进化分析将菌株聚为3个主要类群,包括荧光假单胞菌(P. fluorescens)、韩国丛毛假单胞菌(P.koreensis)和恶臭假单胞菌(P.putida)。gacA基因序列聚类分析图与16S rRNA序列聚类分析图具有拓扑一致性,结果相互支持。Tajima's D统计分析发现FormⅠ菌群间有低频率的多态性,说明发生了群落膨胀或正向选择,这与16S rRNABLAST结果发现FormⅠ菌株遗传差异大相吻合。不同研究方法的结果均显示可能存在一种新的假单胞菌基因型。聚类分析也发现在加拿大农业土壤中可能存在新近确定的,只在韩国土壤环境中发现的韩国假单胞菌(Pseudomonas koreensis),这证明该种群具有更广泛的地理分布性。
4.通过对143株分离菌进行平板拮抗反应,筛选得到12株对三种小麦真菌病害的病原菌具有抑制作用的潜在生防菌株。实验对筛选获得的生防菌株设计了充足的鉴定试验,将分离所得拮抗菌株与已知并深入研究的生防细菌Pf-5,从生化与分子生物学各方面进行比较。通过Biolog phenotype microarrays和API 20NE试剂盒,并送公司检测磷酸脂肪酸图谱(FAME),以及运用gacA基因和16S rRNA基因全长序列,通过系统进化树分析,对生防菌株的分子生物学和生物化学多样性进行了详尽的描述。拮抗细菌的生物化学试验结果相似,除了一些小分歧以外,显示其与Pf-5菌株具有接近的表型关系,这与gacA基因序列分析结果一致。实验结果证明,BIOLOG PM板、API 20NE试剂盒和脂肪酸甲酯分析(FAME)更适用于研究菌株生化特性,而16S rRNA基因全长序列系统进化在鉴定新分离菌株种属方面更具有优势。
Wheat is one of the most important grain crops in the world with its strong adaptability, wide spreading and multi-purpose. Its total growth area, yield and trade took the first place among all the grain crops respectively. There is 1/3 of world population fed on wheat product. In China, wheat is the dominating crops just on the neck of rice. Nowadays, more and more researchers focused on the study of wheat seed quality and disease resistant. Our study is mainly about the molecular characterization of Pina gene of einkorn wheat and the potential biocontrol agent of wheat pathogens-Pseudomonas fluorescens. The following are key results:
1. Fifty-six sequences encoding the pina protein were characterized from three species or subspecies of einkorn wheat. These sequences contained 1,595 nucleotides, including 1,270 conserved sites,21 single nucleotide polymorphisms (SNPs), and 16 indels. The average frequency of SNPs and indels was one out of 76.1 and 99.9 bases, respectively. Five SNPs and no indels were found in the translated sequences. Fourteen haplotypes were defined, and the accessions in each haplotype ranged froml to 18. There were nine haplotypes in Triticum monococcum ssp. aegilopoides, eight in T. monococcum ssp. monococcum, and two in T. urartu. Phylogenetic analysis showed that Pina genes from different species or subspecies could be clearly differentiated based on the open reading frame. Genes from T. urartu grouped together, whereas genes from T. monococcum ssp. aegilopoides and T. monococcum ssp. monococcum were shared by three and two clusters, respectively. Both the haplotype and phylogenetic analyses indicated that T. monococcum ssp. aegilopoides was more diverse. These results would contribute to the understanding of functional aspects and efficient utilization of Pina genes.
2. In our study, a polyphasic molecular approach was used to study the biodiversity of close to 300 fluorescent Pseudomonas strains isolated from long-term (>30 years) mineral fertilization experimental agricultural plots.
3. One hundred and forty-three pseudomonads were isolated from one of the field plot were chosen for more intensive studies. The strains were grouped into two distinct phenotypes (Form I and II) based on colony morphology and growth patterns on King's B medium. Further characterization was performed by electron microscopy, BOX, ERIC, Miniprimer PCR and 16S rRNA and gacA sequencing. The genomic fingerprints from ERIC and BOX were similar but not identical. As expected, fingerprints derived from miniprimer PCR exhibited comparatively lower correlations to ERIC and BOX. Sequencing and phylogenetic analyses of the 16S rRNA gene identified three major groups, consisting of P. fluorescens, P. koreensis and P. putida. Tajima's D statistics applied to the 16S rRNA sequences of the two subpopulations indicated an excess of low frequency polymorphisms within strains of form I, suggesting a population size expansion and or positive selection. This is corroborated by the 16S rRNA BLAST search in that the form I strains are more diverse. The methods employed allowed for the characterization of the bacterial strains leading to a tentative identification of potentially a new species within the genus Pseudomonas. The characterization also revealed the potential presence in Canadian soils of Pseudomonas koreensis, a recently described new species, leading to the conclusion that this new species could have a wider distribution.
4. The characterized bacterial strains were evaluated in vitro for their ability to suppress major soilborne fungal pathogens of wheat, leading to the identification of 12 effective bacterial biocontrol agents. The potential biocontrol strains phenotypically and genotypically characterized relative to a well-known biological control strain pf-5. Phenotypic methods used included Biolog, API 20NE assays and FAMEs. Genotypic methods used included REP-PCR and gacA gene sequencing, the phylogenetic tree inferred from all the 52 type strains of P. fluorescens subgroup from flurescent Pseudomonas genus and the isolated strains. By using phenotypic tests, the cluster results were similar with each other except some small differences by dividing these strains into two groups with a close relation with Pf-5 together, which was supported by gacA sequence analysis. API 20NE, Biolog PM and FAME proved to be the best for characterization of isolates, whereas 16S rRNA sequence analysis were found to be the best resolution for preliminarily identification of these newly isolated strains.
1.以一粒系小麦为载体,采用PCR方法分离克隆一粒系小麦籽粒硬度蛋白Puroindoline a基因。得到了56份一粒系小麦影响籽粒硬度的Pina基因序列,并进行了单核苷酸多态性(SNP)分析和单倍型分析。在获得的1595 bp核苷酸序列中,有1270个保守位点,21个SNP位点和16个插入缺失(Indels)。分析得到14种单倍型,其发生频率由1到18不等。在野生一粒、栽培一粒和乌拉尔图小麦中,分别有9、8和2种单倍型。系统发育分析发现来自不同染色体组来源(Am and Au)的Pina能通过开放阅读框分析清楚地区分开来,这些研究为进一步研究一粒系小麦Pina基因功能和利用打好了基础。
2.针对小麦真菌病害(全蚀病、立枯病、赤霉病)的生物防治细菌筛选鉴定试验,以分离自加拿大渥太华地区5个试验点土样的约300株荧光假单胞菌为研究对象,运用分子生物学技术,系统的研究荧光假单胞菌的遗传多样性。
3.针对其中一土样分离得到的143株假单胞菌株中可分为两种表型群体FormⅠ和Ⅱ。运用电子显微镜和BOX-PCR、ERIC-PCR和新的Miniprimer-PCR技术以及16S rRNA和gacA基因序列测序,系统研究分离菌株不同表型和基因型的特性。基因组指纹图谱BOX-和ERIC-PCR的结果相近而不完全相同。与预期结果一致,新的图谱分析方法Miniprimer-PCR与BOX-和ERIC-PCR的相似性则较低,因其是基于16S rRNA序列设计的引物。16S rRNA基因测序及系统进化分析将菌株聚为3个主要类群,包括荧光假单胞菌(P. fluorescens)、韩国丛毛假单胞菌(P.koreensis)和恶臭假单胞菌(P.putida)。gacA基因序列聚类分析图与16S rRNA序列聚类分析图具有拓扑一致性,结果相互支持。Tajima's D统计分析发现FormⅠ菌群间有低频率的多态性,说明发生了群落膨胀或正向选择,这与16S rRNABLAST结果发现FormⅠ菌株遗传差异大相吻合。不同研究方法的结果均显示可能存在一种新的假单胞菌基因型。聚类分析也发现在加拿大农业土壤中可能存在新近确定的,只在韩国土壤环境中发现的韩国假单胞菌(Pseudomonas koreensis),这证明该种群具有更广泛的地理分布性。
4.通过对143株分离菌进行平板拮抗反应,筛选得到12株对三种小麦真菌病害的病原菌具有抑制作用的潜在生防菌株。实验对筛选获得的生防菌株设计了充足的鉴定试验,将分离所得拮抗菌株与已知并深入研究的生防细菌Pf-5,从生化与分子生物学各方面进行比较。通过Biolog phenotype microarrays和API 20NE试剂盒,并送公司检测磷酸脂肪酸图谱(FAME),以及运用gacA基因和16S rRNA基因全长序列,通过系统进化树分析,对生防菌株的分子生物学和生物化学多样性进行了详尽的描述。拮抗细菌的生物化学试验结果相似,除了一些小分歧以外,显示其与Pf-5菌株具有接近的表型关系,这与gacA基因序列分析结果一致。实验结果证明,BIOLOG PM板、API 20NE试剂盒和脂肪酸甲酯分析(FAME)更适用于研究菌株生化特性,而16S rRNA基因全长序列系统进化在鉴定新分离菌株种属方面更具有优势。
Wheat is one of the most important grain crops in the world with its strong adaptability, wide spreading and multi-purpose. Its total growth area, yield and trade took the first place among all the grain crops respectively. There is 1/3 of world population fed on wheat product. In China, wheat is the dominating crops just on the neck of rice. Nowadays, more and more researchers focused on the study of wheat seed quality and disease resistant. Our study is mainly about the molecular characterization of Pina gene of einkorn wheat and the potential biocontrol agent of wheat pathogens-Pseudomonas fluorescens. The following are key results:
1. Fifty-six sequences encoding the pina protein were characterized from three species or subspecies of einkorn wheat. These sequences contained 1,595 nucleotides, including 1,270 conserved sites,21 single nucleotide polymorphisms (SNPs), and 16 indels. The average frequency of SNPs and indels was one out of 76.1 and 99.9 bases, respectively. Five SNPs and no indels were found in the translated sequences. Fourteen haplotypes were defined, and the accessions in each haplotype ranged froml to 18. There were nine haplotypes in Triticum monococcum ssp. aegilopoides, eight in T. monococcum ssp. monococcum, and two in T. urartu. Phylogenetic analysis showed that Pina genes from different species or subspecies could be clearly differentiated based on the open reading frame. Genes from T. urartu grouped together, whereas genes from T. monococcum ssp. aegilopoides and T. monococcum ssp. monococcum were shared by three and two clusters, respectively. Both the haplotype and phylogenetic analyses indicated that T. monococcum ssp. aegilopoides was more diverse. These results would contribute to the understanding of functional aspects and efficient utilization of Pina genes.
2. In our study, a polyphasic molecular approach was used to study the biodiversity of close to 300 fluorescent Pseudomonas strains isolated from long-term (>30 years) mineral fertilization experimental agricultural plots.
3. One hundred and forty-three pseudomonads were isolated from one of the field plot were chosen for more intensive studies. The strains were grouped into two distinct phenotypes (Form I and II) based on colony morphology and growth patterns on King's B medium. Further characterization was performed by electron microscopy, BOX, ERIC, Miniprimer PCR and 16S rRNA and gacA sequencing. The genomic fingerprints from ERIC and BOX were similar but not identical. As expected, fingerprints derived from miniprimer PCR exhibited comparatively lower correlations to ERIC and BOX. Sequencing and phylogenetic analyses of the 16S rRNA gene identified three major groups, consisting of P. fluorescens, P. koreensis and P. putida. Tajima's D statistics applied to the 16S rRNA sequences of the two subpopulations indicated an excess of low frequency polymorphisms within strains of form I, suggesting a population size expansion and or positive selection. This is corroborated by the 16S rRNA BLAST search in that the form I strains are more diverse. The methods employed allowed for the characterization of the bacterial strains leading to a tentative identification of potentially a new species within the genus Pseudomonas. The characterization also revealed the potential presence in Canadian soils of Pseudomonas koreensis, a recently described new species, leading to the conclusion that this new species could have a wider distribution.
4. The characterized bacterial strains were evaluated in vitro for their ability to suppress major soilborne fungal pathogens of wheat, leading to the identification of 12 effective bacterial biocontrol agents. The potential biocontrol strains phenotypically and genotypically characterized relative to a well-known biological control strain pf-5. Phenotypic methods used included Biolog, API 20NE assays and FAMEs. Genotypic methods used included REP-PCR and gacA gene sequencing, the phylogenetic tree inferred from all the 52 type strains of P. fluorescens subgroup from flurescent Pseudomonas genus and the isolated strains. By using phenotypic tests, the cluster results were similar with each other except some small differences by dividing these strains into two groups with a close relation with Pf-5 together, which was supported by gacA sequence analysis. API 20NE, Biolog PM and FAME proved to be the best for characterization of isolates, whereas 16S rRNA sequence analysis were found to be the best resolution for preliminarily identification of these newly isolated strains.
引文
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