香蕉枯萎病菌进化研究和Foc TR4致病相关效应蛋白的鉴定及功能分析
发布时间：2019-07-06 20:00
【摘要】：香蕉是世界上最重要的水果之一,也是仅次于水稻、小麦和玉米的第四大粮食作物,因此香蕉生产攸关世界粮食安全、地区发展和人类健康(Ploetz R C,2006)。但是,香蕉枯萎病菌热带4号生理小种(Fusarium oxysporum f.sp.cubense Tropical race 4)的大规模爆发和流行,已严重危及到该产业的可持续发展。为了防控该病害,本课题对来自不同香蕉生产国的56个Foc菌株(涵盖4个生理小种、24个营养体亲合群(Vegetative Compatibility Groups,VCGs))进行了基因组重测序,利用比较基因组学研究了全球范围内病原菌的进化规律、鉴定了Foc TR4效应蛋白,并对重要的蛋白开展功能分析,具体结果如下:1香蕉枯萎病菌基因组重测序及生物信息学分析(1)基因组重测序及组装:对来自不同香蕉生产国的55个Foc菌株(涵盖4个生理小种、24个VCGs),包括6株Foc TR4,8株Foc STR4(亚热带4号小种),35株Foc race 1,2株Foc race2,2株大蕉Foc,2株非致病性尖孢镰刀菌,进行了基因组重测序,测序覆盖度均在50倍以上。测序的原始数据经过滤后组装成Contigs和Supercontigs。(2)基因组Core和LS-Region的区分结合公布在Broad Institute网站上的14种镰刀菌基因组序列,预测了Foc TR4菌株II5的核心基因组(Core genome或者regions)和Lineage-specific regions(LS-Regions),并以此为参考,采用“剔除核心基因组”(Eliminating the Core Genome)的方法鉴定各个测序菌株的Core regiongs和LS regions,结果显示不同类型菌株间的Core和LS-Region的大小存在显著差异。(3)全球范围内的香蕉枯萎病菌的进化规律利用3种方法研究了全球范围内的Foc的进化规律:(1)根据10个看家基因绘制的进化树;(2)全基因组范围内的SNP绘制的进化树;(3)主成分分析法(Principal Component Analysis,PCA),发现Foc TR4菌株进化非常保守,是单一起源的,并且进化速度非常快;其它类型菌株如Foc STR4和Race 1则进化比较复杂,有多个进化源头;大蕉枯萎病菌由Race 1进化而来,克服了对Race 1、Foc TR4和Foc STR4有高度抗性的大蕉的免疫系统。(4)香蕉枯萎病菌交配型的分析收集交配型位点MAT1-1和MAT1-2基因序列作为对照,将组装的序列进行Mapping,获得不同类型菌株的交配型基因,结果显示香蕉枯萎病菌为异宗配合真菌,而Foc TR4(VCG01213,01216和01213/16)为MAT1-1类型。(5)香蕉枯萎病菌效应蛋白的预测根据结构特征,预测了各测序菌株的效应蛋白。结合公布在Broad Institute网站上的14种镰刀菌基因组序列,利用比较基因组学方法鉴定到各VCGs特异的效应蛋白,也对各生理小种特异的效应蛋白进行分析,鉴定到Foc Race1有24个、Foc STR4有47个、Foc TR4有37个。2 Foc TR4侵染过程中效应蛋白的表达规律将Foc TR4接种广粉1号、巴西蕉和抗病品种中蕉6号组培苗,将其侵染过程分为附着、共生、共生向腐生转化、腐生等4个阶段。对侵染过程中表达的效应蛋白进行鉴定。鉴定出的效应蛋白的功能主要有:(1)降解植物细胞壁:如果胶酶、纤维素酶、角质酶、糖基水解酶等;(2)结合自身细胞壁几丁质;(3)细胞自噬,参与香蕉枯萎病菌的形态建成;(4)胞吞作用;(5)清除寄主释放的ROS;(6)其它功能。3 Foc TR4重要效应蛋白的功能分析对前期Foc TR4接种巴西蕉组培苗的蛋白质组学数据进行分析,结合比较基因组学鉴定结果,挑选107个效应蛋白基因进行研究,挑选标准为侵染过程中高表达或者Foc TR4特异效应蛋白;研究内容包括:(1)筛选坏死效应蛋白;(2)基因敲除,筛选对致病性影响较大的效应蛋白。坏死效应蛋白的筛选:将挑选的效应蛋白基因在大肠杆菌中进行表达,再将表达的蛋白接种于香蕉组培苗叶片上,筛选到20个能引起HR反应的效应蛋白。对致病性影响较大的效应蛋白的筛选:对各个阶段高表达的效应蛋白,以及能使香蕉叶片产生HR反应的效应蛋白进行基因敲除,接种香蕉后,调查了发病率,结果如下:FOIG_00012,03734,07201敲除突变体完全丧失致病力;FOIG_01651,03512,03690,04451,07803,10415,01077,01919敲除突变体有弱致病力;FOIG_04525,07508,15400,15834敲除突变体有中度致病力,说明这些效应蛋白对病原菌的致病性有重要影响。4白僵菌素是Foc TR4一种重要的毒性因子本课题组的前期研究已经表明:白僵菌素(BEA)能够导致假茎腐烂、细胞死亡、香蕉幼苗的腐烂和死亡,转录组学结果显示BEA能够抑制寄主DNA复制。为进一步确定BEA对病原菌致病力的影响,构建了BEA生物合成途径的限速酶Bbeas的基因敲除突变体。测定了突变体的BEA生物合成和致病力变化,与野生型菌株相比较,Bbeas敲除突变体的BEA的生物合成量显著降低,致病性完全丧失,而回补菌株Bbeas-complement能够恢复BEA的生物合成及致病性,表明Bbeas基因是病原菌的关键因素之一或者BEA是病原菌致病性不可或缺的因素。
文内图片：
图片说明：香蕉枯萎病菌随着寄主向世界各地扩散过程
[Abstract]:The banana is one of the most important fruit in the world, and is the fourth largest food crop next to rice, wheat and corn, so banana production is a world food security, regional development and human health (Ploetz R C,2006). However, the large-scale outbreak and prevalence of Fusarium oxysporum f. sp. ceuense Trological Trace 4, Fusarium oxysporum f. sp., has seriously endangered the sustainable development of the industry. In order to prevent and control the disease,56 Foc strains from different banana-producing countries (four physiological races and 24 vegetative compatibility groups (VCGs)) were re-sequenced, and the evolution of the pathogenic bacteria in the world was studied by using comparative genomics. Foc TR4 effect protein was identified and functional analysis was carried out on important protein. The results were as follows:1. The genome re-sequencing and bioinformatics analysis of the Fusarium oxysporum (1) genome re-sequencing and assembly: The genomic re-sequencing was carried out for 55 Foc strains from different banana-producing countries (including 4 physiological races,24 VCGs), including 6 Foc TR4,8 Foc STR4 (4 of subtropics),35 Foc race 1,2 Foc race2,2 large banana Foc,2 non-pathogenic Fusarium oxysporum, And the sequencing coverage is more than 50 times. The sequenced raw data were filtered and assembled to Contigs and Superconstigs. (2) The partition of the genome Core and LS-Region is published on the genome sequence of 14 fusarium of the Bad Institute website, and the core genome or regions of the Foc TR4 strain II5 and the Lineage-specific regions (LS-Regions) are predicted, Core regions and LS regions of the various sequencing strains were identified using the method of the "removal of the core genome" of the Core Genome, and the results showed that there was a significant difference in the size of the Core and LS-Region between the different types of strains. (3) The evolution rule of the Fusarium oxysporum in the world is studied by three methods: (1) the evolutionary tree drawn according to the 10 look-at genes; (2) the evolutionary tree of the SNP mapping in the whole genome range; (3) Principal Component Analysis (PCA), found that the evolution of the Foc TR4 strain is very conservative and is of a single origin, and the evolution speed is very fast; other types of strains such as Foc STR4 and Race 1 are more complex and have a plurality of evolutionary sources; the Fusarium oxysporum is evolved from the Race 1, The immune system of a large banana with high resistance to Race 1, Foc TR4 and Foc STR4 was overcome. (4) The mating type of the fusarium wilt of the banana collected the MAT1-1 and the MAT1-2 gene sequence of the mating type as the control, and the assembled sequence was mapped to obtain the mating type genes of different types of strains. The results showed that the fusarium wilt of the banana was an alien complex, and the Foc TR4 (VCG01213,01216, and 01213/16) was of the MAT1-1 type. (5) The prediction of the effect protein of the fusarium wilt of the banana is based on the structural characteristics, and the effect protein of each sequencing strain is predicted. In combination with the 14 fusarium genome sequences published on the Broad Institute's website, the specific effector proteins were identified by the comparative genomic method, and the specific effector proteins of the various physiological races were also analyzed,24 of the Foc Race1 were identified, and 47 of the Foc STR4 were identified. The expression of effector protein in Foc TR4 was 37. The expression of effector protein in Foc TR4 was divided into four stages, such as adhesion, symbiosis, symbiosis, saprophytic transformation, saprophytic and saprophytic. The effect protein expressed in the infection process is identified. The function of the identified effect protein is mainly as follows: (1) degrading plant cell wall: such as pectase, cellulase, cutinase, glycosyl hydrolase and the like; (2) binding to the cell wall chitin; (3) autophagy of the cell, and participating in the morphological construction of the banana wilt pathogen; (4) endocytosis; (5) removing the ROS of the host release; (6) other functions. The functional analysis of the important effector protein of the 3-Foc TR4 is carried out on the proteomics data of the tissue culture tissue culture seedlings of the Brazilian banana in the early stage Foc TR4, and the result of the comparative genomics identification is combined. 107 effector protein genes were selected and selected as high expression or Foc TR4 specific effector protein in the infection process; the study included: (1) screening the necrosis effect protein; (2) gene knockout, and screening the effect protein with great influence on the pathogenicity. Screening of the necrosis effect protein: the selected effector protein gene is expressed in the escherichia coli, the expressed protein is inoculated on the banana tissue culture seedling leaf, and the effect protein capable of causing the HR reaction is screened. Screening of effector proteins with a large effect on pathogenicity: the high expression of the effector proteins in each stage, and the gene knock-out of the effector proteins that can cause the HR reaction of the banana leaves, and after inoculation, the incidence was investigated and the results were as follows: FOG _ 00012,03734,07201 knock-out mutant completely lost the pathogenic force; FOG _ 01651,03512,03690,04451,07803,10415,01077,01919 knockout mutant has weak pathogenic force; FOG _ 04525,07508,15400,15834 knockout mutant has moderate pathogenic force, which indicates that these effect proteins have an important influence on the pathogenicity of pathogenic bacteria. The BEA can lead to the decay of the pseudostem, the cell death, the decay and death of the banana seedlings, and the results of the transcriptome show that the BEA can inhibit the replication of the host DNA. In order to further determine the influence of BEA on the pathogenic force of the pathogenic bacteria, the gene knock-out mutant of the speed-limiting enzyme Bbeas of the BEA biosynthesis pathway was constructed. Compared with the wild-type strain, the biosynthetic quantity of the BEA of the Bbeas knockout mutant is obviously reduced, the pathogenicity is completely lost, and the back-up strain Bbeas-cement can restore the biosynthesis and the pathogenicity of the BEA, It is shown that the Bbeas gene is one of the key factors in the pathogenic bacteria, or the BEA is an essential factor in the pathogenicity of the pathogenic bacteria.
【学位授予单位】：华南农业大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：S436.68
本文编号：2511288
文内图片：
图片说明：香蕉枯萎病菌随着寄主向世界各地扩散过程
[Abstract]:The banana is one of the most important fruit in the world, and is the fourth largest food crop next to rice, wheat and corn, so banana production is a world food security, regional development and human health (Ploetz R C,2006). However, the large-scale outbreak and prevalence of Fusarium oxysporum f. sp. ceuense Trological Trace 4, Fusarium oxysporum f. sp., has seriously endangered the sustainable development of the industry. In order to prevent and control the disease,56 Foc strains from different banana-producing countries (four physiological races and 24 vegetative compatibility groups (VCGs)) were re-sequenced, and the evolution of the pathogenic bacteria in the world was studied by using comparative genomics. Foc TR4 effect protein was identified and functional analysis was carried out on important protein. The results were as follows:1. The genome re-sequencing and bioinformatics analysis of the Fusarium oxysporum (1) genome re-sequencing and assembly: The genomic re-sequencing was carried out for 55 Foc strains from different banana-producing countries (including 4 physiological races,24 VCGs), including 6 Foc TR4,8 Foc STR4 (4 of subtropics),35 Foc race 1,2 Foc race2,2 large banana Foc,2 non-pathogenic Fusarium oxysporum, And the sequencing coverage is more than 50 times. The sequenced raw data were filtered and assembled to Contigs and Superconstigs. (2) The partition of the genome Core and LS-Region is published on the genome sequence of 14 fusarium of the Bad Institute website, and the core genome or regions of the Foc TR4 strain II5 and the Lineage-specific regions (LS-Regions) are predicted, Core regions and LS regions of the various sequencing strains were identified using the method of the "removal of the core genome" of the Core Genome, and the results showed that there was a significant difference in the size of the Core and LS-Region between the different types of strains. (3) The evolution rule of the Fusarium oxysporum in the world is studied by three methods: (1) the evolutionary tree drawn according to the 10 look-at genes; (2) the evolutionary tree of the SNP mapping in the whole genome range; (3) Principal Component Analysis (PCA), found that the evolution of the Foc TR4 strain is very conservative and is of a single origin, and the evolution speed is very fast; other types of strains such as Foc STR4 and Race 1 are more complex and have a plurality of evolutionary sources; the Fusarium oxysporum is evolved from the Race 1, The immune system of a large banana with high resistance to Race 1, Foc TR4 and Foc STR4 was overcome. (4) The mating type of the fusarium wilt of the banana collected the MAT1-1 and the MAT1-2 gene sequence of the mating type as the control, and the assembled sequence was mapped to obtain the mating type genes of different types of strains. The results showed that the fusarium wilt of the banana was an alien complex, and the Foc TR4 (VCG01213,01216, and 01213/16) was of the MAT1-1 type. (5) The prediction of the effect protein of the fusarium wilt of the banana is based on the structural characteristics, and the effect protein of each sequencing strain is predicted. In combination with the 14 fusarium genome sequences published on the Broad Institute's website, the specific effector proteins were identified by the comparative genomic method, and the specific effector proteins of the various physiological races were also analyzed,24 of the Foc Race1 were identified, and 47 of the Foc STR4 were identified. The expression of effector protein in Foc TR4 was 37. The expression of effector protein in Foc TR4 was divided into four stages, such as adhesion, symbiosis, symbiosis, saprophytic transformation, saprophytic and saprophytic. The effect protein expressed in the infection process is identified. The function of the identified effect protein is mainly as follows: (1) degrading plant cell wall: such as pectase, cellulase, cutinase, glycosyl hydrolase and the like; (2) binding to the cell wall chitin; (3) autophagy of the cell, and participating in the morphological construction of the banana wilt pathogen; (4) endocytosis; (5) removing the ROS of the host release; (6) other functions. The functional analysis of the important effector protein of the 3-Foc TR4 is carried out on the proteomics data of the tissue culture tissue culture seedlings of the Brazilian banana in the early stage Foc TR4, and the result of the comparative genomics identification is combined. 107 effector protein genes were selected and selected as high expression or Foc TR4 specific effector protein in the infection process; the study included: (1) screening the necrosis effect protein; (2) gene knockout, and screening the effect protein with great influence on the pathogenicity. Screening of the necrosis effect protein: the selected effector protein gene is expressed in the escherichia coli, the expressed protein is inoculated on the banana tissue culture seedling leaf, and the effect protein capable of causing the HR reaction is screened. Screening of effector proteins with a large effect on pathogenicity: the high expression of the effector proteins in each stage, and the gene knock-out of the effector proteins that can cause the HR reaction of the banana leaves, and after inoculation, the incidence was investigated and the results were as follows: FOG _ 00012,03734,07201 knock-out mutant completely lost the pathogenic force; FOG _ 01651,03512,03690,04451,07803,10415,01077,01919 knockout mutant has weak pathogenic force; FOG _ 04525,07508,15400,15834 knockout mutant has moderate pathogenic force, which indicates that these effect proteins have an important influence on the pathogenicity of pathogenic bacteria. The BEA can lead to the decay of the pseudostem, the cell death, the decay and death of the banana seedlings, and the results of the transcriptome show that the BEA can inhibit the replication of the host DNA. In order to further determine the influence of BEA on the pathogenic force of the pathogenic bacteria, the gene knock-out mutant of the speed-limiting enzyme Bbeas of the BEA biosynthesis pathway was constructed. Compared with the wild-type strain, the biosynthetic quantity of the BEA of the Bbeas knockout mutant is obviously reduced, the pathogenicity is completely lost, and the back-up strain Bbeas-cement can restore the biosynthesis and the pathogenicity of the BEA, It is shown that the Bbeas gene is one of the key factors in the pathogenic bacteria, or the BEA is an essential factor in the pathogenicity of the pathogenic bacteria.
【学位授予单位】：华南农业大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：S436.68
【参考文献】
相关期刊论文 前1条
1 邝瑞彬;李春雨;杨静;魏岳荣;杨乔松;胡春华;盛鸥;易干军;;抗感枯萎病香蕉的细胞结构抗性研究[J];分子植物育种;2013年02期
，本文编号：2511288
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