香蕉枯萎病菌進(jìn)化研究和Foc TR4致病相關(guān)效應(yīng)蛋白的鑒定及功能分析
發(fā)布時(shí)間：2019-07-06 20:00
【摘要】：香蕉是世界上最重要的水果之一,也是僅次于水稻、小麥和玉米的第四大糧食作物,因此香蕉生產(chǎn)攸關(guān)世界糧食安全、地區(qū)發(fā)展和人類健康(Ploetz R C,2006)。但是,香蕉枯萎病菌熱帶4號(hào)生理小種(Fusarium oxysporum f.sp.cubense Tropical race 4)的大規(guī)模爆發(fā)和流行,已嚴(yán)重危及到該產(chǎn)業(yè)的可持續(xù)發(fā)展。為了防控該病害,本課題對(duì)來(lái)自不同香蕉生產(chǎn)國(guó)的56個(gè)Foc菌株(涵蓋4個(gè)生理小種、24個(gè)營(yíng)養(yǎng)體親合群(Vegetative Compatibility Groups,VCGs))進(jìn)行了基因組重測(cè)序,利用比較基因組學(xué)研究了全球范圍內(nèi)病原菌的進(jìn)化規(guī)律、鑒定了Foc TR4效應(yīng)蛋白,并對(duì)重要的蛋白開(kāi)展功能分析,具體結(jié)果如下:1香蕉枯萎病菌基因組重測(cè)序及生物信息學(xué)分析(1)基因組重測(cè)序及組裝:對(duì)來(lái)自不同香蕉生產(chǎn)國(guó)的55個(gè)Foc菌株(涵蓋4個(gè)生理小種、24個(gè)VCGs),包括6株Foc TR4,8株Foc STR4(亞熱帶4號(hào)小種),35株Foc race 1,2株Foc race2,2株大蕉Foc,2株非致病性尖孢鐮刀菌,進(jìn)行了基因組重測(cè)序,測(cè)序覆蓋度均在50倍以上。測(cè)序的原始數(shù)據(jù)經(jīng)過(guò)濾后組裝成Contigs和Supercontigs。(2)基因組Core和LS-Region的區(qū)分結(jié)合公布在Broad Institute網(wǎng)站上的14種鐮刀菌基因組序列,預(yù)測(cè)了Foc TR4菌株II5的核心基因組(Core genome或者regions)和Lineage-specific regions(LS-Regions),并以此為參考,采用“剔除核心基因組”(Eliminating the Core Genome)的方法鑒定各個(gè)測(cè)序菌株的Core regiongs和LS regions,結(jié)果顯示不同類型菌株間的Core和LS-Region的大小存在顯著差異。(3)全球范圍內(nèi)的香蕉枯萎病菌的進(jìn)化規(guī)律利用3種方法研究了全球范圍內(nèi)的Foc的進(jìn)化規(guī)律:(1)根據(jù)10個(gè)看家基因繪制的進(jìn)化樹(shù);(2)全基因組范圍內(nèi)的SNP繪制的進(jìn)化樹(shù);(3)主成分分析法(Principal Component Analysis,PCA),發(fā)現(xiàn)Foc TR4菌株進(jìn)化非常保守,是單一起源的,并且進(jìn)化速度非�？�;其它類型菌株如Foc STR4和Race 1則進(jìn)化比較復(fù)雜,有多個(gè)進(jìn)化源頭;大蕉枯萎病菌由Race 1進(jìn)化而來(lái),克服了對(duì)Race 1、Foc TR4和Foc STR4有高度抗性的大蕉的免疫系統(tǒng)。(4)香蕉枯萎病菌交配型的分析收集交配型位點(diǎn)MAT1-1和MAT1-2基因序列作為對(duì)照,將組裝的序列進(jìn)行Mapping,獲得不同類型菌株的交配型基因,結(jié)果顯示香蕉枯萎病菌為異宗配合真菌,而Foc TR4(VCG01213,01216和01213/16)為MAT1-1類型。(5)香蕉枯萎病菌效應(yīng)蛋白的預(yù)測(cè)根據(jù)結(jié)構(gòu)特征,預(yù)測(cè)了各測(cè)序菌株的效應(yīng)蛋白。結(jié)合公布在Broad Institute網(wǎng)站上的14種鐮刀菌基因組序列,利用比較基因組學(xué)方法鑒定到各VCGs特異的效應(yīng)蛋白,也對(duì)各生理小種特異的效應(yīng)蛋白進(jìn)行分析,鑒定到Foc Race1有24個(gè)、Foc STR4有47個(gè)、Foc TR4有37個(gè)。2 Foc TR4侵染過(guò)程中效應(yīng)蛋白的表達(dá)規(guī)律將Foc TR4接種廣粉1號(hào)、巴西蕉和抗病品種中蕉6號(hào)組培苗,將其侵染過(guò)程分為附著、共生、共生向腐生轉(zhuǎn)化、腐生等4個(gè)階段。對(duì)侵染過(guò)程中表達(dá)的效應(yīng)蛋白進(jìn)行鑒定。鑒定出的效應(yīng)蛋白的功能主要有:(1)降解植物細(xì)胞壁:如果膠酶、纖維素酶、角質(zhì)酶、糖基水解酶等;(2)結(jié)合自身細(xì)胞壁幾丁質(zhì);(3)細(xì)胞自噬,參與香蕉枯萎病菌的形態(tài)建成;(4)胞吞作用;(5)清除寄主釋放的ROS;(6)其它功能。3 Foc TR4重要效應(yīng)蛋白的功能分析對(duì)前期Foc TR4接種巴西蕉組培苗的蛋白質(zhì)組學(xué)數(shù)據(jù)進(jìn)行分析,結(jié)合比較基因組學(xué)鑒定結(jié)果,挑選107個(gè)效應(yīng)蛋白基因進(jìn)行研究,挑選標(biāo)準(zhǔn)為侵染過(guò)程中高表達(dá)或者Foc TR4特異效應(yīng)蛋白;研究?jī)?nèi)容包括:(1)篩選壞死效應(yīng)蛋白;(2)基因敲除,篩選對(duì)致病性影響較大的效應(yīng)蛋白。壞死效應(yīng)蛋白的篩選:將挑選的效應(yīng)蛋白基因在大腸桿菌中進(jìn)行表達(dá),再將表達(dá)的蛋白接種于香蕉組培苗葉片上,篩選到20個(gè)能引起HR反應(yīng)的效應(yīng)蛋白。對(duì)致病性影響較大的效應(yīng)蛋白的篩選:對(duì)各個(gè)階段高表達(dá)的效應(yīng)蛋白,以及能使香蕉葉片產(chǎn)生HR反應(yīng)的效應(yīng)蛋白進(jìn)行基因敲除,接種香蕉后,調(diào)查了發(fā)病率,結(jié)果如下:FOIG_00012,03734,07201敲除突變體完全喪失致病力;FOIG_01651,03512,03690,04451,07803,10415,01077,01919敲除突變體有弱致病力;FOIG_04525,07508,15400,15834敲除突變體有中度致病力,說(shuō)明這些效應(yīng)蛋白對(duì)病原菌的致病性有重要影響。4白僵菌素是Foc TR4一種重要的毒性因子本課題組的前期研究已經(jīng)表明:白僵菌素(BEA)能夠?qū)е录偾o腐爛、細(xì)胞死亡、香蕉幼苗的腐爛和死亡,轉(zhuǎn)錄組學(xué)結(jié)果顯示BEA能夠抑制寄主DNA復(fù)制。為進(jìn)一步確定BEA對(duì)病原菌致病力的影響,構(gòu)建了BEA生物合成途徑的限速酶Bbeas的基因敲除突變體。測(cè)定了突變體的BEA生物合成和致病力變化,與野生型菌株相比較,Bbeas敲除突變體的BEA的生物合成量顯著降低,致病性完全喪失,而回補(bǔ)菌株Bbeas-complement能夠恢復(fù)BEA的生物合成及致病性,表明Bbeas基因是病原菌的關(guān)鍵因素之一或者BEA是病原菌致病性不可或缺的因素。
文內(nèi)圖片：
圖片說(shuō)明：香蕉枯萎病菌隨著寄主向世界各地?cái)U(kuò)散過(guò)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.
【學(xué)位授予單位】：華南農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2016
【分類號(hào)】：S436.68
本文編號(hào)：2511288
文內(nèi)圖片：
圖片說(shuō)明：香蕉枯萎病菌隨著寄主向世界各地?cái)U(kuò)散過(guò)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.
【學(xué)位授予單位】：華南農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2016
【分類號(hào)】：S436.68
【參考文獻(xiàn)】
相關(guān)期刊論文 前1條
1 鄺瑞彬;李春雨;楊靜;魏岳榮;楊喬松;胡春華;盛鷗;易干軍;;抗感枯萎病香蕉的細(xì)胞結(jié)構(gòu)抗性研究[J];分子植物育種;2013年02期
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