番茄抗叶霉病Cf-12候选基因的筛选及抗性应答机制分析
发布时间：2018-08-03 19:18
【摘要】：番茄(Solanum lycopersicum)作为我国乃至世界上重要的蔬菜作物,其在农业产业结构以及人们的日常生活中扮演着重要的角色。然而在番茄保护地栽培日常生产过程中易受到多种病原物的侵扰,尤其在我国中部番茄主产区夏季高温高湿的栽培环境下极易引起番茄叶霉病的大面积发生,造成果实产量、品质的严重下降,威胁当地番茄的安全生产。叶霉病菌(Cladosporium fulvum[syn.Passalora fulva])与番茄植株发生的亲和性互作是番茄叶霉病发生的根源,因此以叶霉病抗性基因Cf为主导的抗叶霉病育种工作便成为工作的重点。目前,已鉴定出Cf-1、Cf-2、Cf-4、Cf-4E、Cf-5、Cf-9、Cf-11、Cf-9DC以及多个Cf-ECPs抗性基因,并成功的将Cf-4、Cf-5、Cf-9应用于生产。含Cf基因的番茄能够抵抗C.fulvum的关键在于,Cf基因编码的类受体蛋白能够特异性识别C.fulvum所分泌的效应因子,从而介导植株产生HR(Hypersensitive response)。但目前存在的新问题是,随着具不同Cf基因的商业番茄品种的长期、大面积推广栽培,C.fulvum生理小种也发生了选择性的激烈分化,其中新近发现的C.fulvum小种2.4克服了Cf-2、Cf-4基因的抗性,C.fulvum小种2.5和2.5.9克服了Cf-5、Cf-9基因的抗性,这些具有广谱或者复合毒性的C.fulvum生理小种使其对应的番茄抗病品种失去抗病性,同时也使番茄主产区C.fulvum小种的流行趋势变的更加多态化。为此,急需挖掘新的、抗性更广更强的Cf基因应用于生产。已经发现的Cf基因都来源于野生番茄,因此挖掘新的Cf抗性基因,并通过转基因或者远缘杂交等手段,将Cf基因导入番茄常规栽培种,继而培育出具叶霉病抗性的植株是番茄抗叶霉病育种的关键。通过多年的、多地的抗病性鉴定,CGN7495材料具有优良的C.fulvum抗性,由Kanwar的鉴定结果得知该材料含有Cf-12基因,来源于醋栗番茄(L.pimpinellifolium),该基因目前并没有研究基础。因此,本试验主要以番茄叶霉病新抗性候选基因Cf-12的挖掘和筛选,以及Cf-12对C.fulvum的抗性应答机制为主要研究重点,同时也对Cf-12与C.fulvum非亲和互作的组织学特征和抗性遗传规律进行了分析。主要研究结果如下:(1)对Cf-12抗性番茄材料与C.fulvum的非亲和互作过程的详细的显微观察,明确了Cf-12抵御C.fulvum侵染的组织学特征,如C.fulvum孢子在抗性和感病材料上的萌发以及从叶背气孔侵入的过程并没有表现出差异性,但是当C.fulvum菌丝侵入Cf-5、Cf-12等的抗性材料气孔2d后便停止生长,且菌丝肿胀、弯曲,与寄主叶肉细胞接触的菌丝和叶肉细胞彼此都相继崩溃,从而将C.fulvum侵入的菌丝限制在一个很小的有限空间内,使其无法再侵染,且叶片上坏死斑点的产生逐渐增多和扩大。将Cf-12与Cf-5等基因对C.fulvum的抗性特征进行了比较分析后,发现不同的Cf基因对C.fulvum的抗性组织学特征差异表现在过敏性坏死斑点的发生数量和面积等方面。(2)Cf基因对C.fulvum的抗性范围调查结果表明,不同的Cf基因能够抵抗的C.fulvum生理小种的种类也具有较大差异,含Cf-12基因的番茄材料能够抵御1.2、1.2.4、1.5、1.2.4.5、1.2.5、1.2.4.9等多种C.fulvum生理小种的侵染,但并不抗生理小种1.2.3.4,抗性范围的明确对含Cf-12基因番茄有针对性的在不同C.fulvum生理小种流行区合理种植提供参考。(3)依据构建的S.pennellii LA0716与CGN7495的6世代群体,对群体中Cf-12基因的抗性遗传规律的分析结果表明,F2群体中Cf-12基因的分离比符合3:1的分离定律,BC1P1群体中Cf-12基因的实际分离比符合1:1的孟德尔分离定律,由此可知Cf-12基因对C.fulvum的抗性符合单基因显性遗传规律,这与已鉴定的其它Cf基因为单基因显性遗传的规律相一致。(4)在明确Cf-12基因为单基因显性遗传的基础上,通过采用父母本重测序,F2代抗、感群体SLAF简化测序技术进行基于ED(Euclidean Distance)和SNP-index的联合分析,对番茄叶霉病抗性基因Cf-12进行筛选,并成功将其关联到番茄第6号染色体,对目标关联区域所包含基因进行NR、NT、trEMBL、Swiss-prot、GO、KEGG和COG等多数据库的功能注释和LRR-TM-sCT结构预测确定了3个候选基因,在进一步通过Proparam、pfam、SMART、SignalP、ProtComp、SOPMA、Swiss-Model等程序对候选基因编码蛋白的结构域、理化性质、亚细胞定位、二级和三级结构、PPI网络进行了详细分析,以及基于qRT-PCR的表达模式分析和共表达分析的基础上,确定位于CGN7495抗性材料上Solyc06g008270.2基因位点的72 bp碱基片段插入后的基因,即Ccg2基因为所筛选的Cf-12基因。此外,通过对番茄全基因组进行GO分类中定义为结合活性功能(GO:0005515)且具有eLRR-TM结构的基因进行番茄全基因组搜索,结果发现已报道的所有Hcr2s、Hcr9s及Cf-Ecp基因均被覆盖到,这一思路可为研究其它未知的Cf基因的定位工作提供新的途径。(5)对Cf-12对C.fulvum的抗性应答机制研究,采用基于高通量测序的RNA-seq技术,对多时间点的非亲和互作样本进行转录组学分析,通过对测序样本间差异表达基因(DEGs)的k-mean聚类分析,总共发现了3类表达模式,其中最值得注意的是subcluster_4中持续高表达的34个DEGs,以及subcluster_3中先低表达后又剧烈高表达的41个DEGs,GO分析显示它们富集于氧化还原、氮代谢、磷代谢、结合活性、转移酶活性、激素应答、离子转运以及防御反应相关等类别,这对于后续试验中筛选特异性表达的基因起到重要的辅助作用;可变剪接分析(AS)显示,非亲和互作过程主要发生SE和MXE事件,随着时间的延长SE和MXE两AS事件的总数量呈现增加的趋势,说明这些显著差异的可变剪接在Cf-12基因调控对C.fulvum抗性的转录水平的基因表达的复杂程度以及蛋白质功能的多样性正在发生巨大的变化;GO和Pathway分析表明苯丙氨酸代谢途径、植物—病原菌互作、苯丙烷生物合成、谷胱甘肽代谢,次生代谢产物的生物合成等生物过程中的基因被上调显著富集,对植物激素信号转导途径分析表明SA和ET对Cf-12的抗病防卫反应起重要的调控作用,而JA可能在Cf-12对C.fulvum的抗病防卫反应信号转导途径中起到交叉、辅助的作用。此外,还富集到大量的氧化物酶体,谷胱甘肽过氧化物酶基因,植保素合成基因,与苯丙素和黄酮类代谢途径相关的苯丙氨酸解氨酶基因、4CL、TC4M、F5H,有关黄酮类合成的CHSs、CHI、DFR、FLS,与细胞壁合成相关的纤维素合成酶基因和果胶酶活性的果胶(甲)酯酶基因等也均被富集到呈现上调表达的趋势,与蛋白泛素化相关的E1、E2、E3等多数基因呈上调表达趋势,说明蛋白的泛素化被积极响应,总的来看与SA和ET、PR蛋白、植保素、防卫素、硫堇、木质素、渗调蛋白、过氧化物酶以及黄酮类等的大量合成以及细胞壁的加强都使得Cf-12番茄对C.fulvum产生较好的抗性;在转录因子(TFs)的分析中,首次发现多个新的ARF、MADS、G2-like、G3H、C2H2、b HLH等TFs家族参与到Cf-12对C.fulvum的抗性防卫反应中,且检测到的这60个TFs家族呈现两种表达模式,如bZIP、C2H2、HSF、MADS、NAC等在早期阶段先上调表达,然后在后期下调表达,而bHLH、GRAS、HB、SBP、MYB、WRKY等在抗性反应的后期呈高表达的趋势;通过全面的Cf-12对C.fulvum非亲和互作的转录组学分析,对Cf-12与C.fulvum的抗性应答机制也有了一定的了解。(6)对Cf-12与C.fulvum抗性应答反应中的部分基因的VIGS的初步功能验证,Ccg2候选基因以及NPR1(Solyc07g044980.2)、CDPK(Solyc03g113390.2)基因沉默后的部分植株出现了感病症状,病情指数统计发现沉默NPR1和CDPK的植株仍表现为中抗,而沉默Ccg2的植株表现为中感,说明Ccg2、NPR1(Solyc07g044980.2)、CDPK(Solyc03g113390.2)基因与Cf-12番茄对C.fulvum的抗性应答反应过程相关。
[Abstract]:Solanum lycopersicum, as an important vegetable crop in our country and in the world, plays an important role in the structure of agricultural industry and people's daily life. However, it is easily disturbed by various pathogens during the daily production of tomato cultivated land, especially in the main tomato producing area in the middle of our country. The large area of tomato leaf mould was easily caused by the cultivation environment, resulting in the fruit yield, the serious decline in quality and the safety of the local tomato. The affinity interaction between Cladosporium fulvum[syn.Passalora fulva] and tomato plants was the root of tomato leaf mold disease. Therefore, the leaf mould resistance gene Cf was used as the root cause. At present, Cf-1, Cf-2, Cf-4, Cf-4E, Cf-5, Cf-9, Cf-11, Cf-9DC and multiple Cf-ECPs resistance genes have been identified, and Cf-4, Cf-5, and Cf-9 should be used in production. The key to the resistance of tomato containing genes is that the gene encoded receptor protein can be specific. Sex recognizes the effect factors secreted by C.fulvum, which mediates the production of HR (Hypersensitive response) in the plant, but the new problem is that, with the long term of the commercial tomato varieties with different Cf genes, large area cultivation and the selective intense differentiation of the C.fulvum physiological races, of which the newly discovered 2.4 grams of C.fulvum species are newly discovered. The resistance of Cf-2, Cf-4 gene, C.fulvum small species 2.5 and 2.5.9 overcame the resistance of Cf-5 and Cf-9 genes. These broad-spectrum or compound toxic C.fulvum species make their corresponding tomato resistant varieties lose their resistance to disease, and also make the flow trend of C.fulvum small species in tomato main producing area become more polymorphic. The Cf gene, which has a more extensive and stronger resistance, is used in production. The Cf gene has been found from the wild tomato. Therefore, it is the key to develop a new Cf resistance gene and transfer the Cf gene into the conventional cultivation of Tomato by transgene or distant hybridization. After many years of identification of resistance to disease, CGN7495 material has excellent C.fulvum resistance. It is found that the material contains Cf-12 gene from the results of Kanwar identification, which is derived from gooseberry tomato (L.pimpinellifolium). This gene has no research basis at present. Therefore, this experiment is mainly based on the mining and screening of the candidate gene of tomato leaf mould resistance candidate gene Cf-12. Selection, and the main research focus on the resistance response mechanism of Cf-12 to C.fulvum, and the analysis of the histologic characteristics and resistance genetic rules of Cf-12 and C.fulvum incompatibility interaction are also analyzed. The main results are as follows: (1) the detailed microscopic observation of the non affinity interaction process of Cf-12 resistant tomato materials and C.fulvum is made clear that Cf-12 is offset. The histological features of the C.fulvum infection, such as the germination of the C.fulvum spores on the resistant and susceptible materials and the invasion of the stomata from the back of the leaf, were not different, but when the C.fulvum mycelium invaded the Cf-5, Cf-12 and other resistant materials, the stomata stopped growing, and the mycelium swelled and curved, and the mycelium contacted with the host mesophyll cells. The mesophyll cells collapse one after another, thus limiting the C.fulvum intruded mycelium to a small limited space, which can not be reinfected, and the production of necrotic spots on the leaves gradually increases and expands. The resistance characteristics of the Cf-12 and Cf-5 genes to the resistance of C.fulvum have been compared, and the resistance of the different Cf genes to the C.fulvum is found. The differences in the characteristics of sexual histology showed in the number and area of the occurrence of anaphylactic necrosis spots. (2) the investigation of the resistance range of the Cf gene to C.fulvum showed that the species of C.fulvum physiological races which were resistant to the Cf genes were also different, and the Cf containing the gene of the gene could resist the 1.2,1.2.4,1.5,1.2.4.5,1.2.5,1.2. .4.9 and other C.fulvum physiological races were infected, but they were not resistant to the physiological race 1.2.3.4, and the range of resistance was clear for the rational planting of the Cf-12 gene tomato in different C.fulvum physiological races. (3) the resistance inheritance of the Cf-12 gene in the population based on the constructed S.pennellii LA0716 and the 6 generation of CGN7495. The results of regular analysis show that the separation ratio of Cf-12 gene in F2 population is consistent with the law of separation of 3:1. The actual separation ratio of Cf-12 gene in BC1P1 population conforms to the Mendel law of 1:1, thus the resistance of the Cf-12 gene to C.fulvum is consistent with the dominant inheritance law of the single gene, and the other Cf genes identified are the dominant inheritance of the single gene. (4) (4) on the basis of the explicit genetic dominance of the single gene, the genetic analysis of ED (Euclidean Distance) and SNP-index was carried out by the combined analysis of ED (Euclidean Distance) and SNP-index by the parental re sequencing, the SLAF sequencing technology of the susceptible population and the susceptible population, and was screened for tomato leaf mould resistance based on Cf-12, and was successfully associated with tomato No. sixth. Chromosomes, the functional annotations of NR, NT, trEMBL, Swiss-prot, GO, KEGG, and COG, including the functional annotations and the structural prediction of LRR-TM-sCT, determine the 3 candidate genes. The properties, subcellular localization, two and three level structures, PPI networks were analyzed in detail, and based on qRT-PCR expression pattern analysis and co expression analysis, the base of the 72 BP base fragment inserted at the Solyc06g008270.2 locus on the CGN7495 resistant material was determined, that is, the Ccg2 gene was selected as the Cf-12 gene. The whole genome of tomato was classified by GO classification, which was defined as a gene that combines active function (GO:0005515) with eLRR-TM structure to search the whole genome of tomato. The results showed that all of the reported Hcr2s, Hcr9s and Cf-Ecp genes were covered. This idea can provide a new way for the study of other unknown Cf genes. (5) In the study of the resistance response mechanism of Cf-12 to C.fulvum, the RNA-seq technology based on high throughput sequencing was used to make a transcriptional analysis of the non compatible interaction samples of multiple time points. Through the K-mean clustering analysis of the differentially expressed genes (DEGs) among the sequencing samples, 3 types of expression patterns were found, among which the most noteworthy was in subcluster_4. Continuous high expression of 34 DEGs, and 41 DEGs with low expression and intense high expression in subcluster_3, GO analysis shows that they are enriched in redox, nitrogen metabolism, phosphorus metabolism, binding activity, transferase activity, hormone response, ion transport and defense response related categories, which can be used to screen genes for specific expression in follow-up tests. The variable splicing analysis (AS) showed that the SE and MXE events occurred mainly in the incompatible interaction process, and the total number of SE and MXE two AS events increased with time, indicating that the variable splicing of these significant differences in the Cf-12 gene regulates the complex process of gene expression at the transcriptional level of C.fulvum resistance. The diversity of degree and protein function is undergoing great changes; GO and Pathway analysis show that phenylalanine metabolic pathway, plant pathogen interaction, phenylpropane biosynthesis, glutathione metabolism, and biosynthesis of secondary metabolites are significantly enriched, and the plant hormone signal transduction pathway is divided. The analysis shows that SA and ET play an important role in regulating the resistance and defense response of Cf-12, and JA may play a cross and auxiliary role in Cf-12 on the signal transduction pathway of the resistance defense response of C.fulvum. In addition, a large number of oxide enzymes, glutathione peroxidase (GSH PX), pod synthesis gene, and phenylpropanol and flavonoid generation are also enriched. The phenylalanine ammonia lyase gene, 4CL, TC4M, F5H, related to the flavonoid synthesis of CHSs, CHI, DFR, FLS, and the cellulose synthetase gene related to cell wall synthesis and pectinase activity of pectin (a) esterase gene are also enriched to present the trend of up-regulated expression, and most of the genes related to protein ubiquitination, E1, E2, E3, etc. The up-regulated trend indicates that the ubiquitination of protein is actively responsive. In general, a large number of synthesis of SA and ET, PR protein, flocking, defensin, thio, lignin, peroxidase, and flavonoids, and the enhancement of cell wall, make Cf-12 tomatoes have a better resistance to C.fulvum; in the analysis of the transcription factor (TFs), It is the first time that a number of new ARF, MADS, G2-like, G3H, C2H2, B HLH and other TFs families are involved in the resistance defense response of Cf-12 to C.fulvum, and the detected 60 TFs families present two kinds of expression patterns. A high expression trend in the later stage of sexual response; the transcriptional analysis of C.fulvum incompatibility interaction by a comprehensive Cf-12 also has a certain understanding of the resistance response mechanism of Cf-12 and C.fulvum. (6) preliminary functional verification of the VIGS of some genes in the response to Cf-12 and C.fulvum responses, the Ccg2 candidate genes and NPR1 (Solyc07g0449). 80.2) a part of the plant with CDPK (Solyc03g113390.2) gene was silent, and the disease index showed that the plants with silent NPR1 and CDPK still showed moderate resistance, while the silent Ccg2 plants showed medium sense, indicating that Ccg2, NPR1 (Solyc07g044980.2), CDPK (Solyc03g113390.2) gene and Cf-12 tomato responded to the resistance response to C.fulvum. Course related.
【学位授予单位】：东北农业大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：S436.412
本文编号：2162749
[Abstract]:Solanum lycopersicum, as an important vegetable crop in our country and in the world, plays an important role in the structure of agricultural industry and people's daily life. However, it is easily disturbed by various pathogens during the daily production of tomato cultivated land, especially in the main tomato producing area in the middle of our country. The large area of tomato leaf mould was easily caused by the cultivation environment, resulting in the fruit yield, the serious decline in quality and the safety of the local tomato. The affinity interaction between Cladosporium fulvum[syn.Passalora fulva] and tomato plants was the root of tomato leaf mold disease. Therefore, the leaf mould resistance gene Cf was used as the root cause. At present, Cf-1, Cf-2, Cf-4, Cf-4E, Cf-5, Cf-9, Cf-11, Cf-9DC and multiple Cf-ECPs resistance genes have been identified, and Cf-4, Cf-5, and Cf-9 should be used in production. The key to the resistance of tomato containing genes is that the gene encoded receptor protein can be specific. Sex recognizes the effect factors secreted by C.fulvum, which mediates the production of HR (Hypersensitive response) in the plant, but the new problem is that, with the long term of the commercial tomato varieties with different Cf genes, large area cultivation and the selective intense differentiation of the C.fulvum physiological races, of which the newly discovered 2.4 grams of C.fulvum species are newly discovered. The resistance of Cf-2, Cf-4 gene, C.fulvum small species 2.5 and 2.5.9 overcame the resistance of Cf-5 and Cf-9 genes. These broad-spectrum or compound toxic C.fulvum species make their corresponding tomato resistant varieties lose their resistance to disease, and also make the flow trend of C.fulvum small species in tomato main producing area become more polymorphic. The Cf gene, which has a more extensive and stronger resistance, is used in production. The Cf gene has been found from the wild tomato. Therefore, it is the key to develop a new Cf resistance gene and transfer the Cf gene into the conventional cultivation of Tomato by transgene or distant hybridization. After many years of identification of resistance to disease, CGN7495 material has excellent C.fulvum resistance. It is found that the material contains Cf-12 gene from the results of Kanwar identification, which is derived from gooseberry tomato (L.pimpinellifolium). This gene has no research basis at present. Therefore, this experiment is mainly based on the mining and screening of the candidate gene of tomato leaf mould resistance candidate gene Cf-12. Selection, and the main research focus on the resistance response mechanism of Cf-12 to C.fulvum, and the analysis of the histologic characteristics and resistance genetic rules of Cf-12 and C.fulvum incompatibility interaction are also analyzed. The main results are as follows: (1) the detailed microscopic observation of the non affinity interaction process of Cf-12 resistant tomato materials and C.fulvum is made clear that Cf-12 is offset. The histological features of the C.fulvum infection, such as the germination of the C.fulvum spores on the resistant and susceptible materials and the invasion of the stomata from the back of the leaf, were not different, but when the C.fulvum mycelium invaded the Cf-5, Cf-12 and other resistant materials, the stomata stopped growing, and the mycelium swelled and curved, and the mycelium contacted with the host mesophyll cells. The mesophyll cells collapse one after another, thus limiting the C.fulvum intruded mycelium to a small limited space, which can not be reinfected, and the production of necrotic spots on the leaves gradually increases and expands. The resistance characteristics of the Cf-12 and Cf-5 genes to the resistance of C.fulvum have been compared, and the resistance of the different Cf genes to the C.fulvum is found. The differences in the characteristics of sexual histology showed in the number and area of the occurrence of anaphylactic necrosis spots. (2) the investigation of the resistance range of the Cf gene to C.fulvum showed that the species of C.fulvum physiological races which were resistant to the Cf genes were also different, and the Cf containing the gene of the gene could resist the 1.2,1.2.4,1.5,1.2.4.5,1.2.5,1.2. .4.9 and other C.fulvum physiological races were infected, but they were not resistant to the physiological race 1.2.3.4, and the range of resistance was clear for the rational planting of the Cf-12 gene tomato in different C.fulvum physiological races. (3) the resistance inheritance of the Cf-12 gene in the population based on the constructed S.pennellii LA0716 and the 6 generation of CGN7495. The results of regular analysis show that the separation ratio of Cf-12 gene in F2 population is consistent with the law of separation of 3:1. The actual separation ratio of Cf-12 gene in BC1P1 population conforms to the Mendel law of 1:1, thus the resistance of the Cf-12 gene to C.fulvum is consistent with the dominant inheritance law of the single gene, and the other Cf genes identified are the dominant inheritance of the single gene. (4) (4) on the basis of the explicit genetic dominance of the single gene, the genetic analysis of ED (Euclidean Distance) and SNP-index was carried out by the combined analysis of ED (Euclidean Distance) and SNP-index by the parental re sequencing, the SLAF sequencing technology of the susceptible population and the susceptible population, and was screened for tomato leaf mould resistance based on Cf-12, and was successfully associated with tomato No. sixth. Chromosomes, the functional annotations of NR, NT, trEMBL, Swiss-prot, GO, KEGG, and COG, including the functional annotations and the structural prediction of LRR-TM-sCT, determine the 3 candidate genes. The properties, subcellular localization, two and three level structures, PPI networks were analyzed in detail, and based on qRT-PCR expression pattern analysis and co expression analysis, the base of the 72 BP base fragment inserted at the Solyc06g008270.2 locus on the CGN7495 resistant material was determined, that is, the Ccg2 gene was selected as the Cf-12 gene. The whole genome of tomato was classified by GO classification, which was defined as a gene that combines active function (GO:0005515) with eLRR-TM structure to search the whole genome of tomato. The results showed that all of the reported Hcr2s, Hcr9s and Cf-Ecp genes were covered. This idea can provide a new way for the study of other unknown Cf genes. (5) In the study of the resistance response mechanism of Cf-12 to C.fulvum, the RNA-seq technology based on high throughput sequencing was used to make a transcriptional analysis of the non compatible interaction samples of multiple time points. Through the K-mean clustering analysis of the differentially expressed genes (DEGs) among the sequencing samples, 3 types of expression patterns were found, among which the most noteworthy was in subcluster_4. Continuous high expression of 34 DEGs, and 41 DEGs with low expression and intense high expression in subcluster_3, GO analysis shows that they are enriched in redox, nitrogen metabolism, phosphorus metabolism, binding activity, transferase activity, hormone response, ion transport and defense response related categories, which can be used to screen genes for specific expression in follow-up tests. The variable splicing analysis (AS) showed that the SE and MXE events occurred mainly in the incompatible interaction process, and the total number of SE and MXE two AS events increased with time, indicating that the variable splicing of these significant differences in the Cf-12 gene regulates the complex process of gene expression at the transcriptional level of C.fulvum resistance. The diversity of degree and protein function is undergoing great changes; GO and Pathway analysis show that phenylalanine metabolic pathway, plant pathogen interaction, phenylpropane biosynthesis, glutathione metabolism, and biosynthesis of secondary metabolites are significantly enriched, and the plant hormone signal transduction pathway is divided. The analysis shows that SA and ET play an important role in regulating the resistance and defense response of Cf-12, and JA may play a cross and auxiliary role in Cf-12 on the signal transduction pathway of the resistance defense response of C.fulvum. In addition, a large number of oxide enzymes, glutathione peroxidase (GSH PX), pod synthesis gene, and phenylpropanol and flavonoid generation are also enriched. The phenylalanine ammonia lyase gene, 4CL, TC4M, F5H, related to the flavonoid synthesis of CHSs, CHI, DFR, FLS, and the cellulose synthetase gene related to cell wall synthesis and pectinase activity of pectin (a) esterase gene are also enriched to present the trend of up-regulated expression, and most of the genes related to protein ubiquitination, E1, E2, E3, etc. The up-regulated trend indicates that the ubiquitination of protein is actively responsive. In general, a large number of synthesis of SA and ET, PR protein, flocking, defensin, thio, lignin, peroxidase, and flavonoids, and the enhancement of cell wall, make Cf-12 tomatoes have a better resistance to C.fulvum; in the analysis of the transcription factor (TFs), It is the first time that a number of new ARF, MADS, G2-like, G3H, C2H2, B HLH and other TFs families are involved in the resistance defense response of Cf-12 to C.fulvum, and the detected 60 TFs families present two kinds of expression patterns. A high expression trend in the later stage of sexual response; the transcriptional analysis of C.fulvum incompatibility interaction by a comprehensive Cf-12 also has a certain understanding of the resistance response mechanism of Cf-12 and C.fulvum. (6) preliminary functional verification of the VIGS of some genes in the response to Cf-12 and C.fulvum responses, the Ccg2 candidate genes and NPR1 (Solyc07g0449). 80.2) a part of the plant with CDPK (Solyc03g113390.2) gene was silent, and the disease index showed that the plants with silent NPR1 and CDPK still showed moderate resistance, while the silent Ccg2 plants showed medium sense, indicating that Ccg2, NPR1 (Solyc07g044980.2), CDPK (Solyc03g113390.2) gene and Cf-12 tomato responded to the resistance response to C.fulvum. Course related.
【学位授予单位】：东北农业大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：S436.412
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