【作者】 茆振川；

【导师】 杨之为； 谢丙炎；

【作者基本信息】 西北农林科技大学 ， 植物病理学， 2007， 博士

【摘要】 根结线虫(Meloidogyne spp.)是辣椒主要病原物之一,每年造成巨大损失。Carolina Wonder (Capsicum annuum L.)和HDA149 (Capsicum annuum L.)为抗根结线虫的辣椒品种,分别含有单显性抗根结线虫基因N、Me3,抗南方根结线虫(Meloidogyne incognita)、爪哇根结线虫(Meloidogyne javanica)、花生根结线虫(Meloidogyne arenaria)。通过SSH方法分析辣椒与根结线虫之间的不亲和互作可以初步的了解N基因和Me3基因介导的抗根结线虫机制,并分离抗性相关基因,对于辣椒抗根结线虫育种有重要意义。以Carolina Wonder和HDA149为试验材料,以接种南方根结线虫12、24、36 h的根尖材料作为测验方(tester),未接种根尖材料作为驱动方(driver),分别构建了N基因和Me3基因介导早期表达的抑制消减杂交cDNA文库,并结合文库高密度点阵膜杂交差异筛选,构建了相应的EST表达谱。在N基因介导的表达基因谱中获得了237条表达序列标签(EST)片段。在GenBank上进行BLASTn与BLASTx分析,得到148条已知的EST序列,获得已知的上调抗性相关EST 68个。分离出了编码抗线虫蛋白和类LRR抗性蛋白的基因;与过敏性坏死反应和系统获得性抗性相关的类萌芽素(GLP)、HSR203J蛋白、蜜腺蛋白、蛇毒素肽、Kuntiz蛋白酶抑制剂等基因;多种信号蛋白基因,如ATP酶、钙结合蛋白、14-3-3蛋白、离子通道、水通道蛋白、渗透蛋白、泛素蛋白;以及与抗性相关的WRKY、乙烯转录因子(ERFBP)等转录因子的基因。在Me3基因所介导的早期抗根结线虫基因表达库中,经过点阵膜杂交筛选到309个克隆为阳性表达。通过NCBI基因库BLASTn和BLASTx同源对比,共有229个可以找到已知的同源基因,其中71个为功能未知的基因。30个没有发现与其匹配的同源序列。将所得的已知的158个EST序进行功能分类,可知表达的抗根结线虫相关基因涉及抗病反应的全过程,包括与病原菌相互识别作用,防御相关基因的调控,信号转导,转录因子、代谢调控以及细胞组分等多个方面。分离到三个编码NBS-LRR结构的基因,H-40、H-634、H-614,分别与抗黄萎病(Verticillium dahliae)、BS2抗细菌(Xanthomonas campestris)、Mi-1抗根结线虫(Meloidogyne spp.)基因具有同源性,且相似度很高。同时分离出了WRKY转录因子、乙烯转录因子、MYB型转录因子,以及介导基因沉默的AGO1-2基因、介导过敏性坏死的Avr9/Cf-9快速激发蛋白、hin1蛋白等重要防御相关的基因。通过根结线虫侵染组织的显微观察和消减文库中EST功能分类方法对所得基因在分子功能、细胞组成及生物过程等方面进行功能分类和分析,初步推断认为N基因,Me3基因介导的早期抗根结线虫作用具有相似的反应体系:具有NBS-LRR结构的蛋白识别线虫的入侵,激活抗性信号的传递,使WRKY、EREBP及非毒性基因快速激发蛋白等重要抗性相关反应因子的表达,使寄主植物的HR、SAR防御反应和保护机制适当地表达,并且,这些反应是基于寄主的初生代谢和次生代谢的调控体系。通过消减杂交,在根结线虫诱导的Carolina wonder辣椒根尖中分离出了CaRKNIF (WRKY)、Ca-372(Snakin2)、CaERF基因,并通过RACE方法确定了这三个基因的全长。通过Southern和Northern杂交的方法确定了这些基因的部分特性。Ca-372基因全长为707 bp,开放阅读框为315 bp,编码具有104氨基酸的小分子蛋白。这个基因编码蛋白与来自马铃薯(Solanum tuberosum)蛇毒素蛋白(Snakin2)具有同源性,确定相似度达到89% (93/104),而蛇毒素蛋白在马铃薯抵御逆境作用中起着重要作用。通过Southern和Northern杂交证实,在Carolina wonder辣椒中Ca-372是一个单拷贝基因,并且在接种根结线虫6 h时表达量明显增加,在48 h时达到最高。Ca-372分离自根结线虫诱导抗根结线虫辣椒表达的差显文库,所以推测Ca-372在抗根结线虫中起着一定作用。CaRKNIF基因全长为2139 bp,开放阅读框为1473 bp,编码具有490氨基酸的蛋白,它与来自烟草的(Nicotiana tabacum)DNA绑定蛋白2(WRKY2)具有同源性,相似度达到68% (371/538),期望值达到1e-179,通过基因对比分析CaRKNIF属于WRKY家族,具有两个保守的WRKY区域。通过杂交证实,在Carolina wonder辣椒中CaRKNIF是一个单拷贝基因。在接种根结线虫6 h时CaRKNIF即可被诱导表达,12 h时强烈表达。CaERF基因全长为1528 bp的全长基因,开放阅读框为1128 bp, CaERF编码375个氨基酸,CaERF的分子量为41.5 kd,具有一个由59个氨基酸构成的保守的ERF结构域(104-162),和一段可能作为核定位信号的富含K、R氨基酸的碱性结构域。属于乙烯反应因子家族的第Ⅳ次家族。Southern和Northern杂交证实,在HDA149辣椒中,CaERF是一个单拷贝基因,并且在接种根结线虫6 h时被强烈的诱导表达。总之,试验采用SSH方法初步的探索了辣椒N基因和Me3基因的抗根结线虫机制,分离到许多抗性相关基因,同时对Ca-372、CaRKNIF、CaERF基因全长的分离及功能分析有助于进一步的认识并利用N基因和Me3基因对根结线虫的抗性奠定了良好的基础。在研究中分离出的许多抗根结线虫基因EST片段,以及在试验中发现的许多功能未知的基因,可能代表着新的抗根结线虫基因,有待于进一步的研究。更多还原

【Abstract】 The root knot nematodes (Meloidogyne spp.) are obligate endoparasitic nematode, cause severe losses in yield throughout the world. Carolina Wonder (Capsicum annuum L) and HDA149 (Capsicum annuum L) are the nematode resistant cultivar pepper, which have a single dominant gene N and Me3 respectively, and resistance to root knot nematode Meloidogyne incognita, Meloidogyne javanica, and Meloidogyne arenaria. Through the SSH analysis of pepper and root knot nematode, it can be known primerly that the rule mechanisms of pepper resistant to root knot nematode mediated by N and Me3,and isolated the resistant related genes. This will be good at pepper breed.To investigate the early up-regulated expression gene profile in cultivar Carolina Wonder and HDA149 induced by root-knot nematode (RKN), the cDNA of pepper seedling root tips inoculated with J2 RKN 12, 24, and 36h were used as tester and that from untreated root tips as driver, two forward subtracted cDNA library was constructed using suppression subtractive hybridization (SSH) respectively.Totally 237 RKN-induced EST fragments were selected with DIG Nonradioactive Nucleic Acid Labeling and Detection System from the Carolina Wonder SSH cDNA library. Based on the sequencing and Blast analyzing of 148 fragments, 68 up-regulated expression EST fragments were identified, including the genes encode disease resistant proteins and LRR resistance protein-like, both of them have nucleotide–binding /leucine-rich repeat (NBS-LRR) or LRR domain; some defense proteins, such as Germin-like protein, HSR203J protein-like, Nectarin, and Snakin-2; some biostress resistance related transcription factor, like WRKY and ethylene-responsive element binding protein (EREBP); some signal proteins, such as GRP-like protein and 14-3-3 family protein. The Gene Ontology analysis revealed that the up-regulated ESTs involved in the recognition to nematode, signal transduction, protection mechanism, hypersensitive response (HR) and systemic acquired resistance (SAR).We got 309 expressed EST fragments in the SSH cDNA library of HDA149 with DIG Nonradioactive Nucleic Acid Labeling and Detection System. Base on the sequencing and Blast analyzing, 229 expression EST fragments can find homology genes in the NCBI gene library, but in which 71 ESTs fragments were encode unknown proteins. 30 ESTs fragments are no match. The Gene Ontology analysis also revealed that the up-regulated ESTs involved in the recognition to nematode, signal transduction, protection mechanism, HR and SAR. H-40、H-634、H-614 were isolated and identified, which encode proteins have NBS-LRR structure, and are homologous with the Verticillium wilt disease resistance protein, Disease resistance protein BS2, Disease resistance gene homolog Mi-copy1 respectively. In addition, some important defense gene were isolated, such as Double WRKY type transcription factor, Ethylene-responsive element binding protein, Avr9/Cf-9 rapidly elicited protein, MYB transcription factor, and Argonaute protein Gene et al.Base on the observing with microscope and the SSH ESTs function analyzing, we putative that the genes encode NBS-LRR proteins have a key rule to recognizes the root knot nematode, and result in the signal transduction-related genes expressed, such as WRKY transcription factors and EREBP, activated a suitable defensive response and protection mechanism system, this often including HR and SAR. In addition, all of these defensive responses are base on the first and secondary metabolites.With the RACE technology, we got the all length of gene CaRKNIF (WRKY type transcription factor 2) and Ca-372 (Snakin2) from the RKN induced Carolina wonder pepper tips, and using Southern and Northern blot, some characters of the both genes were identified.The all length of Ca-372 is 707 bp, and the open reading frame (ORF) is 315 bp, encode a 104 amino acides small protein. The protein is homologous with Snakin2 protein from potato (Solanum tuberosum), the identify percent is 89% (93/104). The snakin2 protein has an important role in the potato defense to the pathogens, cold, salt and other unbiostress. The southern and northern bolt revealed that gene Ca-372 is a single copy in Carolina wonder, and was strongly expressed after inoculated RKN 6 hours, and more strongly at 48 hours. Ca-372 was isolated from the SSH cDNA library of Carolina wonder and combine with the results of Southern and Southern blot, it was putative that Ca-372 have a rule in defense against the RKN.The all length of CaRKNIF is 2139 bp, the ORF is 1473 bp, and encode 490 amino acides protein. It is homologous with DNA-binding protein 2(WRKY2)from Tobaco (Nicotiana tabacum) , the identify percent is 68% (371/538), and the except is 1e-179. Through the compare of gene function classification, CaRKNIF belong to wrky gene family, it has two conservative WRKY domains. The Southern and Northern blots show that there is a single copy, it was expressed after inoculated RKN 6 hours, and strongly expressed after inoculated RKN 12 hours.The all length of CaERF is 1528 bp, the ORF is 1128 bp, and the encoded protein has 375 amino acides, the molecular weight is 41.5 kd. It is homologous with Ethylene-responsive element binding protein, belong toⅣfamily. CaERF has a conservative Ethylene-responsive domain, which include 59 amino acides. The Southern and Northern blots show that CaERF is a single copy in HDA149 pepper and it was strongly expressed after inoculated RKN 6 hours.In a conclusion, with the SSH, we understand the rule mechanisms of pepper resistant to root knot nematode mediated by N and Me3, and isolated many resistant related genes. The isolation of all length gene Ca-372、CaRKNIF、CaERF and the function analysis of them is a better base to research and apply the gene N and Me3. In addition, there are many resistant nematode EST fragments, unknown protein genes and no match genes in Carolina wonder and HDA149 SSH cDNA library, these may be novel genes and should be researched further.更多还原