Isolated And Analysis Of Resistance Related Genes In The Interaction Of Root Knot Nematode And Pepper

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.