| Peanut(Arachis hypogaea)is an important oil and economic crop in China.Bacterial wilt disease caused by Ralstonia solanacearum is one of the most destructive soil-borne bacterial diseases of peanut,is the key limiting factor for the yield and quality of peanut.There are not any efficient chemical methods or other methods to control it so far.Developing disease-resistant peanut cultivars is an efficient strategy to solve bacterial wilt.But there are some problems in the traditional disease-resistant cross-breeding,such as long cycle breeding,negative correlation between resistence and high yield and quality and so on.The effective way to solve R solanacearum infection is breeding high yield and quality peanut varieties with resistance to bacterial wilt disease through molecular breeding technology.There are few studies on the molecular mechanism of resistence to bacterial wilt in peanut,the number of resistence genes to bacterial wilt,peanut-R.solanacearum interaction mechanism is unclear.With whole-genome sequencing and functional genomics rapid development,it is inevitable that cloning resistance gene to bacterial wilt and analyzing peanut-Rsolanacearum interaction mechanisms through forward and reverse genetics approach.But that will be more difficult to solve above problems through forward genetics approach due to large peanut genome.Therefore,the objective of this research is cloning resistence-related genes and analyzing resistence regulation network through reverse genetics approach.It will provide a theoretical basis for molecular mechanism of disease resistance in peanut and promote genetic improvement of disease resistance in peanut.The main findings are as follows:1.A proteomic approach was adopted to compare the protemes of susceptible and resistant cultivars of peanut(Arachis hypogaea L.).Two cultivars of peanut were selected on the basis of their response to bacterial(Ralstonia solanacearum)inoculation wherein cultivar Yueyou 92,and cultivar Xinhuixiaoli were considered as hyper-resistent and hyper-susceptible cultivars respectively.Proteins were extracted from leaves of 4-week-old seedlings of the four cultivars after leafcutting inoculation,Two-dimensional gel electrophoresis(2-DE),colloidal Coomassie staining,coupled with mass spectroscope and protein database searching,A total of 14 proteins were found to be differentially expressed between the susceptible and resistant cultivars compared to their control.Among these proteins,5 induced,4 upregulated,2 downregulated and 3 differentially expressed contrary.The identified proteins belong to the categories of photosynthesis;energy metabolism,signal transduction and defense.Taken together,this study provides new insights into understanding of the molecular mechanism of resistance to bacterial wilt in peanut.2.The objective of this research was to identify resistance genes in response to bacterial wilt using microarray technology.To identify transcripts involved in disease resistance,we studied the gene expression profiles in two peanut genotypes,resistant or susceptible to bacterial wilt,using high-density cDNA microarray(Roche NimbleGen poplar gene expression microarrays)containing 101,344 unigenes integrated from 454 sequencing challenged by abiotic and biotic stresses and ESTs from genebank.Expression profile differences analyzed by comparing microarray hybridization results of two peanut varieties after Ralstonia solanacearum challenge.Differentially expressed genes were selected by t-test(FDR<0.05)and log2?1 or log2?-1.2,638 genes upregulated and 1,410 genes downregulated in the resistent peanut variety Yueyou 92 after inoculated by Ralstonia solanacearum,and susceptible variety Xinhuixiaoli showed 1,551 upregulated and 2,054 downregulated genes.The reliability of the microarray was validated through qRT-PCR of five differentially expressed genes(DEGs).Gene function annotation differences was significant by GO annotation between the resistant and susceptible varieties,and KEGG pathway analysis showed DEGs involved in metabolic pathway in the susceptible variety were more than in the resistent variety.Some important genes associated with the resistence of bacterial wilt,such as NBS-LRR class resistance genes,transcription factors and kinases genes were expressed differentially in the two varieties.There were more upregulated genes in resistant variety and more downregulated gene in the susceptible variety.These DEGs may be associated with defense answer in peanut after Ralstonia solanacearum infection which may be involved in resistance to bacterial wilt.These information can provide a theoretical basis for the resistent molecular mechanism of peanut bacterial wilt.It will provide a guide for disease-resistant molecular breeding in peanut.3.Leucine-rich repeat receptor protein kinase(LRR-RLK)homologous sequence as candidate genes,such genes have been shown to have important physiological functions,such as involved in the regulation of plant growth and development,participate in stress tolerance response and defense response.It is a prerequisite to understand the molecular mechanism of defense response to Ralstonia solanacearum infection in peanut through cloning LRR-RLKs.In this study,an up-regulated differential LRR-RLK gene named AhRLK1 was screened by microarray anylisis.This gene was induced by Ralstonia solanacearum after inoculation.The full-length cDNA was cloned by RACE,the cDNA sequences of full length is 3,292 bp in length,including 2,976 bp open reading frame and encoding 992 amino,After analysis,AhRLK1 gene contained a signal peptide,nine LRR domains and extracellular kinase conserved motifs.Subcellular localization of AhRLK1 suggested 35S::AhRLK1-GFP fusion protein was localized in the membrane through agroinfiltrating leaf epidermal cells in Nicotina benthamiana.According to analyze the expression pattern by Real time PCR results,AhRLK1 was constantly up-regulated in the suscepitible peanut cultivar Xinhuixiaoli significantly after inoculated Ralstonia solanacearum,and no change in the resistent peanut cultivar Yueyou92.This gene was induced by different homones such as SA,ABA,ET,JA and PAC,but down-regulated after cold and dry treatments.Transient AhRLK1 overexpression can induce HR response by agroinfiltration in Nicotina benthamiana.By constructing overexpression vector and Agrobacterium-mediated transforming into Nicotiana tabacum cv CB-1,The trangenic tobacco significantly enhanced resistance to Ralstonia solanacearum.In contrast to CB-1 plants,the expression level of several representative stress-responsive genes like NtH1N1,NtHSR201,NtHSR515,NtHPR3,NtHPR4,NtCHN50,NtPR1b,NtPR2,NtEFE26 and NtAcs6 were significantly up-regulated in the overexpression AhRLK1 transgenic line.It suggested AhRLK1 may involved in defense response to Ralstonia solanacearum in plant,and may be involved pathogen recognition in the early infection,and it may participated in the abiotic stress defense response of plant.The cloning of AhRLK1 gene provides a basis for studying the structure and function of peanut disease-resistance relating genes and bacterial wilt disease resistant genetic breeding in peanut.4.The nucleotide-binding site(NBS)-Leucine-rich repeat(LRR)gene family accounts for the largest number of known disease resistance genes,these R genes play an important role in plant defense against pathogenic diseases.In this study,an up-regulated differential NBS-LRR resistent gene named AhRRS5 was screened by microarray anylisis.This gene was induced by Ralstonia solanacearum after inoculation.The full-length cDNA was cloned by RACE,the cDNA sequences of full length is 3,157 bp in length,including 2,829 bp open reading frame and encoding 943 amino,after analysis,AhRRS5 gene contained all the typical NBS-LRR resistent gene conserved motifs.Transient expression analysis in onion epidermal cells suggested 35S::AhRRS5-GFP fusion protein was localized in the nucleus.According to Real time PCR analysis,AhRRS5 was up-regulated in the resistent and suscepitible peanut cultivars to bacterial wilt,The magnitude of upregulation was larger in susceptible peanut than resistent one.This gene was induced by different homones such as SA,ABA,ET,JA and PAC,as well as cold and dry treatments.Transient overexpression can induce HR response by agroinfiltration in Nicotina benthamiana.By constructing overexpression vector and Agrobacterium-mediated transforming into Nicotiana tabacum cv CB-1,The trangenic tobacco significantly enhanced resistance to Ralstonia solanacearum.In contrast to CB-1 plants,the expression level of several representative stress-responsive genes like NtHIN1,NtHSR201,NtHSR515,NtPRlb,Ntla/c,NtNPR1,NtEFE26 and NtAcs6 were significantly up-regulated in the overexpression AhRRS5 transgenic line.It suggested AhRRS5 may involved in defense response to Ralstonia solanacearum in plant,and the resistence achieved through multiple signaling complex regulatory networks.The cloning of AhRRS5 gene provides a basis for studying the structure and function of peanut disease-resistance relating genes and bacterial wilt disease resistant genetic breeding in peanut. |
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