Resistance Identification Of Bacterial Wilt In RILs And Resistance Gene AhqBW3 Cloning And Analysis Of Peanut

As one of the main edible oil and protein crops of China,peanut(Arachis hypogaea L.)is seriously influenced by bacterial wilt disease(BW)which was caused by soil-borne Ralstonia solanacearum.No efficient chemical control or other approaches have so far been developed.Developing disease-resistant-peanut cultivars is known as an efficient strategy to solve bacterial wilt.As the molecular mechanism underlining the resistence to bacterial wilt in peanut is not reported,the number of the resistence genes and the mechanism of Peanut-R.solanacearum interaction are not clarified.Therefore,developing varieties with high resistance to bacterial wilt together with high yield and quality is difficult.The objective of this research is to characterize a RIL(Recombinant inbred line)population for screening resistance germplasms and for cloning and analyzing resistence-related gene.It will provide genetic resources and provide an insight into the molecular mechanism of disease resistance,which should promote genetic improvement of disease resistance with ideal characters in peanut.The main findings are as follows:1.In this research,the peanut RILs including 300 F9 progenies derived from Yueyou 92 X Xinhuixiaoli were investigated six agronomic traits and disease resistance were tested through inoculation with Ralstonia solanacearum in fields.The results showed that six agronomic traits(main stem height,lateral branch length,the total number of branches,fruit number per plant,full fruit number per plant,fruit weight per plant)are quantitative traits of continuous traits values and polygenic inheritance.Significant differences of the resistance to Ralstonia solanacearum were observed among the RILs,and some lines showed more resistant than the parental line.Resistance to Ralstonia solanacearum is quantitative trait,and it is not tightly linked with the lateral branch length and fruit weight per plant.According to the disease index,there are 58 lines resistant to Ralstonia solanacearum,and 125 lines high susceptible to Ralstonia solanacearum.Ten high resistant lines(16,108,130,131,251,261,336,362,475,646)and 10 high susceptible lines(4,13,35,151,161,265,287,317,463,552)to BW were obtained by comparation.Considered with the results of the resistance to Aspergillus flavus identifed in early time,3 lines(16,251,475)resistant to both BW and aflatoxin were identified.2 lines(475,251)of which are with ideal traits in yields(fruit weight per plant is 38g,43g respectively),BW and aflatoxin resistance,and can be used as fine quality germplasm materials in peanut bacterial wilt resistance breeding.2.The NBS(nucleotide-binding site)-LRR(Leucine-rich repeat)gene family is known as the biggest disease-resistance gene family which plays a key role in plant defense responses.In this study,an NBS-LRR resistance gene AhqBW3 was found linked with a BW resistance molecular marker SNP79,it was down regulated 4 times in resistance peanut Yueyou92.The full-length cDNA was cloned by RACE.It contained 2,998bp encoding 855 amino acids.The AhqBW3 gene contained all the typical NBS-LRR resistence gene conserved motifs.Agrobacterium mediated transient expression analysis in onion epidermal cells suggested 35::AhqBW3-GFP fusion protein was localized in the membrane and cytoplasm.By Real time PCR analysis,AhqBW3 was down-regulated in the resistent peanut cultivars to bacterial wilt,and appeared up-regulated firstly,then down-regulated in suscepitible peanut cultivars.This gene was induced by different hormones such as ABA,ET and JA,demonstrating down-regulated in the resistent peanut cultivars,but up-regulated in the suscepitible peanut cultivars.Transient overexpression can induce hypersensitive response(HR)by agroinfiltration in Nicotina benthamiana.Overexpression AhqBW3 in Nicotiana tabacum cv hongda significantly enhanced resistance to Ralstonia soanaeearum.In contrast to wild hongda plants,the expression level of several representative stress-responsive genes like NtH1N1,NtHSR515,NtPR1b,NtPR2,NtPR4,Ntlak,NtNPR1 and NtAcs6 were significantly up-regulated in the AhqBW3 overexpression transgenic line.It suggested AhqBW3 may involved in defense response to Ralstonia solanacearum in plant,and the resistence achieved through complex multiple signaling regulatory networks.The cloning of AhqBW3 gene provides a basis for the study of disease-resistance mechanism of the candidate genes and a benefit to genetic enhancement to BW in peanut.