Selection Of NBS-LRR Genes And Verification Of Function Against Powdery Mildew In Woodland Strawberry Based On RNA-seq

Strawberry powdery mildew(Sphaerotheca macular is(S.humuli))is a common and universal fungal disease.In recent years,strawberry powdery mildew was serious in the production process,and it was increasing year by year.In particular,strawberry powdery mildew in protected areas occurred seriously,the rate of diseased leaves was above 45%,the rate of diseased fruit was more than 50%,and the yield was generally reduced by 20%,which seriously affected the yield,quality and economic benefit of strawberries.At present,the main control methods of strawberry powdery mildew are chemical control,but basically more than 70%of the chemicals will have residues on strawberries after application,which will cause harm to the human body.Therefore,it is essential to excavate disease-resistance genes and develop disease-resistant varieties.Strawberry(Fragaria ananassa)is a herbaceous plant of the genus(Fragaria)of Rosaceae,which has the reputation of "fruit queen".Because of the high heterozygosity and polyploidy of octoploid strawberry,it brings a series of difficult problems to its breeding work,such as long cultivation cycle and so on.However,diploid woodland strawberry is an excellent experimental material with small genome and short life cycle.In addition,woodland strawberry and octaploid cultivated strawberry have a large number of sequence similarity and fragment collinearity,and the study of woodland strawberry can provide a basis for revealing the mechanism of resistance to disease of cultivated strawberry.Diploid woodland strawberry is a excellent experimental material with small genome and short life cycle.In addition,woodland strawberry and octaploid cultivated strawberry have a lot of sequence similarity and segment collinearity.The study of woodland strawberry provide basis for revealing the disease resistance mechanism of cultivated strawberry.Plant NBS-LRR gene family is the most numerous in plant disease resistance gene family.NBS-LRR(nucleotide binding site plus leucine-rich repeat)is an intracellular receptor protein that repeats nucleotide binding sites and leucine-rich repeats.Some plant NBS-LRR proteins can bind to pathogenic proteins directly,while others NBS-LRR proteins can indirectly feel pathogenic toxic proteins.NBS-LRR gene cloning is the basis of an in-depth understanding of plant disease resistance mechanism and disease resistance breeding by genetic engineering,which are of great significance for plant disease resistance research.1.In this paper,the RNA-seq data of two Fragaria vesca accessions(Hawaii 4(HW)and Yellow Wonder 5AF7(YW))infected by powdery mildew(PM)were analyzed,and we identified 2310 and 1681 differentially expressed genes(DEGs)in HW and YW,respectively.In addition,compared with YW(high-susceptible accession),more DEGs and higher expression levels were detected in HW(medium-susceptible accession).2.In HW and YW,the DEGs contained 935 and 728 kinds of domains,respectively,and many of these high frequency domains were associated with response to abiotic/biotic stimuli.Beside,the DEGs both have 16 expression profiles in two accessions,and the genes in four profiles were significantly enriched in "Plant-pathogen interaction" pathway.Three candidate disease resistance genes of PM,mrna13687,mrna15044,and mrna24119 were screened by the co-expression network analysis of differentially expressed NBS-LRR genes in two accessions.And the expression levels of the three genes were significantly up-regulated.3.We constructed the over-expression recombinant vectors of the three differentially expressed NBS-LRR genes(mrna13687,mrna15044,and mrna24119),and successfully transferred the over-expression recombinant vectors into the tobacco(Nicotiana tabacum L.)through genetic transformation by agrobacterium-mediated method.A total of 90 transgenic tobacco resistant lines were obtained,and the transgenic tobacco lines with mrna13687,mrna15044 and mrna24119 over-expression lines were 13,33,and 43,respectively.4.Through the inoculation with powdery mildew(Erysiphe cichoracearum),with non-transgenic tobacco as control,it was found that non-transgenic tobacco leaves were seriously affected,the disease area index reaching 94.54%after 15 days.Besides,some leaves appeared grayish brown spots,and white filaments covered the leaves surface.However,for transgenic tobacco,the disease area index of transgenic tobacco with the plant over-expression recombinant vectors of mrna24119 reached 78.53%,which was lower than that of the control group.Part of the leaves of transgenic tobacco with the plant over-expression recombinant vectors of mrna13687 were infected,and the disease area index was 30.46%.In addition,the disease area index of transgenic tobacco with the plant over-expression recombinant vectors of mrna15044 was only 18.9%,which was evidently lower than that of the control group.Therefore,mrna13687 and mrna15044 could be used as candidate genes for further transgenic experiments of strawberry.