Study On Function Of WRKY70 And LRR-RLK Genes Related To Disease Resistance In Brassica Napus By VIGS

WRKY70 and LRR-RLK are involved in plant disease resistance reaction in many plants,WRKY70 factor is the important member in plant signaling transduction process,plays an important role for plant resistance,mainly involved in the jasmonic acid(JA)and salicylic acid(SA)approach,LRR-RLK can be directly or indirectly identify of pathogens,and then transmission signals,make the plant obtain a series of disease resistance response.In this study,we used transcriptome sequencing data based Leptosphaeria biglobosa infect oilseed rape,screening of high expression and differentially expressed genes,in combination with whole genome sequencing data of oilseed rape,we obtained two genes of WRKY70 and LRR-RLK,in order to have a thorough understanding of the two genes resistant function and provide data information for oilseed rape important gene in disease resistance pathways,sequence information and function verification were studied by using bioinformatics analysis,and VIGS technique,we carried the cDNA of foreign genes into host,homologous gene silencing induced by VIGS to achieve the purpose of the validation of gene function.The research results are as follows:1.The full-length CDS sequences of the WRKY70 and LRR-RLK gene s were sequenced according to the sequence of the transcriptome obtained fro m the infected oilseed rape by a weakly strain Leptosphaeria biglobosa NM-1 infected.Using CLC SequenceViewer 6 deduced WRKY70 and LRR-RLK ami no acid sequence,and analysis of the WRKY70 and LRR-RLK amino acid seq uences for protein size,isoelectric point,hydrophobic domain,phosphorylatio n,N-.glycosylation.The WRKY70 gene CDS is 858bp,encoding 285 amino aci ds,containing a conserved WRKY domain,which belongs to the class III WR KY transcription factor;LRR-RLK gene CDS is 2775bp,encoding 924 amino acids,containing serine threonine protein kinase active site.2.The partial CDS sequences of WRKY70 and LRR-RLK genes were cloned,the size of WRKY70 fragments was 308bp,and the size of LRR-RLK gene fragments was 394bp.After sequencing,sequence alignment was compare d with that of transcriptome sequence 100%.3.Construction of successful VIGS vector pTRV2-WRKY70 and pTRV 2-LRR-RLK,switch to E.coli MC1061 carry on Bacterial fluid PCR and singl e and double enzyme digestion,the plasmid was transformed into Agrobacteriu m tumefaciens GV3101,which was used to infect the leaves of rape.4.Agrobacterium mediated silencing vector infection of rapeseed leave s after 14d inoculation of blackleg pathogen NM-1,collection of 4h,12h,24h,36h,48h,96h leaves,extraction of RNA expression detected by qPCR gene,an d to observe the incidence of plants.The results showed that the relative expr ession of target gene silencing plants was lower than non silencing plants,an dsilencing the leaf lesion larger,Leaf Chlorosis was yellow.