Preliminary Analysis Of Comparative Transcriptome And Vf/VmLRR-RLKs In Vernicia Fordii And Vernicia Montana Infected With Fusarium Oxysporum

Tung trees(Vernicia fordii),native to China,are famous for tung oil as an industrial oil plant in the world.Tung oil is widely used in industry,fishery,military,medicine and other fields.Meanwhile,the characteristic of clean,security and renewable make the tung trees be considered to be an important biological energy source.However,the outbreak of tung wilt dieases destroyed tung trees and caused enormous losses to tung tree planting since 1939.In 2007,the tung wilt dieases made heavy lost again in tung industry.With the characteristic of long duration,wide distribution,heavy damage and enormous losses,it is difficult to control the tung wilt disease,the resistant mechanis are far beyond our knowledge.Study on the resistance related gene of tung wilt dieases from a molecular level will be conduce to parse the resistant mechanism to the tung wilt dieases.The tung wilt dieases mainly lead damages in V.fordii,while the other species in Vernicia(V.montana)is resistant to tung wilt dieases.The article utilized an RNA sequencing-based approach to generate comparative transcriptomic atlases for different stages of F.oxysporum infected Vernicia root,and make an analysis on the gene expression pattern and differences of the twoVernicia species,response to tung wilt dieases,found that as hub genes,LRR-RLK gene family played an important role in the response of V.montana to tung wilt dieases.The main results were as follows:(1)RNA-seq of different stages of F.oxysporum infected V.fordii root.We have got 258,435 transcripts,133,304 genes.63.41%,56.68% and 40.66% genes were annotated for their functions by BLASTA search against NR database,KOG database and GO database,respectively.Besides,34.00% genes were annotated to 1,021 enzymes and 279 pathways to KEGG database.The analysis on differential expression gene(DEG)[(|log2foldchange|>=1,P<0.05)] in different stages of F.oxysporum infected V.fordii root revealed that much genes respond to the tung wilt dieases in early stage(F0-F1).GO term enrichment analysis revealed these genes were gathered in ATP combination,serine/threonine protein kinase activity and DNA binding.(2)RNA-seq of different stages of F.oxysporum infected V.montana root.We have got 245,240 transcripts,111,278 genes.70.26%,58.53% and 39.06% genes were annotated for their functions by BLASTA search against NR database,KOG database and GO database,respectively.Besides,67.43% genes were annotated to 956 enzymes and 278 pathways to KEGG database.The analysis on DEG in different stages of F.oxysporum infected V.montana root revealed that much genes respond to the tung wilt dieases in the middle stage(M0-M2).GO term enrichment analysis revealed these genes were gathered in ATP combination,serine/threonine protein kinase activity and receptor activity.(3)Comparative transcriptome analysis on V.fordii and V.montana infected by F.osysporum.Most genes in V.fordii responded to the F.osysporum infection in early stage and many of them were down-regulated,while a large proportion of genes in V.montana were induced in the middle stage(M0-M2)and most of them were up-regulated.The DEGs of V.montana were gathered to defense reactions,the cell walls of multicellular organization,carbohydrate metabolism and protein hydrolysis.44,310 pairs of orthologous genes were found in V.fordii(57.60%,44,310 / 76,924)and V.montana(72.83%,44,310 / 60,842)based on RNA-seq.K-means analysis of orthologous genes in V.fordii and V.montana during F.osysporum infection found that majority of orthologous genes showed the same expression patterns,and a handful of genes exhibited different expression patterns.The result of qRT-PCR verification of 30 orthologous genes in V.fordii and V.montana which exhibed different expression pattern to F.osysporum infection was roughly consistent with that of RNA-seq.The analysis on correlation of the DEGs in two Vernicia species reaveled that 450 orthologous genes showed a significant correlation(r > 0.65 or r <-0.65,p < 0.05)during F.osysporum infection..The GO enrichment analysis of them gathered in defense reaction,jasmonic and salicylate.Gene co-expression network analysis of V.montana revealed that leucine-rich repeat receptor protein CLAVATA2 and the serine/threonine protein kinase D6 PK were hub gene,the resistance to tung wilt dieases mediated by them mainly involves MAPK signaling pathways,plant pathogen interactions,circadian rhythm,calcium and signaling pathways.(4)Preliminary analysis of Vf/VmLRR-RLKs.We identified 236 and 230 LRR-RLKs in V.fordii and V.montana,respectively.Sequence alignment analyses showed that the RLK domains were more conserved than these of LRR domain among the Vf/VmLRR-RLKs.Phylogenetic analysis based on the RLK domain revealed that Vf/VmLRR-RLKs were grouped into 16 subclades.The conserved motif of the LRR domain in Vf/VmLRR-RLKs is LxxLxLxxNxLx GxIPxxLxxW/Lxx,which matched well with the known plant LRR consensus sequence LxxLxLxxNxLxGxIPxxLxxLxx,but differed at the third last amino acid(W or L).The expression analysis revealed that Vf/VmLRR-RLK orthologous genes mainly showed similar expression patterns in response to tung wilt disease,and a handful of gene exhibited different expression patterns.Tissue-specific expression analysis showed that the orthologous genes VfLRR-RLK256 and VmLRR RLK206 responsed to the F.oxysporum differentlly,but exhibited high expression in kernel and in root,respectively.(5)We cloned the VfLRR-RLK1 gene with 678 bp by RT-PCR,successfully constructed the plant expression vector VfLRR-RLK1-pBI121,and then transfered it into the agrobacterium EHA105.Infected the mutants Arabidopsis plants using the transformant agrobacterium EHA105.After resistance screening and PCR identification,finally we obtained the positive transgenic plants.The experiment laid foundation for identification function of VfLRR-RLK1.