Conjoint Analysis Of Expression Profiling And Proteomics And Analysis And Function Verification Of TNL & SNARE Gene Families In Chinese Cabbage Under TuMV Infection

Brassica species can serve as food,oil,seasoning,and fodder.Some of Brassica species are also developed to study genomic evolution.Chinese cabbage(Brassica rapa ssp.Pekinensis)is an important member of the Brassica genus in Brassicaceae family.Its yield and quality can be significantly influenced by biological stress,including a kind of Potyvirus,Turnip mosaic virus(TuMV).TuMV can infect many plants and usually causes severe symptoms,such as vein clearing,necrosis,and dwarfing.Plant viruses need to hijack enormous host factors,which could be protein,nucleic acids,and lipids,etc.,to support their own full lifecycle.Hence,it would be a new trend of modern plant disease resistance breeding through discovering new host factors and disease resistance genes and then applying them to production by modern molecular biological techniques.Herein,we identified many differentially expressed genes and protiens by conjoint analysis of RNASeq and iTRAQ-LC-MS/MS in TuMV challenged Chinses cabbage.Then we examined the functions of one candidate gene,BrLTP3.Our data suggest that BrLTP3 is associated with TuMV infection.Moreover,TIR-NBS-LRR(TNL)gene family,which is an R gene family,is analyzed thoroughly.One of the candidate genes Bra008055 is verified to confer resistance to plants.Finally,the SNARE gene family,which is closely related to plant virus infection,is also analyzed in detail.Two related genes,BrSYP31 and BrSEC22,are proved to be related to the formation of virus replication complexes(VRCs).Our study offers a theoretical basis for elucidation of the anti-TuMV mechanism of Chinese cabbage and also provides several candidate genes that are related to virus infection.1.We performed RNA-Seq and iTRAQ-LC-MS/MS analysis.757 differentially expressed genes(DEGs)and 75 differently expressed proteins were identified in Chinese cabbage infected by TuMV.Conjoint analysis between iTRAQ-LC-MS/MS and RNA-Seq was further conducted.DEGs are enriched in many pathways and among them,plant hormone signal transduction pathway,plant-pathogen interaction pathway,and protein processing in the endoplasmic reticulum pathway are thought to be the TuMV infection closely related pathways.Combined with previous studies,the regulatory network related to TuMV in Chinese cabbage is summarized.In the regulatory network,four non-specific lipid transfer proteins are highly expressed,and one translation elongation factor(EF1B)is up-regulated.In the pathway of heat shock protein regulation,the heat shock protein factor 4(HSF4)is down-regulated,many heat shock proteins and their interacting proteins are upregulated and heat shock proteins can promote virus replication.Further,WRKY23 and WRKY33 are up-regulated in the nucleus.WRKY22 and WRKY33 can activate the expression of resistance genes and participate in the pathway of calcium regulation in disease resistance.In addition,CML genes with calcium recognition function also significantly respond to TuMV infection.Finally,we identify some candidate genes associated with TuMV infection.After series of verification,it is suggested that BrLTP3 is associated to TuMV infection.2.90 TNL-type genes are identified and characterized in Chinese cabbage(Brassica rapa ssp.Pekinensis).It reveals that these genes express deferentially in tissues by performing tissue-expression profiling.qRT-PCR analysis reveals that the expression patterns of 69 genes are changed after TuMV challenge,with 42 genes up-regulated and 11 genes down-regulated.These genes are further grouped according to their different expression patterns.16 candidate genes are identified as responding to TuMV infection.The functional verification of a candidate gene screened from the genome-wide analysis is conducted.The protein encoded by Bra008055 is located in the nucleus.And it can interact with TuMV-VPg,which is related to translation.As it is hard to get the Chinese cabbage mutant,instead,the homologous mutant is used in TuMV challenge.Compared with wild type,the mutant accumulates more viruses,while the overexpressed lines accumulate fewer viruses.These results indicate that the Bra008055 is a resistant gene against TuMV.3.78 BrSNARE genes are identified in Chinese cabbage.We analyzed the phylogenetic relationships(including their orthologous and paralogous relationships with those in Arabidopsis and rice).Then we detected the expression of 55 BrSNAREs and grouped them according to their change trends and sub-cellular location in different organelles.On the basis of our analysis,we conclude that nine BrSNAREs might be associated with cell-to-cell movement and 15 BrSNAREs might be associated with longdistance transport.The functional verification of a candidate gene screened from the genome-wide analysis is identified.BrSYP31 and BrSEC22 can be located in the cytoplasm as scattered punctate.Based on the co-localization with the Golgi marker,it is suggest that these two proteins are located on Golgi.Furthermore,these two proteins can co-localize with VRC.Different from BrSYP31,BrSEC22 can interact with 6K2.Taken together,it is revealed that BrSEC22 is closely related to TuMV infection.