Molecular Cloning Of MAPK C Group Gene Families From Brassica Napus,Brassica Rapa,Brassica Oleracea And Functional Identification Of MAPK1 From Brassica Napus

The genus Brassica and Arabidopsis thaliana are two model systems in Cruciferous plants.Arabidopsis thaliana is an important model plant,and the genus Brassica contains a number of economically important crops,which play a significantly role in agricultural economics.Arabidopsis and Brassica originated from a common ancestor with a closer relationship.Their highly consistency in gene sequence made it possible to analysis some functionally important genes of Brassica by using Arabidopsis thaliana genome information as reference.As early as the 1920s and 1930s,studies in cytology revealed that the genus Brassica contained three basic species as B.rapa(AA,2n=20),B.nigra(BB,2n=16),B.oleracea(2n=18,CC)and three complex species included B.juncea(AABB,2n=36),B.napus(AACC,2n=38)and B.carinata(BBCC,2n=34).Interspecific crosses between basic species formed tetraploid species.U's triangle was generally acknowledged for it's explanation about the relationship between Brassica allopolyploid and it's diploid ancestors,and the speculation that B.napus originated from natural interspecific crosses of B.rapa and B.oleracea had been confirmed by more evidence.Comparative genomics studies between Brassica and Arabidopsis thaliana showed that Brassica ancestor species genomes had triploidization.MAPK(mitogen-activated protein kinase)was a large serine and/or threonine protein kinase family in plants.MAPK cascade was constituted by MAPK,MKK(MAPKK)and MEKK(MAPKKK),and signals were amplified along with the transduction from upstream to downstream receptors by protein phosphorylation(MEKK?MKK?MAPK).It played a central role in mediating responses to extracellular signals,developmental regulation and stress resistance.Based on the RACE technology,full length cDNA and genome DNA of MAPK1,MAPK2 and MAPK7 gene familys(MAPK C group gene families)were cloned from B.napus and its parental species,B.rapa and B.oleracea,respectively.Comparative genomics evidence acquired from bioinformation analysis between Arabidopsis thaliana and these species revealed some important molecular evolutional characteristic of MAPK C group gene families.We also identified the function of MAPK1 through biotic/abiotic induction and transgenic methods.The main results are as follows:1.Cloning the main members of MAPK C group gene families in BrassicaBased on RACE homology cloning technology,we separated four MAPK1 and four MAPK2,respectively,from B.napus,B.rapa and B.oleracea,named BnMAPK1-1l,BnMAPK1-2,BrMAPKl,BoMAPK1 and BnMAPK2-1,BnMAPK2-2,BrMAPK2,BoMAPK2.Five MAPK7 were cloned,called BnMAPK7-1,BnMAPK7-2,BrMAPK7-1,BrMAPK7-2 and BoMAPK7.No vertical homology genes of AtMAPK14 in Arabidopsis thaliana were separated from B.napus,B.rapa and B.oleracea.Except intron,all cDNA were well corresponding to its genomic DNA.Some "TC" repeats which extremely not conservative were found in 5' UTR region of MAPK C subfamilies,speculating that the repeats could be one of regulatory sites.Moreover,the UTR region of MAPK C subfamilies was polymorphism,and one more forms of UTR existed in the ends of ORF,which probably one way to achiveving regulatory function of MAPK genes.Most of MAPK C subfamily genes contained variable transcriptional initiation site and poly(A)site.Except MAPK7,some poly(A)signals were detected in 3'UTR,such as AATAA,AAATAA,AAATATAA,AAATAATAA.2.The evolutional relationship of MAPK C group genes in BrassicaPhylogenetic analysis of the genus Brassica and Arabidopsis thaliana indicated that BnMAPKl-1 and BnMAPK2-1 derived from the basic species,B.rapa genome A,while BnMAPK1-2 and BnMAPK2-2 from B.oleracea genome C on the MAPK C1(MAPK1 and MAPK2)site.In this case,B.napus was equal to the sum of B.rapa and B.oleracea.Differently,on the MAPK C2(MAPK7)site,BnMAPK7-1 came from BrMAPK7-1,while BnMAPK7-2 from BoMAPK7.Simultaneously,BnMAPK7-2 and BoMAPK7 were phylogenetically closer to BrMAPK7-2,but farther to BrMAPK7-1.It showed that the relationship between BnMAPK7-2,BoMAPK7,BrMAPK7-2 and AtMAPK7 is closer than BnMAPK7-1 and BrMAPK7-1,which might be caused by some conservative evolution(the ancestor of BrMAPK7-2 and BoMAPK7)occurred when one MAPK7 ancestor duplicated after separating with Arabidopsis thaliana ancestor,while large changes in other menbers(the ancestor of BrMAPK7-1),both conservative and largely changed members existed in B.napus after natural hybridization between B.rapa and B.oleracea.B.napus was equal to the sum of B.rapa and B.oleracea on MAPK7 site,which was similar to MAPK C1 site.3.BnMAPK1 may involve in a variety of signaling pathwaysThe promoters of MAPK1 gene family were cloned from B.napus,which contained multiple hormone and stress response elements.Analysis of BnMAPK1 expression patterns induced by wounding stress or pathogen attack showed that BnMAPK1 could be activated transcriptionally by plant hormones,such as MeJA(methyl jasmonate),SA(salicylic acid),ABA(abscisic acid),H2O2,wounding and sclerotinia sclerotiorum,speculating that BnMAPK1 played a role in various signaling pathways relative to plant hormones(MeJA,SA,ABA),signaling molecular(H2O2)and extracellular stress resistance.JA signaling pathway was mainly responsible for the response process involving wounding and pathogen invason,and inhibited SA signaling pathway.4.Generation of transgenic B.napus with overexpression of BnMAPK1We constructed over-expression vector of BnMAPK1 gene and transferred it into B.napus ZY821 DH lines with hypocotyls infected by Agrobacterium,and aquired twelve transgenic plants.qRT-PCR results showed that the expression level of BnMAPK1 genes in T0 generation were all improved,the increased multiples ranged from 2 to 13.5.BnMAPK1 may involve in the development of B.napusThe accumulations of biomass were higher in positive transgenic lines compared with white type for each stage.The observation results of leaf surface showed that the number of cells was more in bud leaves,and bigger in Juvenile leaves and mature leaves in transgenic plants,compared with white type.Speculating that BnMAPK1 might be interrelated with the growth and cell division,the up-regulation of gene expression might play a role in improving plant development.6.BnMAPKl plays a role in response to pathogenThe BnMAPK1 gene over-expressing leaves were inoculated with sclerotinia sclerotiorum in vitro,it was found that the diffusion degree of the plaque is far smaller compared with white type after 72 hours,the resistance to S.sclerotiorum was obviously improved,speculating that BnMAPKl gene could regulate the resistance ability to pathogen positively.7.Obtaining differentially expressed genes in overexpression BnMAPKl plants based on high-throughput sequencingWe got differential expression genes of transgenic lines and white type based on high-throughput sequencing,and in generally,acquired amount of information about biological process,cellular components,molecular function and signaling pathway by GO functional significant enrichment analysis and KEGG pathway significant enrichment analysis in differential expression genes.At the same time,differential expression analysis of the three MAPK cascade gene families showed that the expression of other MAPK cascade genes varied along with the elevation of MAPK1,while the range was limited in double multiples,indicating that MAPK cascade pathway was transcriptionally stable,to ensue the stability of the entire cascade pathway.8.Obtaining interaction protein with BnMAPKl by yeast two-hybrid of B.napus.Genes interacting with MAPK1 were preliminary obtained in B.napus by yeast two-hybrid.Further analysis indicated that they were mainly related to plant development,biotic and abiotic stress.Meanwhile,the expression level varied in degree in BnMAPKl overexpressional transgenic plants.We got a general knowledge about the signaling pathway and biological processes involved by BnMAPKl though yeast two-hybrid.