Cloning,expression Profiling And Expression Analysis Of Annexin Gene Family In Epidermis And Seedlings Of Brassica Rapa 'Tsuda'

Brassica rapa'Tsuda' Turnip anthocyanin synthesis is induced by UV-A light,synergies by UV-B/blue complex light.Therefore,we believe that the UV-A signal receptoe of Brassica rapa 'Tsuda' Turnip is different from blue/UV-A receptor(cryptochrome)in Arabidopsis.It is a new specific receptor induced by UV-A.The 2D-DIGE(Two-dimensional fluorescence difference gel electrophoresis)proteomic analysis of UV-A light treatments Brassica rapa‘Tsuda'Turnip white taproot epidermis showed increased expression of annexin family BrANNEXIN after UV-A light treatment.In this research,we cloned annexin family genes BrANNEXIN1,2,3,4 from Brassica rapa'Tsuda' Turnip and analysised the expression characteristics of different organizations and different treatments in Brassica rapa 'Tsuda'Turnip.We built overexpression vectors and trans the BrANNEXIN4 into Arabidopsis thaliana to research overexpression BrANNEXIN4 whether could affect anthocyanin synthesis,and screening and identification of specific anthocyanin biosynthesis signal transduction factors.Mainly the results of this research were as follows:1.The cloning of BrANNEXIN family genesWe cloned in to four annexin genes from Brassica rapa 'Tsuda' Turnip,named BrANNEXINl,2,3,4.ORF lengths are 954 bp,951 bp,960 bp and 948 bp.3'UTR lengths are 171 bp,74 bp,130 bp and 151 bp.Southern blotting analysis showed BrANNEXIN1,3 have two copies at least in Brassica rapa 'Tsuda' Turnip genome,and BrANNEXIN2,4 have at least one copy.Bioinformatics phylogenetic tree showed BrANNEXINl,2,4 close genetic relationship,BrANNEXIN3 distants genetic relationship.Brassica rapa 'Tsuda' Turnip close genetic relationship with Brassica napus,Brassica oleracea,Arabidopsis thaliana.2.BrANNEXIN1,2,3,4 expression characteristics analysis(1)Tissue-specific expressionThe highest expression levels of BrANNEXIN1,2,3,4 is in buds and freshy root.The higher expression level of BrANNEXIN1 is in leaves and the lower expression of BrANNEXIN2,3,4 in leaves.The expression of BrANNEXINl,4 in the petals is also higher,but the expression of BrANNEXIN2.3 can not be detected in petals.The expression of BrANNEXIN4 in the red root epidermis is higher than the expression in white root epidermis,The expression of BrANNEXIN1,2,3 in the red root epidermis is lower than the expression in white root epidermis.(2)BrANNEXINI,2,3,4 expression characteristics under treated by different lightsThe white root epidermis of Brassica rapa 'Tsuda' Turnip was treated by different lengths time UV-A.The expression levels of BrANNEXIN1,3dropped substantially,and the expression slight recovery in 12-24 h.The expression of BrANNEXIN2 was decreased in a short amount.then returned to normal levels.The expression of BrANNEXIN4 was significantly up-regulated under the UV-A treatment.In the Brassica rapa 'Tsuda' Turnip seedlings.UV-A can induce BrANNEXIN1,2,3,4 expression substantial increased in cotyledons and HI.The expression of BrANNEXIN1,2,3,4 increase rate decreased in H2.Blu-ray induced the expression levels of BrANNEXIN1,2,4 greatly increased in cotyledons and H1 but the expression of BrANNEXIN3 decreased.The expression levelsof BrANNEXIN1,2,3 increased,while BrANNEXIN4 essentially unchanged in H2.The expression levels of BrANNEXIN1,2,3,4 were increased by the red/far-red light treated in cotyledons and HI.The expression levels of BrANNEXIN1,2,4 were also increased in H2,but the expression levels of BrANNEXIN3was decreased in H2.(3)Different stresses induce the BrANNEXIN family genesCold stress(4?)can increase the expression of BrANNEXIN1,2,3,4 and the reduce the expression.Hot stress(37?)can induce the expression of BrANNEXIN3 a significant reduction,but the expression of BrANNEXINl,2,4 had no significant effect.The expression of BrANNEXIN1 was reduced by dried stress(25?),but the expression level of BrANNEXIN2,3,4 had no significant effect.Osmotic stress(PEG mannitol,lithium ions,sodium ions,cesium ions)have some effects on the expression of BrANNEXIN family genes.The expression levels of BrANNEKIN1,4 were increased by ABA treated,but no significant effect in expression levels of BrANNEXIN2,3.3.BrANNEXIN1,2,3,4 Arabidopsis transgene studiesBrANNEXIN1,2,3,4 genes were constructed to excessive expression vector pH7WG2D by Gateway technology.By Agrobacterium-mediated tidbits dip method,overexpress BrANNEXIN4 genetic transformation.We got plenty of transgenic candidates by hygomycin selection system.