Cloning And Functional Analysis Of BnDREPP Gene In Brassica Napus

DREPP(Developmentally regulated plasma membrane polypeptides)protein is widely present in plants.Previous studies showed that plant DREPP family members are involved in regulating plant responses to biotic and abiotic stresses.In this study,the full length CDS sequences of two BnDREPP protein genes were cloned from B.napus "Zhongshuang 11".We constructed overexpression vector and transformed into A.thaliana.We studied the tissue expression patterns of BnDREPPs and the expression patterns of BnDREPPs under different stress treatments.The main work is summarized as followings:1.Bioinformatics analysis of B.napus BnDREPPs genesIn this study,the DREPP family member of A.thaliana MDP25 protein sequence was firstly compared with the genome database of B.napus by using BLAST method.We obtained four candidate genes homologous to MDP25,named BnDREPP1,BnDREPP2,BnDREPP3 and BnDREPP4,respectively.BnDREPP1 is identical to BnaA01g35060D of the genome database of B.napus,BnDREPP2 is identical to BnaC07g36310D,BnDREPP 3 is identical to BnaA03g44480D,and BnDREPP4 is located in an unknown chromosomal region of the B.napus.The genetic structure of BnDREPPs consists of four exons and three introns.Bioinformatics analysis of BnDREPPs genes revealed that BnDREPP 1 ORF is 729 bp,encoding 243 amino acids;BnDREPP2 ORF is 678 bp,encoding 226 amino acids;BnDREPP3 ORF is 711 bp long and encoded 237 amino acids;BnDREPP4 ORF is 711 bp and encoded 237 amino acids.The amino acid sequences deduced from the four genens contain each the DREPP domain and the characteristic features VEExK motif of MDP25.We speculated that the BnDREPP proteins in B.napus should also function as the microtubule-binding proteins.However,the three-dimensional structures of BnDREPP protein family members in B.napus were found to be all different between them,as well as from this of the MDP25 in A.thaliana,suggesting that their function in cells may be different.2.Construction of overexpression vector of BnDREPPs genes in B.napus and transformation of A.thalianaWe successfully cloned BnDREPP2 and BnDREPP4 from"Zhongshuang 11".The overexpression vector pCXSN::BnDREPP2 was constructed and transformed into A.thaliana by floral dip method via Agrobacterium-mediated transformation procedure.Transgenic T0 and T1 seeds were obtained,the T0 and T1 plants did not show obvious phenotypic changes under normal growth conditions compared to the wild controls.The further work is carrying on and the salt and drought tolerance tests will be carried out on the T2 plants.3.Expression pattern analysis of B.napus BnDREPPs genesAnalysis of the tissue expression profiles of BnDREPPs genes showed that,these four genes constitutively expressed in all the examined tissues,their transcription levels are highest in roots,followed by flowers,and lowest in siliques.Expression profile analysis of BnDREPPs genes under different stress treatments showed that the four BnDREPPs genes of B.napus have different responses to these stresses.Under the treatment of 200 mM mannitol,the expression of BnDREPP1 was inhibited,BnDREPP2 was induced,while BnDREPP3 and BnDREPP4 were inhibited at first and then induced.Under the treatment of 200 mM NaCl,the expression of BnDREPP1,BnDREPP2 and BnDREPP3 were inhibited at first and then induced,while BnDREPP4 showed an expression pattern of"inhibited-induced-inhibited".These results suggest that they played a more or less significant role in the salt and drought tolerance of B.napus.Under the treatment of 100 ?M ABA,BnDREPPl,BnDREPP3 and BnDREPP4 showed an expression pattern of"inhibited-induced-inhibited",while the expression of BnDREPP2 was induced at first and then inhibited.Under the treatment of 200 ?M GA,BnDREPPl,BnDREPP2 and BnDREPP3 showed an expression pattern of "inhibited-induced-inhibited",while the expression of BnDREPP4 was induced at first and then inhibited.Under the treatment of 200 ?M SA,the four BnDREPPs showed different expression patterns,the expression of BnDREPP1 reached the highest level at 6h,and then down-regulated.The expression of BnDREPP2 and BnDREPP4 reached the highest level at 12 h,and decreased at 24 h,the expression of BnDREPP3 was inhibited and decreased to a level of 0.12 times of the control at 3 h,but was upregulated after 3 h.4.Construction of BnDREPP2 promoter analysis vector for B.napusWe have successfully isolated the promoter region of BnDREPP2 and cloned it into the pCXGUS-P vector,which will be useful for further study of the expression patterns of BnDREPP2 in A.thaliana and Brassica crops.