Clonging And Characterization Of BnXTH1 And BnEXPA4 Genes In Brasscia Napus

The xylophones endotransglucosylase/hydrolase(XTH)and expansin(EXP)are the plant cell wall loosening factors.They are known to have plant cell-wall loosening activity and to be involved in cell expansion and other developmental envents during which cell-wall modification occurs.To date,there was no research report on XTH or EXP genes in Brassica napus.In order to investigate the functions of XTH and EXP in B.napus,we cloned BnXTH1 and BnEXPA4,analyzed the sequences and expression patterns of these two genes.We constructed RNAi and overexpression vector of BnXTH1 and BnEXPA4,respectively.The recombinant overexpression vectors were transferred into Arabid.opsis thaliana.The main results are as follows:(1)Xylophones endotransglucosylase/hydrolase(BnXTH1):XTHs have the ability to change cell wall structures,thus control the growth of plant cells,also they also involve in processes such as seed germination,root development,and stress resistence.① We cloned BnXTHl with a full length of 1173 bp containing 3 exons and 2 introns.The length of its ORF is 855 bp,encoding a protein with 284 amino acids,which consists of an N terminal signal peptide and two domains belonging to XTH class II.② BnXTH1 is expressed constitutivly in all tissues of B.napus.③ Expression of BnXTHl mRNA levels in hypocotyls are upregulated when treated with GA3,BR,IAA,ABA,ACC and pH 4.5 but downregulated when treated with 15%PEG6000,CaC12and pH 7.2.The expression levels of BnXTH1 mRNA are decreased both in stems and roots under the treatment with CuSO4,decreased in hypocotyls but increased in roots under the treatment with CdCl2.Thus,These results indicated that the BnXTHl is regulated by regulators and abiotic stresses.④ The RNA interference vector and over-expression vector of BnXTHl were constructed.The BnXTH1 over-expression vector was transferred into A.thaliana mediated by the Agrobacterium tumefaciens,and 4 T1 plants were obtained.(2)Expansin(BnEXPA4):EXPs can induce the heat-inactived cell wall extension,but do not affect the plasticity of elasticity of cell wall.EXPs are involved in plant processes such as seed germination,stem elongation,fruit ripening,morphogenesis and stree response.① We cloned BnEXPA4 with 1238 bp in length containing 3 exons and 2 introns.The length of ORF was 777 bp,which encoded a protein composed of 258 amino acids.The protein named as BnEXPA4 contains a signal peptide and two domainsbelonging to a-expansin.② BnEXPA4 is expressed in all tissues of B.napus.③ Expression of BnEXPA4 mRNA levels in hypocotyls are upregulated when treated with ACC and NaCl but downregulated by GA3、BR、IAA、pH 7.2 and 15%PEG6000.The expression levels of BnEXPA4 mRNA are decreased both in stems and roots under the treatment with CuSO4,did not change in hypocotyls but decreased in roots under the treatment with CdCl2.The expression of BnEXPA4 is regulated by phytohormones and abiotic stress,suggesting that it may be involved in the hormone signaling and stress resistance.④ The RNA interference vector and over-expression vector of BnEXPA4 were constructd.The BnEXPA4 over-expression vector was transferred into A.thaliana mediated by the Agrobacterium tumefaciens,and 6 TO plants were obtained.