Cloning Of Brxet Gene In Chinese Cabbage And Genetic Transformation Into Arabidopsis Thaliana

The temperature sensitive characteristic of thermo-sensitive male sterile line of Chinese cabbage(Brassica campestris L.ssp.Pekinensis)plays an important role in the Chinese cabbage breeding practice.But how is the fertility affected by temperature and which genes involve in the regulation of the process is still not clear.We get the xyloglucan endotransglucosylase(XET)gene BrXET from the thermo-sensitive male sterile line 94C9 of Chinese cabbage,which has obvious expression difference before and after the fertility transformation.We also constructed the expression vector of the BrXET gene and transformed it into Arabidopsis.thaliana by agrobacterium-mediated transformation.Preliminary observation were taken on the transgenic Arabidopsis thaliana.The results are as followings.1.The DNA sequences of BrXET gene in the thermo-sensitive male sterile line 94C9 and its maintainer line 94B7 were identical to each other,1610 bp in length.The complete BrXET gene cloned from 94C9 is composed of 879 nucleotides from codon ATG to TAG and encoding 292 amino acids.2.The genomic structure analysis indicated that the full-length gDNA of the BrXET gene is comprised of 4 exons and 3 introns.The protein sequence deduced from the cDNA sequence not only has the specific conserved region of the XTH gene family but also contains potential signal peptide sequence and potential transmembrane.3.With total DNA of 94C9,the promoter of BrXET gene was cloned by PCR.The results of blast and bioinformatics analysis showed that the cloned sequence has characters of promoter.Except for the basic elements such as TATA-box and CAAT-box,there also include some light responsive element?defense and stress responsive element?hormone response element and element invoves in the plant morphogenesis.4.The expression vector p2301-BrXET was constructed successfully and transfered to wild type Arabidopsis thaliana by using the method of agrobacterium-mediated transformation.Eleven T2 generation transgenic lines were obtained by antibiotic resistance screening,PCR confirmation and GUS histochemical staining.Lastly five homozygous transgenic lines were got from the twenty T3 generations.5.From the phonetype observation of the T2 generation transgenic Arabidopsis thaliana,it was speculate that the over expression of BrXET gene may cause the increase in the number of the rosette leaves and the fold of the leaves on flower branch,and also inhibit the development of main flower branch and influence the development of lateral branches.