Cloning And Sequence Analysis Of CDNA Encoding Xyloglucan Endotransglycosylase From Anthocephalus Chinensis

Xyloglucan endotransglycosylase (XET) is a protein responsible for the extension of cell walls, which leading to the enlargement of the plant cells. To further understand the function, expression and mechanism of XET during wood formation and to provide the basis for improving wood quality by genetic engineering, We cloned the XET gene from A. chinensis by the methods of RACE and chromosome walking, and analyzed it from several aspects of Bioinformatics. Results of this study are summarized as the followings:1. The RNAs of A. chinensis extracted by the methods of poplar extraction and RNAplant Reagent were rarely degenerated and showed clear bands of 28S rRNA and 18S rRNA, the value of OD260/OD280 reached 1.8~2.0 and the yield was 39.40μg/100 mg after purification. The RNAs may meet the requirements of RT-PCR reaction and RACE experiment, laying a basis for the successful cloning of A. chinensis genes.2. Genomic DNAs were extracted successfully from A. chinensis by the methods of Reagent kit and CTAB. The software of Gene Tools estimates that the yield of DNA are 29~117 ng/μL and 183~243 ng/μL respectively. The DNA quality extracted by CTAB method is better.3. Conserved sequence of XET gene in A. chinensis was cloned by designing degenerate primers, including 307 nucleic acids. 3'-end sequence and 5'-end sequence of XET gene were obtained by the methods of RACE and chromosome walking, including 949 and 603 nucleic acids respectively. We got full-length XET gene of 1396 bp from A. chinensis, which including a 882bp complete ORF and encoding a protein sequence of 294 amino acids. The amino acids were highly homologous to amino acids encoded by XET gene of other species in GenBank, and the homology to Populus tremula×Populus tremuloides, Arabidopsis thaliana, Asparagus officinalis, Litchi chinensis and Cucumis sativus were higher than 60%. Further more, its protein sequence contained two conserved domains related to XET gene, which show that the gene sequences we cloned is XET gene of A. chinensis.4. The protein sequence we got was analyzed from several aspects of Bioinformatics, such as amino acid composition, hydrophobicity profile, trans-membrane domain, signal peptide, secondary structure and tertiary structure.