Cloning And Analyzing Of CAD And CCoAOMT Genes From Pinus Massoniana

CAD and CCoAOMT are two enzymes in the lignin synthesization pathway. They also playan important role in lignin synthesis. Pinus massoniana is an important timber tree species insouthern China, but there is no report about CAD and CCoAOMT gene from Pinus massoniana.In this paper, These two genes from P. massoniana were cloned and analyzed. The results wereas follows:1.We used CODEHOP to design the degenerate primers online, the CAD and CCoAOMTgenes of P. massoniana(GenBank accession number:KF419291and KF419292) was clonedthrough RT-PCR and SMART RACE RT-PCR. The CAD of P. massoniana, whose length is1450bp, contained an open reading frame of1074bp which encoded a polypeptide of357aminoacids with predicted molecular mass of38945.1u and PI of5.8. The CCoAOMT of P.massoniana, whose length is1126bp, contained an open reading frame of780bp which encodeda polypeptide of259amino acids with predicted molecular mass of29068.3u and PI of5.12.Their nucleotide sequences and the encoded amino acid sequences were analyzed with thesoftware of ClustalX, MEGA5.0, DNAMAN and ANTHEPROT and so on. Tissue specificexpression showed that CAD and CCoAOMT genes expressed in stem were much higher than inleaf, root and shoot from P. massoniana.2. The pBI121-antiCAD and pBI121-antiCCoAOMT plant expression vectors wereconstructed. The expression vectors were then introduced into Agrobacterium tumefaciens strainEHA105by freeze-thaw method and proved by PCR detection. The antisense genes weretransferred to the tobacco using leaf-disc cultivated method and proved by PCR detection. Thelignin content was measured using the method of Klason. The average lignin content oftransgenic plants of pBI121-antiCAD was found to be less reduced and transgenic plants ofpBI121-antiCCoAOMT reduced6.6%as compared to the control plants after1months oftransfer in the greenhouse conditions.3. The pET-28a-CAD prokaryotic expression vector was constructed for PmCAD gene. Theinduction condition of expression vector in BL21(DE3) was optimized.The optimization result ofPmCAD induced expression showed that pH7,37℃and IPTG6.0to induct3hours was thesuitable. And the molecular mass of the protein coded by PmCAD gene is about39kDa.