Clone Of Cinnamyl Alcohol Dehydrogenase Gene In Flax

Flax is a very important source of natural fibres in the textile industry, plays an important role in Chinese economy and people's life. Lignin is difficult to be removed during retting process. Lignin has a greater impact on the fiber quality and performance of the textiles. Improving attribute of flax fibre via gene engineering, carrying out low lignin breeding, decreasing the content of lignin and altering its components seem to be good ways to solver the problems. In this paper, the flax CAD gene was cloned by RT-PCR using a pair of primers were designed from the sequences of existing CAD genes. Antisense CAD gene expression vector of flax has been constructed. The results are as follows:⑴High quality flax RNA was extracted by Plant RNA Isolation Regent and reverse transcribed into cDNA using Superscript RT Kit. This is an efficient method for cloning target genes from plant which contains rich multifarious secondary metabolite such as pectin and polysaccharide.⑵A flax cDNA fragment was amplified by Reverse Transcription Polymers Chain Reaction (RT-PCR) using a pair of primers were designed from the sequences of existing CAD genes. Sequencing analysis showed that the PCR products were 477 bp with 95% identity to the CAD gene registered in the GenBank. The result proved that the cloned gene fragment should be CAD gene fragment of the flax.⑶The full length sequence of falx CAD gene cDNA was cloned by RACE, Sequencing analysis showed that the PCR products were 896 bp with 92% identity to the CAD gene sequence registered in the GenBank.⑷To introduce proper restriction sites for subsequent cloning into the plant expression vector, both the pGEM-7Zf (-) vector and the pGEM-T-CAD were cut by EcoR I to release the CAD gene fragment and the pGEM-7Zf (-) fragment, The two fragment were joined together to produce a new plasmid pGEM-7Zf (-)-CAD.⑸The pGEM-7Zf (-)-CAD plasmid and the binary vector pBI121 were cut by Xba I and BamH I to release the CAD gene fragment and the large fragments of pBI121. After ligation of the CAD gene fragment with pBI121, we obtained an expressional vector, named pBI121-antiNTCAD.