Cloning And Expression Of The Genes Involved In The Lignin Synthesization Of Phyllostachys Edulis

China is one of the most abundant bamboo resource countries in the world. Because of its fast-growing and high cellulose content and its utility in afforestation and paper pulping, Phyllostachys edulis is the most important and widely used bamboo species in China. But the application of P. edulis was restricted by its biological characteristics and the traditional breeding methods. The development of high-speed bamboo process was restricted by the inherent shortcomings of P. edulis and the application in paper pulping industry development was also restricted by the shortages of fiber. So, the most important is how to breed new varieties to meet the growing multi-purpose industrialization of P. edulis at present. Therefore, it is of great significance to use molecular biological technologies to improve lignin content of P. edulis.PAL is one of the key enzymes in the early stages of the phenylpropanoid pathway and has significant effects on the lignin content of plant. CCoAOMT and CAD are another two enzymes in the lignin synthesization pathway. Both of them also play an important role on the regulation of lignin content in plant. In this study, PAL, CCoAOMT, CAD genes in P. edulis were cloned and the expressions were also studied. The results were summarized as follows:(1) The DNA and RNA were isolated respectively from P. edulis leaves by modified CTAB and Trizol methods. The result showed that the quality was good enough for further research.(2) A full length of 2 736 bp DNA fragment encoded PAL of P. edulis was obtained by genome walking, which contained an intron and a forecasted promoter sequences. The PAL fragment encoded 712 amino acids with a molecular weight of 77KD. The deduced amino acid sequence shared 95% homology with the PAL of Bambusa oldhamii and 94% with the PAL of Oryza sativa.(3) A full length of PAL cDNA named PAL1 was obtained by RT-PCR and RACE. The size of the cDNA fragment was 2 678 bp, which included a 2 142 bp open reading frame. It shared 90% homology with PAL of Oryza sativa and Bambusa oldhami. Tissue specific expression test revealed that there were significant differences in roots, stems, leaves and seedlings.The expression of PAL in root was the highest.(4) A 787 bp DNA fragment of CCoAOMT and a 1019 bp DNA fragment of CAD were cloned from the genomic DNA of P. edulis which encoded 197 amino acids and 282 amino acids respectively. The CCoAOMT and CAD DNA fragment in P. edulis shared 90% homology with that of Oryza sativa and Phleum pratens. The deduced amino acids sequence of CCoAOMT and CAD in P. edulis shared 85% homology with that of Oryza sativa and Phleum pratens. GenBank accession numbers were EF549579 and EF549577 respectively.(5) The PAL1 gene was cloned into a prokaryotic expression vector pET-16b and then it was used to transform E.coli. BL21. The Km, the optimum temperature and the optimum pH of purified recombinant protein was (0.421±0.023)×10-3 mol/L, 50℃and 8.8 respectively.(6) The pBI121-PAL1,pBI121-anti PAL1,pBI121-anti CCoAOMT and pBI121-anti CAD plant expression vectors were constructed. The vectors were introduced into tabacoo plant by agrobacterium-mediated transformation. PCR analyses confirmed the integration of the target gene into the genome of the transformed tobacco plant.