Cloning Of Two Key Genes Of Lignin Biosynthesis From Eucalyptus Spp And Functional Study

Eucalyptus is the world's second largest plantation species, Eucalyptus wood is also an important paper-making materials in South China. Lignin is the secondary metabolites that has important biological function in the plant, also is the world's second rich in organic matter, but in chemical process of paper pulping, lignin should be extracted from cellulose fraction that polluting and cost-increasing.Many countries attach importance to cultivate the new low-lignin plants and generalize, and through RNAi transgenic technology is the fastest and the most effective way to cultivate low-lignin plants and reduce lignin content of plants fundamentally. As a result, cloning key genes of Eucalyptus lignin synthesis is a foundation for eucalyptus lignin genetic engineering, it is of great significance. Following is major results of this paper:1. Full-length clone of CAD and 4CL genes from Eucalyptus.Making Eucalyptus camaldulensis leaves and stems as materials, extracted DNA and RNA respectively, and making DNA and the reverse transcription cDNA looks on RNA as template. DNA fragments and cDNA fragments were amplified by PCR and RT-PCR using the primer designed from conservative regions of existing homologous gene sequences.The fragments from these bands were ligated to pMD18-T vector. The recombinant plasmids were transformed into E.coli. DH5a. The separate clones transformed from each band were chosen to be sequenced.2. Bioinformatic analysis of CAD and 4CL genes from Eucalyptus camaldulensis. Full-length of genome from CAD is 2304 bp, the cDNA sequence is 1102bp in length, and has a 1068bp ORF coding 356 amino acids; Full-length of genome from 4CL is 5481 bp, the cDNA sequence is 1670bp in length, and has a 1632bp ORF coding 544 amino acids by using Vector NTI 9.0. The homology percentage of the sequences from Eucalyptus with other species in Genbank is over 97%, so we can conclude the sequence is full-length of CAD and 4CL gene. We find that the two genes have 5 exon and 4 intron through alignment Geneome and cDNA sequence. The Physics and chemistry character,second structure, Three-level structure and so on of the deduced amimo acid sequence was predicted and analyzed by Protein Analysis Software, and the results are:CAD protein pI value is 5.705, molecular weight is 38.775KDa, three transmembrane domain, one signal peptide, and the CAD protein is hydrophilic, be rich in alpha helix and beta enfoldment.4CL protein pI value is 5.095,molecular weight is 59.332 KDa, four transmembrane domain, one signal peptide, the protein is rich in alpha helix, its amimo acid sequence exist two conservative area, namely SSGTTGLPKGV and GEICIRG, and SSGTTGLPKGV was deemed to conservative AMP Binding Site in catalyse reaction.3. Prokaryotic expression of CAD and 4CL fusion Protein Gene from Eucalyptus camaldulensis. We design two couples Expression primer, and choose restriction endonuclease BamH I and Sal I, EcoRâ… and Hindâ…¢respectively. Expression Vector of pET-32a-CAD and pET-32a-4CL were constructed and successfully transformed into E.coli. The result is:when 3h, the CAD fused proteins reach the maximum; when 2h, the 4CL fused proteins express more, but when 3h and 4h, the expression reach placidity and keep stable.