The Research On Eucalyptus Camaldulensis Gene Familys In Monolignol Biosynthesis

The plantation-grown Eucalyptus is the largest species in the world. In the industrial production process of pulp and biomass fuels, the rich amount of lignin in plants reduces the extraction efficiency of cellulose, and increasing the cost of production. Furthermore, we know little about the gene familys involve in monolignol biosynthesis pathway, especially those Non-Model Plants and the plantation-grown Eucalyptus. Therefore, in this dissertation, we established a Eucalyptus spp. EST assembly database, and used the tblastn program and the sequence of the genes involve in Arabidopsis thaliana’s monolignol synthesis pathway, to search the homologous sequences in Eucalyptus. By the results of tblastn program, we used the RACE clone technology to obtain the full-length cDNA fragments of those A. thaliana’s genes, and also cloned their full-length genomic fragments in the cold-tolerance Eucalyptus camaldulensis which usually planting in Hunan, Jiangxi and Sichuan provinces. And then, by using the semi-quantitative RT-PCR technology, we detected and analyzed the spatio-temporal expression profiles of the key genes in regulations of the lignin S/G ratio. The following is the achievements in this study:(1) In Arabidopsis monolignol biosynthesis pathway, there’re11kinds of synthetic enzymes coded by a number of genefamily members and including a total of35genes. In this dissertation, by the homologous clone technology and RACE cloning technology, a total of22full-length cDNAs and their full-length genomic fragments, encoding the11kinds of monolignol catalytic enzymes in E. camaldulensis, has been obtained. They’re three genes of the PAL gene family, two genes of the4CL gene family, one C4H gene, two genes of the C3H gene family, one HCT gene, one CSE gene, four genes of the CCoAOMT gene family, one COMT gene, two genes of the F5H gene family, two genes of the C3H gene family and three genes of the CAD gene family. The bioinformatic analysis show that, the21genes we cloned above are highly homologous genes to the Arabidopsis, and can be translated into a protein having the correct catalytic domain, except for the E. camaldulensis CCoAOMT2genes, which encoding a CCoAOMT-like enzyme.(2) By semi-quantitative RT-PCR, we detected and analyzed the spatio-temporal expression profiles of the key genes in regulations of the lignin S/G ratio. The results showed that the E. camaldulensis4CL1gene gradually increased the expression level from the beginning of April, and at last reach the highest expression level in Augest. While the4CL2gene maximum expression levels is in June. Combined the results of bioinformatics gene function analysis, suggesting the4CL2gene is associated with the G lignin synthesis in vivo. In the E. camaldulensis xylem, the expression level of COMT1gene start expression in April and has an upward trend in August, and the expression of from September to October are reamin a high abundance of expression. And in this period, it was the developing stage of E. camaldulensis to form the summerwood and a large of S lignin was accumulated, which proof a evidence that the COMT1gene is necessary for S lignin biosynthesis. The CCoAOMTl gene and CCoAOMT6gene express through all of years, and main primarily responsible for connecting the phenylalanine biosynthesis pathway to lignin biosynthesis pathway. The CCoAOMT2gene and CCoAOMT5gene appear expression only from May to September, antd it suggest the two CCoAOMT genes specifically involved in the formation of S lignin monomers. The gene expression profiles of E. camaldulensis F5H1and F5H2gene in vivo, is basically similar at whole year. And the F5H gene’s expression ratio in the stage of summerwood forming is high than the stage of springwood forming, suggest those two F5H genes mainly involved in the synthesis of S lignin monomers in vivo. The CAD1and CAD5genes expression patterns is similarly, has express all of year. The CAD2gene expression level in a year has no obvious genetic variation.(3) There are a alternative splicing occurrence in E. camaldulensis CCoAOMTl gene, which resulting in forming of two mRNA in different length. The normal spliceosome contains738bp ORF (CCoAOMT1-L, HM106291), coding269amino acid. The alternative spliceosome contains696bp ORF (CCoAOMT1-A42, GQ889423), coding232amino acid. The alternative splicing occurrence in the alternative5’splice site of the first intron, while this splicing doesn’t change the reading frame sequence of posterior amino acids. The alternative splicing of CCoAOMTl gene express from May to September, the most vigorous growing stages of Eucalyptus stem, suggesting that this alternative splicing may involve in the development or growth of tissues and organs.