Monolignol Biosynthesis Gene COMT And F5H Cloning And Expression Regulation In Brassica Napus L.

The fungal pathogen Sclerotinia sclerotiorum (Lib) de Bary generally causes rapeseed yield losses 10%-30%, that can be up to 80% when being serious. Because S.scleroliorum belongs to a non-specific pathogen, no germplasm resources are found to be effective to it till now. Lodging causes rapeseed yield losses 10%-30% and oil content decreases 10%-30%. Furthermore, the gloomy environment after lodging is in favor of germ infecting and expanding.Lignin is crucial for structural integrity of the cell wall and stiffness and strength of the stem. In addition, lignin waterproofs the cell wall, enabling transport of water and solutes through the vascular system, and plays a role in protecting plants against pathogens.Although exceptions exist, dicotyledonous angiosperm (hardwood) lignins consist principally of G and S units and traces of H units, whereas gymnosperm (softwood) lignins are composed mostly of G units with low levels of H units. Lignins from grasses (monocots) incorporate G and S units at comparable levels, and more H units than dicots. G unit is composed of a greater proportion of carbon-carbon linkages than S unit. Carbon-carbon linkages are more steady and lead to more highly condensed lignin. COMT and F5H are two rate-limiting enzymes catalyzing syringyl-substitued lignin precursors, they are crucial to the final lignin polymer composition and the extent of cross-linking.In the study, COMT and F5H cDNAs were cloned by RT-PCR from Brassica napus cv. Zhongshuang 9, which is highly resistant to Sclerotinia sclerotionim and lodging. Analyse the expression regulation in plant using antisense transgenes of COMT and F5H. We expect strengthen resistance to lodging and pathogen by increasing the extent of G unit in Brassica napus. The main study results are as follows:1. There were not usable COMT gene information in Brassica napus. The COMT mRNA sequences of many species were compared and analyzed, primarily according to the sequence of Arabidopsis thaliana. Gene specific and degenerate primers were designed in the sides of the coding sequence and the conservative region. Some partial COMT cDNAs were cloned by RT-PCR from Brassica napus cv. Zhongshuang 9, the whole coding sequences were obtained by SOE-PCR and antisense plant expression binary vector pC4HBnC1 was constructed by inserting one of them. All the sequences had the conserved functional domains of COMT, showed over 96% identity each other (Nuances of amino acids were some similar amino acids replacements) and 92% identity with that of Arabidopsis thaliana. It was suggested there were some groups of COMT genes in Brassica napus.2. Primarily according to the COMT gene of Brassica napus cv. Westar. Gene specific and degenerate primers were designed in the sides of the coding region. The whole coding sequences were obtained by RT-PCR from Brassica napus cv. Zhongshuang 9 and antisense plant expression binary vector pC4HBnF5 was constructed by inserting one of them. All the sequences had the conserved functional domains of F5H and showed high identity with those of Brassica napus cv. Westar . Sequenced some clones, a new member of F5H genes obviously distinguished with the published members was found, it was suggested there were at least 3 members of F5H genes in Brassica napus.