Cloning And Transformation Of The CCoAOMT Gene, A Key Enzyme Gene In Boehmeria Nivea Lignin Biosynthesis

Ramie [Boehmeria nivea (Linn.) Gaud.], a cross-pollinated cultivated plant withcomplex genetic background, being selected by nature and improved by breeder, is a realworth fibre crop of China, plays an imptortant role in Chinese economy and people's life.The attribute of fibre has been improved through conventional breeding methods. However,basic study of its genetic and molecular biology is quite insufficient. There still remainsome difficult problems, for example, how to decrease the content of lignin as low aspossible. In practice, control the content of lignin is not maneuverability by culturemeasure and conventional breeding. Improving attribute of Ramie fibre via geneengineering, carrying out low lignin breeding, decreasing the content of lignin and alteringits components seem to be good ways to solve the problems.In this paper, the Ramie CCoAOMT gene was cloned by RT-PCR firstly. Thesequence of CCoAOMT gene was analyzed, antisense CCoAOMT gene expression vectorof Ramie and tobacco was constructed and transformed into Boehmeria nivea byAgrobacterium tumefaciens. The main results of this study are as follows,(1) The method of extraction of high quality total RNA was meliorated. Proceeding ofRT-PCR and RACE have been optimized. It provides a convenient and applied method ofcloning target gene from plant which contains rich multifarious secondary metabolite suchas polyphenol and polysaccharide.(2) The full length of Ramie CCoAOMT cDNA was cloned for the first time, submitted to GenBank and the GenBank accession number is AY651026. The result ofdomain analysis showed that it is a member of O-methyltransferases family. Phylogenetictree of CCoAOMT generated by multiple alignment between Boehmeria nivea and otherhigher plants at three levels, mRNA sequence(including 3'UTR, 5'UTR and CDS), mRNACDS sequence and amino acid sequence. The result at mRNA level showed that Ramiecann't be clustered into any other plant, but the result on mRNA CDS showed Ramie andZea mays (ZMA242980,ZMA242981) have higher homology than with any other highplant, while the result at amino acid level revealed that Ramie and Populus (AJ224894,AJ224896, PTU27116) have higher homology.(3) High-level expression vector pQE-30-BNCCOAOMT was constructed and ramieCCoAOMT was expressed in E.coli. whose MW is 29.4 kD.(4) The new genome sequences of CCoAOMT gene of four Ramie cultivars havebeen cloned for the first time, submitted to GenBank and the GenBank accession number isAY818191 (Xiangzhu No.3), AY822619 (Yuanma), AY822620 (Luzhuqing) andAY822622 (Qingma). The four sequences are different in length and there are three intronsin each sequence. The sequences of introns are different. In the coding sequence there areonly five polymorphic bases. The homology of these four cultivars are more than 96ï¼….(5) The full length of tobacco CCoAOMT cDNA has been cloned which including thewhole coding sequences of 862 bp. This sequnce is 98ï¼…homogentic with tobaccoCCoAOMT-2 (the GenBank accession number is U62734).(6) Antisense CCoAOMT gene expression vector of Ramie and tobacco have beenconstructed and transformed into Agrobacterium tumefaciens. Engineering bacteriumLBA4404 (pBI121-anti NTCCoAOMT) and LBA4404 (pBI121-antiBNCCoAOMT), areusable in plant genetic transformation.(7) An efficient regeneration system has been set up. The concentration of Kan hasbeen ensured as 25 mg/L, to screen the transformed and non-transformed Ramie plant.Transformation system has been set up.(8) The antisense expression gene of tobacco CCoAOMT was constructed and transformed into Boehmeria nivea via Agrobacterium tumefaciens (LBA4404).Transgenetic plants were identified with PCR. The antisense expression gene of RamieCCoAOMT was constructed and transformed into Boehmeria nivea via Agrobacteriumtumefaciens (LBA4404). Transgenetic calli have antibiotic resistance. The antisenseexpression gene of Ramie CCoAOMT was constructed and transformed into tobacco W38via Agrobacterium tumefaciens (LBA4404). Transgenetic plants have antibiotic resistance.The results provided a exprerimental and theoretical basis for further research on theRamie caffeoyl-CoA 3-O-methyltransferase biology that may involve the gene encodingproteins and their functions in lignin metabolism. Transgenetic Ramie with decreasedlignin-content or altered components shall be obtained, lower lignin transgenetic Ramiegreen variety will be created which adapt to the situations in China. It probes a way tobreeding Ramie with low lignin content to facilitate Ramie textile industry.