Cloning Of Two CDNA Encoding CCoAOMT And 4CL And Their Transformation In Amorpha Fruitcosa

Lignin is a second abundant macromolecular secondary metabolite in plants which ranks only next to cellulose. It is a complex aromatic polymer of vascular plants with important biological functions, such as providing mechanical strength to the stem, improving the ability of cell transport and protecting cellulose fibres from chemical and biological degradation. However, its presence in plants has negative effects on the industrial and agricultural applications. For example, lignin is the main cause of papermaking pollution during wood pulping, and it also degrades the forage digestibility and nutrition absorption of livestocks. According to the long growth period, the traditional crop improvement is not suitable for plants like trees whose resources still come from natural vegetation. With the development of modern genetic engineering, human modification of the trees becomes easier. Using modern biotechnology to regulate lignin biosynthesis can not only elucidate the proper molecular mechanism of lignin and other phenylpropionic acid derivatives, but also has significant meanings in the production of papermaking materials and forage germplasm resources. In some cases, like tobacco and aspen, modified lignin plants with low cost and low pollution beneficial for pulping has been obtained successfully. Thus, the potential value of using genetic engineering methods to regulate lignin biosynthesis has been demonstrated. There are many enzymes involved in lignin biosynthesis including the key enzyme caffeoyl CoA O-methyltransferase CCoAOMT. Antisense inhibition of this gene has been done in transgenic tobacco and aspen, and S/G (syringyl/guaiacyl) ratio was increased accompanied with less condensed and more extractable lignin. 4-coumarate: CoA Ligase (4CL) is another key enzyme in lignin biosynthesis. Different 4CL isoenzymes participate in the synthesis of flavonoid, phytoalexin and many more phenylpropanoid secondary metabolites. The lignin content in 4CL expression inhibited tobacco and aspen decreased, and cellulose in aspen increased. Till now none of abnormal growth in transgenic plants has been found. The transgenic effects are all suitable for paper pulping. Thus, inhibition of CCoAOMT and 4CL's expression could effectively improve trees'efficiency for papermaking.In this paper, we mainly discussed the papermaking material tree Amorpha frucicosa L, and started research as follows:1. The cDNA encoding caffeoyl CoA O-methyltransferase (CCoAOMT), one of the key enzymes in lignin biosynthesis, was cloned by degenerate oligo-nucleotide primed-polymerase chain reaction (PCR) and rapid-amplification of cDNA ends (RACE) PCR from Amorpha fruitcosa, bypassing the complicated lab-work of constructing and screening cDNA library. Designed expressed primers, inserted the cloned CCoAOMT gene into plant binary expression vector pBI121 in reverse direction, and constructed the antisense expression vector AP-CCoAOMT.2. Designed expressed primers according to the published cDNA encoding 4CL sequence on Genebank, inserted the cloned gene into plant binary expression vector pBI121 in reverse direction, and constructed the antisense expression vector AP-4CL.3. Construction of the binary expression vector AP-CC.The antisense expression vector AP-4CL was digested with the restriction enzymes EcoR I and Hindâ…¢, and fragments with 4CL gene were purified via agarose-gel electrophoresis. These fragments were inserted into the expression vector pBluescriptâ…¡(+) which had been cleaved with the same enzymes and purified to produce the new plasmid, intermediate vector pBS-4CL. The vectors pBS-4CL and AP-CCoAOMT were digested with PstI and Sse8387I respectively. (The two restriction enzymes are a pair of isocaudamer, having different recognition sequences but producing compatible cohesive ends.) Thus, the two fragments were ligated with T4 DNA Ligase, and the binary expression vector AP-CC was constructed.4. The antisense expression vector AP-CCoAOMT,AP-4CL and the binary expression vector AP-CC were transferred into the by Freeze-Thaw method. The result was confirmed by PCR.5. CCoAOMT and 4CL were mediated into the Amorpha fruitcosa genome in the reverse direction via Agrobacterium tumefaciens. Young stems of transgenic Amorpha fruitcosa were used as the explants to pre-culture,co-culture,selective culture,sub-culture and root induction culture, and finally got the neogenesis young shoots with Kanamycin Resistance. Results of PCR indicated that the two genes are all integrated into the genome successfully.In conclusion, this paper cloned the cDNA encoding caffeoyl CoA O-methyltransferase (CCoAOMT), one of the key enzymes in lignin biosynthesis, the first time. Two single expression vectors for antisense CCoAOMT and antisense 4CL gene and one binary expression vector harboring the two antisense genes were constructed respectively. Via Agrobacterium strains the transgenic Amorpha fruitcosa was obtained. Thus, laid a solid foundation for the downregulation of lignin biosynthesis and breeding genetically modified trees suitable for paper pulping through antisense inhibition mechanism.