Genes Isolation And Genetic Transformation Of Lignin Biosynthesis Enzyme In Betula Platyphylla

The materials of pulp in paper making industry generally include needle-leave timber and broad-leave timber. Of them the needle-leave timber is the best material, but the growth of needle-leave tree species is commonly slower, which limits its application in paper making industry. In order to meet the needs of pulp supply, largely adopting broad-leave timber is an important approach. Betula platyphylla is one of the main broad-leave tree species in Heilongjiang Province and northeast of China. It has better adaptability and resistance, and with a characteristic of rapid growth, moreover the ratio (56.7) of length and width of fiber is also suitable for paper making. But the content of lignin in timber is higher (needle-leave timber: 26%-30%, broad-leave timber: 23%-30%) in generally, the lignin is the main reasons resulting in cost increase of paper making and severe environmental pollution. Thus, decreasing the content of lignin in paper making material is a new method to renovate paper making pollution from the root. This study makes full use of modern biotechnology, isolating the CCoAOMT gene in correlation with lignin biosynthesis of Betula platyphylla and 4CL gene, conducting the control of lignin biosynthesis for CCoAOMT gene in tobacco, and studying its functions and regulation mechanism. This study will make a foundation for cultivating better new Betula platyphylla species of pulp timber by decreasing the content of lignin in tree species or changing its components.Lignin is a kind of important macromolecular organic material and only inferior to cellulose inside plant, with a content of 15%-30%. The biosynthesis of lignin is complex biochemical reaction with a multi-approach and tens of enzymes, of them, caffeoyl-CoA3-O-methyltransferase (CCoAOMT) and 4-coumaric acid CoAligase (4CL) are two the most important enzymes.This study includes two aspects; the first is that the cDNA span sequences of CCoAOMT gene (744bp) and 4CL gene (1629bp) were isolated from Betula platyphylla by the method of RT-PCR. The result of Blastn sequence analysis showed that nucleotide acid sequence of the CCoAOMT cDNA in Betula platyphylla has the highest homology with that of Fragaria vesca (88%), followed by Vitis.vinifera (86%). The nucleotide acid sequence of the 4CL cDNA in Betula platyphylla has the highest homology with that of Juglans (85%), and followed by Rubus (81%), Amorpha fruticosa (80%), Glycine max L. (79%), and Nicotiana tabacum (79%). The result of Blastp sequence analysis showed that the amino acid sequence of the CCoAOMT in Betula platyphylla has the highest homology with that of Broussonetia papyrifera (92%), followed by Populus tremuloides (91%) and Arabidopsis thaliana (91%). The amino acid sequence of 4CL in Betula platyphylla has the highest homology with that of Rubus L. (77%), followed by Populus tomentosa (76%). Moreover, the sequences of CCoAOMT and 4CL genes for many plants were multi-sequence compared by the DNA analysis software, the results indicated that: (1) For a pair of special two plants,the homology of amino acid sequences for the same genes is higher than that of nucleotide acid. (2) The longer gene sequences (including amino acid and nucleotide acid sequences) are, the lower homology is. The nucleotide acid sequence length of 4CL gene of Betula platyphylla is 1629bp, compared with other plants, the homology is in range of 58.2%-73.6%. The nucleotide acid sequence length of CCoAOMT gene is 744bp, the homology is in range of 72.2%-84.4%. (3) The homology and phylogenesis consanguinity of different genera plants will be changed by different compared sequences. For special two plants, we can't draw a conclusion for their consanguinity only depending on the homology of one or several genes. (4) For the same gene in different species of some genera, the homology of the sequence is very high and highly conservative.The second aspect of this study is that the CCoAOMT gene isolated from Betula platyphylla was transformed into tobacco. The span cDNA of CCoAOMT gene was recombined with the pBI121 vector, and the antisense expression vector of recombined CCoAOMT gene was named as pBPCOA. Afterwards, the pBPCOA was transformed into tobacco by the mediated gene transfer of Agrobacterium tumefaciens (EHA105). The main process of tobacco transformation was that: determining the types of differentiation culture medium, no pre-cultured or pre-cultured 2 days, infected 5-15min, and co-cultured 2-4 days. The prophase selection, delayed selection and anaphase selection were adopted. The lamina of tobacco co-cultured 2, 4 days and the control (no co-culture) were detected by the expression of GUS gene, the result showed that the first two treatments also gave rise to an indigo blue material, and the control has nothing. After the sample co-cultured, the selective pressure in selective culture medium is kanamycin of 50-80 mg/L, the ablastin of Agrobacterium tumefaciens is carbenicillin with a concentration of 250-750 mg/L. The normal developing tobacco seedling on the selective culture medium was sampled to isolate DNA, and conducted PCR amplification by the CCoAOMT cDNA primer of Betula platyphylla and using tobacco DNA as template. The result showed that there were two seedlings amplified specific strap of 754bp among detected 5 seedlings. And then, the detection of PCR-Southern was conducted, and the result is accordant with the relative result of PCR. The samples of T4-1, T4-9, and T6-2 produced stronger hybridization signals, though the sample of T3-1 had a weaker PCR electrophoresis, the hybridization signal still very clear. The sample of T3-3 hadn't the hybridization signal, which showed a negative result and matched with the results of GUS detection and PCR detection. So we can conclude that the genomic DNA in transgenic seedlings of T3-1, T4-1, T4-9, and T6-2 tobacco samples have integrated the CCoAOMT gene of Betula platyphylla.