Gene Cloning Of Pinus Massoniana4CL And Construction Of Its RNAi Expression Vector

Pinus massoniana, as longer and wide distribution range of its wood fiber, has become one of the main paper tree species. Lignin's degradation is the biggest technology problem in Paper industry. The process should not only consume a lot of chemicals, more serious is the black liquor will cause great damage to the environment. Therefore, to cultivate low content of lignin paper making materials tree species, it has become a crack of forestry biological technology frontier research trend, and it will produce a huge economic and ecological benefits. Based on the advantage in papermaking of P. massoniana, we cloned lignin synthesis regulation's key genes:4-Coumarate:Coenzyme A Ligase (4CL), constructed the P. massoniana4CL gene RNA interference vector, and achieved the transformation to tobacco, to provide the scientific basis and technical basis for low lignin transgenic breeding of P. massoniana.The main results for the following:(1) Full length clone of P. massoniana4CL gene. With leaves and stems of P. massoniana for materials, respectively, and the total RNA genome DNA extracted, and separately with total DNA and RNA always reverse transcriptase cDNA of as a template, according to4CL respectively to specific design primer, respectively through the long distance PCR and RT-PCR amplification technology out4CL gene segment of DNA and cDNA clips. The fragments were ligated to pMD18-cloning T vector. The transformation of e. coli DH5a strains, and the two-way DNA sequencing, won2696bp the genomic DNA cloned and1642bp the length of cDNA cloned. The genome of P. massoniana4CL DNA sequences are submitting to GenBank.(2) Bioinformatics analysis of4CL gene from P. Massoniana. Through the bioinformatics software Vector NTI10.0, the analysis results show that: the genome of P. massoniana4CL DNA for2696bp contains five extrons, and four introns; the full-length cDNA sequence is1642bp, contains a1440bp ORF. Through the comparison of the GenBank sequence, we can conclude the sequence is the length of P massoniana4CL gene sequence. Protein analysis software application of the cDNA sequence coding480amino acids. The secondary structure prediction's results show that4CL protein isoelectric point of5.84, the molecular weight of51.975KDa, there are2a transmembrane area, for hydrophobic protein, located in the mitochondrial membrane, have a signal peptide. (3) Prokaryotic expression of4CL fusion protein gene from P. massoniana. With4CL cDNA length of P. massoniana for template, design specifically expressed primer, through the double enzymes BamHI-XhoI method, insert to express pET32a carrier building fusion expression plasmid pET32a-4CL, and successfully in e. coli BL21(DE3) to efficiently express, IPTG induction expression results show that P. massoniana4CL proteins in induction4h, express large quantities.(4) Construction of P. massoniana4CL gene of RNA interference expression vector. On the basic of the length of pinus massoniana clone cDNA, design specific primer, through the high fidelity amplification technologies won four of pinus massoniana interference fragments, size are390bp and390bp; As a bridge to pRi35S carrier, successful construction of P. massoniana4CL gene RNA interference vector: pRi35S-4CLzf.(5) Transformation research. Using Agrobacterium tumefaciens-mediated transformation technology, we carried the interference vector: pRi35S-4CLzf into the model plant tobacco and obtained RNAi screening by anti-Kan fragment transgenic plants. Though the observation of growth, we found that there is no bad interfencement, such as dwarfing, to the transgenic tabocco compared with the wild. According to the analysis of Klason lignin, transgenic plant lignin content decreased by an average of3.81%, the relative content decreased up to15.41%compared with wild-type tobacco plants.