Research On Anti-sense 4CL Regulation Synthesis Of Lignin In Tobacco

Lignin presents in the xylem of plants. It is linked together with cellulose and hemicellulose by toluene ether bond. The Lignin monomer connects each other with stable chemical bond. However, the stable structure of lignin is not beneficial to animal’s digestion and absorption of cellulose and hemicellulose.4- Coumaricacid:coenzyme A ligase (4CL) can catalyze hydroxy cinnamic acid generated hydroxycinnamic coenzyme A in lignin monomer synthesis process, which is connecting phenylpropanoid pathway and specificity biosynthesis, and it is key enzyme of lignin synthesis pathway. Some researches shown that inhibiting the gene expression of 4CL could effectively reduce the lignin content of plants. In this study, the antisense 4CL gene was transferred into the model plant tobacco by Agrobacterium-mediated method, leading to regulating the gene expression of 4CL to reduce the lignin content.This research has made the following progresses:1) Based on the general procedure of Agrobacterium mediated transformation method, the antisense 4CL gene has been transferred into tobacco which was confirmed by PCR and sequencing of target fragment in the transformation plants. Through the first generation of plants self-pollinated, we obtained the second generation plants. The results of PCR, RT-PCR and sequencing of the target fragment showed that antisense 4CL gene has been successfully transferred into the second generation tobacco plants and expressed normally.2) The second generation tobacco plants were detected by genetic stabilty. PCR detected one hundred second generation tobacco plants, and electrophoresis of PCR products was verified. The ratio of experimental group and wildtype group was 77:23, which was close to 3: 1. The results shown that antisense 4CL gene genetic complied with Mendel’s genetic law in the second generation tobacco.3) The second generation tobacco plants were tested by qRT-PCR to observing 4CL1 gene expression. The results shown that the expression level of the two samples of second generation plants leaf were reduced by 21.58% and 17.43% respectively. The ELISA result, the assay of the second generation tobacco plants 4CL generation content, shown that the two samples of the second generation tobacco plants steam were reduced by 11.19% and 9.8% respectively, indicating that the antisense gene could effectively regulate the expression of 4CL genes.4) Detection of physiological and biochemical indexes of transgenic plants, the results showed that the lignin content of the second generation tobacco stems decreased by 11.87% and root lignin content decreased 14.23%.There was no obvious differences in plant height, weight, root length and other aspects between Transgenic plant and wild-type plant. This result demonstrated that description antisense 4CL gene transferred into tobacco reduced the lignin content and maintain the regular growth of the tobacco as well.