Cloning And RNAi Vector Construction Of Gene Fragments Related To Lignin Metabolism Of Miscanthus

Miscanthus, originated from China, is one of the new emerging bio-energy plant. It belongs to the C4 perennial grasses and its height is up to 4m with rosette appearance. As a feedstock of second-generation cellulosic ethanol, it has caught much attention because of its fast growth, low mineral content and high biomass yield. Second-generation cellulosic ethanol takes the lignocellulose as raw material, but it is difficult to use lignin which increases the cellulosic ethanol production costs, and therefore it is significant to research the lignin biosynthesis manipulation.RNA interference is the process of post-transcriptional gene silencing (PTGS) by which double stranded RNA (dsRNA) causes sequence-specific degradation of mRNA sequences. The RNAi technology is an effective strategy and widely used to identify the function of genes in plants.In this paper, Miscanthus sinensis was selected as test material,and the lignin synthesis-related key enzymes such as COMT, 4CL, CCR, CAD were used to the targest to down- regulate . In the experiment, the gene fragments were cloned as well as the RNAi vectors were constructed. The main results are as follows:1. Cloning and sequence analysis of gene fragments:The total RNA was extracted using REAGENT Trizol.Based on the regions with relatively high homology in target genes of different plants, specific primer was designed. 4 gene fragments was cloned using RT-PCR. And the length of the nucleotide sequence COMT, 4CL, CCR and CAD were 481,481,448 and 439 bp respectively, sequence analysis proved that these fragments were all the target gene fragments which can be used to construct RNA interference vector.2. The construction of RNA interference vector: After treated by BP reaction, LR reaction, the target gene fragments were ligated to the plasmid pMGRNAi. PCR assay was carried out using three pairs of PCR primers named CaMv35S and Primer-R, CaMv35S-Terrnlnate and Primer-R, Primer-L and Primer-R respectively, and the result showed that the target gene fragments were successfully inserted into the two sites of the vector, which meant the RNA interference vector were constructed successfully.3. The Agrobacterium-mediated transformation of the expression vector was carried out using freeze-thaw method, and the bacterial liquid PCR detection showed that the bacterium Agrobacterium tumefaciens LBA4404 containing expression vector was obtained. After Cultured through 15 generations, the Agrobacterium containing the expression vector was tested by PCR assay. The results indicated that the bacterium Agrobacterium LBA4404 containing expression vector has a good genetic stability.