Overexpression Of OsCesA4 And RNAi Of Three Lignin Biosynthesis Related Genes In Flax

Flax (Linum usitatissimum L.) is the third largest natural fiber crops. Flax fiber is primary phloem fiber, which is characterized by strong pulling force, soft, slender fineness in textile industry. Because it is a self pollinated diploid species and its small genome size, flax has been thought to be the model plant for the study of bast fiber development. In textile industry and paper industry, high lignin content increases the difficulty in degumming and retting process, decreasing the fiber quality and increasing waste pollution. In this study, plant overexpression vector of OsCesA4 gene and RNAi plant expression vectors of three related genes in flax liglin metabolism were constructed. Parameters that affect the efficiency of Agrobacterium tumefaciens-mediate transformation of flax were studied and optimized. The research work provided material basis for increasing the cellulose content and decreasing the liglin content in flax through genetic engineering. The main results are as follows:1. The full length cDNA of OsCesA4 (3462bp) was digested from pFLCI vector which contains the full length cDNA of OsCesA4, and then it was inserted into pCAMBIA1301M plasmid to construct plant expression vectors by blunt end ligation. The constructed plant expression vector pOsCesA4-OE was checked by resitriction enzyme digestion and cDNA sequencing.2. RNAi fragments of flax CCoAOMT,4CL,COMTgenes were amplified by PCR and inserted into plasmid pCAMBIA1301M to construct the plant expression vectors by intermediate vector pB3D-Linker. The constructed RNAi plant expression vectors pCCoAOMT-RNAi,p4CL-RNAi and pCOMT-RNAi were checked by resitriction enzyme digestion.3. Efficient Agrobacterium-mediated flax transformation system was established. The optimal conditions are as follows:precultured explants for 10 days, immerged for 30 min with Agrobacterium, cocultured for 3 days. Concentration of hygromycin B at 10mg/L for selection phase and at 15mg/L for rooting phase had high transformation frequency.4.4CL RNAi transgenic flax plants were obtained and confirmed by PCR assay. The 4CL gene expression in the transgenic flax plants was down-regulated as revealed by RT-PCR analysis. Transgenic shoots of OsCesA4 overexpression and RNAi of CCoAOMT and COMT were also obtained.