RNA Interferession Vector Construction Of Eucalyptus CAD And COMT Gene And Tobacco Transgenic Research

Eucalyptus is the major papermaking tree in the Southern of China, but In the process of paper manufacture, the removal of Eucalyptus lignin need a lot of chemicals and cause environmental pollution problems seriously. Therefore, nurturing a low-lignin Eucalyptus papermaking species with the transgenic technology fundamentally, reducting lignin content, chemicals consumption and pollution from the source, which is significant benefit for economic, social and ecological. In this paper, we picked cinnamyl alcohol dehydrogenase (CAD), caffeic acid-O-methyltransferase (COMT) which are key to control Eucalyptus lignin biosynthesis as the research object respectively, CAD, COMT gene RNAi expression vector pRi-Ec-CAD and pRi-Ec-COMT were constructed to transform tobacco with interference technogy, and achieved the functional verification through transgenic tobacco, which provide the theoretical and material basis for the transgenic breeding of Low-lignin eucalyptus.Main results achieved were as follows:(1) The obtained of Eucalyptus CAD and COMT gene RNA interference fragments Based on full-length CDNA sequences which has been cloned in the Eucalyptus CAD, COMT,CAD, COMT sense and antisense siRNA fragment were cloned by high-fidelity amplification technolgywhit Specific primers, whose size were:CAD550bp,COMT616bp respectively.(2) Constructed CAD, COMT gene RNAi expression vector. The CAD, COMT gene RNAi expression vector pRi-pRi-Ec-CAD and pRi-pRi-Ec-COMT which contined CaMV35S promoter, catalase intron and antibiotics (Kan) selection marker gene were successfully constructed with modern molecular biology methods.(3) transformed Eucalyptus CAD and COMT gene RNA interference into tobacco. Through Agrobacterium-mediated transformation of leaf disc technology, the RNAi vector pRi-pRi-Ec-CAD and pRi-pRi-Ec-COMT were transformed into model plant tobacco, and obtained RNAi screening by anti-Kan fragment transgenic plants, PCR for detection of transgenic plants, those plants amplified the genomic DNA of the kanamycin gene, as a result, we screened out positive transgenic tobacco plants which was introduced into Eucalyptus CAD and COMT gene RNA interference vector. (4) The analysis of Eucalyptus CAD gene RNA interference effect. Haved the observation of tobacco plants phenotype which have transformed CAD gene interference fragment in the different growth periods, transgenic plants'growth and development is normal compared with wild tobacco plant; haved the transgenic plants analysis of Klason lignin after 6 months'cultivation, transgenic plant lignin content decreased by 12.3% to 14.5% compared with control plants. The results showed eucalyptus CAD gene RNA interference vector can effectively reduce the transgenic tobacco's lignin content and does not affect the plant's normal growth and development(5) The analysis of Eucalyptus COMT gene RNA interference effect. Haved the phenotype observation of tobacco plants which ave transformed Eucalyptus COMT gene interference fragmenof in the different periods, transgenic tobacco plants have larger leaf, smaller size, small soft stems, easy lodging, slow growth and low survival rate at different times when compared with wild-type tobacco plants. The transgenic plants which were analyzed for Klason lignin after 6 months'cultivation have the lignin content which decreased by 2.4% to 3.0% compared with control plants. The results show that using COMT gene RNA interference vector to regulate the control of lignin content or composition will lead to the obstacle of normal growth and development of plants. Meanwhile there is no obvious reduction of the lignin.In summary, through the analysis of transgenic tobacco plants and the phenotypic suppression of lignin synthesis, the results showed that Eucalyptus CAD gene RNA interference vector which was consructed in this paper can reduce the content of plant lignin without significantly affection of the normal growth of the plants, CAD gene fragments can be used as the RNAi target genes to lower lignin metabolism of Eucalyptus, these provided material basis and technological support for the transgenic breeding of Low-lignin eucalyptus.