Studies On Antisense Transformation With Lignin Biosynthesis 4CL And CCoAOMT Gene In Amorpha Fruitcosa

Lignin is a complex phenolic polymer and plays important roles in plants, but it is also the main cause of papermaking pollution. Traditional crop improvement is not suitable for plants like trees due to the long growth period. However, with the development of modern genetic engineering, human modification of trees becomes easier. In some pattern plants like tobacco and aspen, modified transgenic plants with low content and loose structure of lignin has been obtained successfully. Thus, using modern biotechnology to regulate lignin biosynthesis in molecular level, cultivate genetically modified trees more suitable for pulping industry, has significant meanings in both economic value and environment protection.The many already researches have demonstrated that inhibition of endogenous 4-coumarate CoA Ligase (4CL) and Caffeoyl CoA O-methyltransferase (CCoAOMT) gene expression could effectively decrease lignin contents. CCoAOMT gene suppression also resulted in changes of lignin composition, accompanied with increased S/G (syringyl/guaiacyl) ratio and less condensed more extractable lignin. Therefore, 4CL and CCoAOMT gene is the ideal target genes to modify and improve plants profit for papermaking.Lignin is filled in cytoderm, its presence in plants provides mechanical strength to the stem and improves water transport and disease resistance. As a result, the decrease of lignin content and inappropriate expression of its synthesis related enzymes could possibly produce negative effects to plants. During the reforming study of resource plants, the constitutive promoter CaMV35S promoter shows poor specificity, which may cause abnormal growth in many transgenic plants. What's more, the CaMV35S activity detected in mammalian cells suggests environment risk in plants'genetic engineering application. Using the natural tissue-specific promoter of plants instead of constitutive promoter can better solve the problem.In this paper, we used antisense RNA technique, focused on the improvement of papermaking resource tree Amorpha frucicosa L, and started research as follows:1. Cloning of GRP1.8 promoter and two cDNA encoding lignin synthesis involved enzymes 4CL and CCoAOMT gene sequences respectively from Pheaseolus vulgaris L and Amorpha frucicosa L. The latter two cDNA were inserted into plant expression vector pBI121 in reverse direction, and constructed the antisense expression vector AP-4CL and AP-CCoAOMT. Vector AP-G-4CL and AP-G-CCoAOMT were constructed using xylem cambium site-specific GRP1.8 promoter instead of constitutive promoter CaMV35S.2. The antisense expression vector AP-4CL,AP-CCoAOMT and the binary expression vector with GRP1.8 promoter AP-G-4CL,AP-G-CCoAOMT were transferred into Agrobacterium tumefaciens EHA105 by Freeze-Thaw method. The results were confirmed via Colony PCR.3. Young stems of transgenic Amorpha fruitcosa were used as the explants to study the proper condition of generation and transformation.4. CCoAOMT and 4CL were mediated into the Amorpha fruitcosa genome in reverse direction via Agrobacterium-mediated transformation. After pre-culture,co-culture,selective culture,sub-culture and root induction culture, neogenesis young shoots with Kanamycin resistance were obtained finally. Results of PCR indicated that the target gene had integrated into the genome successfully.In conclusion, this paper constructed transgenic Amorpha frucicosa L harboring either antisense CCoAOMT or antisense 4CL gene via antisense RNA technique and Agrobacterium-mediated transformation. Meanwhile, the use of xylem cambium site-specific GRP1.8 promoter instead of constitutive promoter CaMV35S in construction of the antisense expression vectors, which is more profit to transgenic plants'normal growth and application security, has laid a solid foundation for the downregulation of lignin biosynthesis and breeding of genetically modified trees suitable for papermaking.