| Phenylpropanoid metabolism products have important biological functions in plants. Lignin is one of the important phenylpropanoid metabolites, which strengthens the biotic and abiotic stress resistance and defense of plants. However, the presence of lignin is a major source of pollution in papermaking. Meanwhile, lignin in forage is not conducive to the animal's digestion and nutrient absorption. In recent years, the genes of main enzyme in lignin biosynthesis have been cloned, and the regulation of plant lignin biosynthesis using modern biotechnology not only clarifies the molecular mechanism in the synthesis of lignin and other phenylpropanoid metabolites, but also has important application value in raw materials of fine paper making and forage production.4-coumarate acyl-CoA ligase (4CL) is the last enzyme of phenylpropanoid pathway turned downstream branch pathways and the key rate-limiting enzyme in the regulation of lignin biosynthesis, which is in the terminal position of phenylpropanoid derivatives metabolic pathway. The investigation of 4CL gene function with genetic engineering is conducive to reveal the mechanism of phenylpropanoid derivatives metabolic pathway and to provide a theoretical basis for improving the content and composition of lignin in vascular plants.Pto4CL2 and Pto4CL3 cDNA fragments of full-length coding for the 4CL enzyme of Poplar were acquired to investigate the functions of Pto4CL2 and Pto4CL3 in phenylpropanoid pathway particularly in the lignin biosynthesis and their possible applications in genetic engineering. The model plant Tobacco and 741 Poplar were used as materials, and we carried out downstream work of genetic engineering using Pto4CL2 and Pto4CL3 gene cDNA. In this paper, sense pBI121-4CL2 and pBI121-4CL3 were constructed and used to mediate the Agrobacterium genetic transformation of tobacco, while antisense pBI121-4CL2 and pBI121-4CL3 were used for mediating Agrobacterium genetic transformation of Populus. The effects of gene excessive or inhibiting expression on transgenic phenotypic characters were analyzed, and the changes in lignin content of different tissues, and the composition of the main changes in the structure were determined. The main results of this paper are described as follows:1)Bioinformatic analysis of 4CL gene sequence: Using the data obtained from NCBI website and the Mega4.0 software, the Blastn poplar 4CL gene sequences were analyzed and compared with other species for multiple alignment analysis. Meanwhile, the phylogenetic tree of 4CL gene family was constructed, which could be divided into two groups: Class I and Class II. The Pto4CL2 used in this study belonged to Class II and it had highest homology with Populus tremuloides Pt4CL2. simultaneously, Giycine max Gm4CL3 and Gm4CL4 also had higher homology with the Pto4CL2. The Pto4CL3 belonged to Class I and it had a high homology with Populus tremuloides Pt4CL1 and Populus tomentosa Pto4CL1. The homology of the cloned 4CL2 and 4CL3 genes with amino acid was analyised using coresponding softwares and the homology was predicted to be 58.52%. Meanwhile, the physicochemical properties and their preliminary functions of the 4CL2 and 4CL3 were predicted.2) The PCR and Southern blot detections: PCR results showed that the specific DNA fragments could be amplified by using the transgenic tobacco genomic DNAs as template and 4CL specific primers, while wild-type plants doesn't have specific band, indicating that the sense pBI121-4CL2 and pBI121-4CL3 gene fragment might have been integrated into tobacco genome respectively. Southern blot results showed that transgenic tobacco plants could show the hybridization signal except the control ones, which proving the recombinant vector gene had been integrated into the tobacco genome DNA. We also successfully obtained the antisense pBI121-4CL2 and pBI121-4CL3 transgenic poplars and got preliminary PCR detections.3) The transgenic tobaccos with sense 4CL2 and 4CL3, and wild-type tobaccos, which growed for 6 weeks, were used as experimental materials to determine 4CL enzyme activities of different organizations and parts. The results showed that 4CL activity from high to low was in the following order: phloem, xylem, root and leaves, and the 4CL activities in the leaves were almost undetectable. The 4CL enzyme activities in phloem and xylem of the sense 4CL2 and 4CL3 transgenic plants were increased compared with the control ones. The phenolic acids content in xylem and leaves of transgenic plants were determined and the results showed that the content of 4-coumaric acid, caffeic acid, ferulic acid, sinapic acid and cinnamic acid in xylem were significantly increased while the content of 4-coumaric acid, caffeic acid and cinnamic acid in leaves were significantly increased compared with the control plants.4) The transgenic tobaccos with sense 4CL2 and 4CL3, and wild-type tobacco plants, which growed for 6 weeks, were used as experimental materials to determine lignin content of different organizations and parts. The results showed that the integration of sense 4CL2/4CL3 gene had affected lignin biosynthesis and distribution in different parts of tobacco.The phloem, xylem and root lignin content of the sense 4CL2 and 4CL3 transgenic plants increased compared with the control ones, especially those in the roots are significantly improved compared with the control ones. Simultaneously, the stem holocellulose content were measured and the results showed that the holocellulose of transgenic plants had no obvious differences compared with the control ones. Stem sections were used to make Wiesner reaction and KMnO4 stain reaction, and microscopic observations showed that transgenic plants had no visable differences compared with the control ones. The morphological characteristics and growth of the control and transgenic tobaccos, which growed for 4 months, were observed. The results revealed that there were no clear differences in external morphology such as height, diameter and water content between the transgenic and wild-type tobacco plants.
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