The Effects Of 4CL Gene On Lignin Metabolism In Plant

The content of lignin consists of 15% to 35% in the plant which is the second main resource of terrestrial polymer only next to cellulose. It can enhance mechanical strength of plant and improve cell transportation capacity, so the synthesis of terrestrial lignin is one of the important evolution characteristics adapted to land environment. However, lignin in the fodder is not conducive to the animal's digestion and absorption and lignin constitutes the major source of pollution in papermaking industry. So reducing content or changing the component of lignin would have important application value in fodder production and papermaking industry. In recent years, the genes of main enzyme concerning lignin biosynthesis have been cloned, and by using modern biotechnology to regulate plant lignin biosynthesis will help clarify the molecular mechanism of lignin synthesis and can use the plant resource more effectively.4-coumarate acyl-CoA ligase (4CL) is the enzyme regulating the pathway of lignin biosynthesis. It is found that by regulating the amount of this enzyme can effectively regulate the lignin content. So the research of 4CL enzymic characteristics and gene function will lay a theoretical foundation in genetic engineering to improve vascular plants.To do research on the functions of the 4CL in lignin biosynthesis and their possible application in genetic engineering breeding, we first did bioinformatic analysis of Pto4CL1 and Pto4CL4 cloned from poplar and by using tobacco NC89 and 741 poplar as materials, we tranformed the 4CL1 and 4CL4 gene into tobacco and poplar through the Agrobacterium mediation, and got transformed plants. We measured and analyzed the effects of excessive expression of these two genes on transgenic phenotypic characters and the changes of enzyme activity, the contents of phenolic acid and lignin. The results of this paper are as following:(1) The bioinformatics analysis of 4CL1 and 4CL4 gene sequences were carried out by using Blastn software from BCBI website. The results indicated that the homology of 4CL1 with AC211731.1(E:0.0), XM 002338702.1(E:0.0), D49366.1(E:2e-113), EZ339134.1(E:4e-75) and EZ369686.1(E:6e-73) was 93%, 89%, 96%, 74%and 79%respectively; the similarity of 4CL4 with XM002329613.1(E:0.0), AF008184.1(E:0.0), AF008183.1(E:0.0), XM 002533140.1(E:0.0), XM002335362.1(E:0.0),GU949552(E:0.0), XM002335188.1(E:2e-83)and DQ124216.1(E:2e-78) was 97%, 97%, 88%, 80%, 98%, 79%, 95% and 76% respectively.(2) We used relevant software to analyze the protein structure of 4CL1 and 4CL4, and preliminarily predict their functions. The results revealed that 4CL1 protein's relative molecular mass was 58.53 KD, and its theoretical pI value was 5.6 . The result of secondary structure analysis showed that theα-helix,β-folding and random coil in 4CL1 protein accounted for 28.73%, 52.05% and 19.22% respectively. It indicated that 4CL1 had no transmembrane helix zone, so it was not a transmembrane protein. In addition, the analysis result revealed that 4CL1 was not a secreted protein because there was no signal peptide in this protein. The relative molecular mass of protein 4CL4 was 61.14 KD, and its theoretical pI value was 5.67. The analysis of 4CL4's secondary structure showed that theα-helix,β-folding and random coil in 4CL4 accounted for 28.73%, 52.72% and 18.88% respectively. 4CL4 was a transmembrane protein because it had transmembrane helix zone. Signal peptide was not found in protein 4CL4, thence we concluded that it was not a secreted protein.(3) The PCR results showed that the specific DNA fragments could be amplified by using the transgenic tobacco genomic DNAs as template and 4CL1 or 4CL4 specific primers, while wild-type plant did not have specific band, indicating that the gene 4CL1 and 4CL4 fragment might have been integrated into tobacco genome respectively. Southern blotting results proved that the recombinant vector gene containing 4CL1 and 4CL4 had been integrated into the tobacco genome DNA respectively. We also successfully obtained the pBI121-4CL4 transgenic poplars.(4) The transgenic tobaccos of 4CL1 and 4CL4 as well as the wild-type ones, which grew for 6 weeks, were used as material to determine 4CL enzyme activities (using 4-coumaric acid as its substrate) in different sections. The results indicated that the 4CL enzyme activities in stem phloem and stem xylem of 4CL1 and 4CL4 transgenic plants were increased compared with the control. The phenolic acids content in stem xylem and leaves of transgenic plants were determined and the results showed that the content of 4-coumaric acid, ferulic acid, sinapic acid in stem xylem were significantly increased while the content of 4-coumaric acid, caffeic acid, ferulic acid, sinapic acid and cinnamic acid in leaves were remarkably increased compared with the control plants. (5) The transgenic tobaccos of 4CL1 and 4CL4 as well as the wild-type ones, which grew for 6 weeks, were used as experimental material to measure the lignin content. The results showed that the lignin contents in stem xylem, stem phloem and root of transgenic tobaccos increased compared with the control ones, especially those in the roots were significantly improved compared with the control.