Cloning Of Betula Platyphylla COMT1and CESA4Gene And Genetic Transformation Of AntiCOMT1Gene

Lignin is phenolic polymer, whose amount is merely less than that of cellulose. Lignin plays an important role in plant cell wall lignification, formation of mechanical tissues, and resistance to the pathogen. COMT(caffeic acid-O-methyltransferase) is the key enzyme in the biosynthesis pathways for lignin monomer, which can catalyze the methylation of caffeic acid,5-hydroxy coniferyl aldehyde,5-hydroxy coniferyl alcohol to produce the ferulic acid,mustard aldehyde and sinapyl alcohol respectively.. Nowadays, people have cloned COMT family from different plants. Betula platyphylla, a very important wood in the northeastern area in China. Whereas, there is no report on COMTl and CESA4gene functions and the role of COMT in lignin biosynthesis process in Betula platyphylla.In our research, we design the degenerate gene primers in accordance with the other plants reported conserved regions of COMT1and CESA4, and isolate the COMT1cDNA(852bp) using RT-PCR from the Betula platyphylla. Then, we utilize this cDNA to design the primers to clone the whole length(1367bp) of the COMT1with the combination of RACE technique. The full coding region is1095bp and encodes365amino acids. The registration number in GenBank is KC292201. The amino acid homology comparison indicates that the homology between the Betula platyphylla and the Gossypium hirsutum, Populus trichocarpa. Eucalyptus camaldulensis and Betula pendula is79.2%、79.2%、77.2%、99.5%respectively. In the similar way, we isolate the CESA4cDNA(3231bp) from the Betula platyphylla. The full coding region is3150bp and encodes1049amino acids. The CESA4gene homology between the Betula platyphylla and Betula luminifera and Betula pendula is98.6%,99.7%respectively.We also construct the COMT1overexpression and antisense vectors utilizing the Gateway technique. The constructed pGWB2-antiCOMTl is transformed into the agrobacterium. we complete the Betula platyphylla genetic transformation using the leaf disc transformation method. After analyzing of the COMT1gene transcription level in these lines, the COMT1expression level in the transformation lines are significantly downregulated compared with the wild type, by the way, there are different reduction degrees in different lines. Our results indicate, the antisense COMT1can significantly reduce the COMT1expression level in the Betula platyphylla. With regard to the lignin biosynthsis metabolism changes because of the COMT1gene expression level reduction, We will do further research afterwards.