Cloning And Expression Analysis Of Lignin Biosynthetic Enzyme Gene Of Ramie

Ramie [Boehmeria nivea (Linn.) Gaud.]falls into the category of Urticaceae herbaceous plants and its fibre is an important textile materials in China. In recent years, the weed fibre is used in the biological fuel ethanol fermentation makes the ramie a more economic value as an energy sources plant. But the existence of lignin with fiber limits the effectivity utilization of ramie in a foundmental cause. Therefore, the study of the molecular biology of lignin synthesis and metabolism has important application value for guiding the selection of low lignin content and artificial metabolic manipulation of ramie.The cinnamyl alcohol dehy.drogenase(CAD),ferulate-5-hydroxylase(F5H), cinnamate-O-methyltrasferase(COMT) and caffeovl-CoA O-methyltransferase (CCoAOMT) are key enzymes for lignin synthesis in plant. The CAD and F5H gene cDNA sequences were identified by transcriptome analysis of ramie(Boehmeria nivea) by provious study. The whole CAD gene cDNA and the coding sequence of F5H gene cDNA are cloned by RT-PCR combined with RACE method. The qRT-PCR were carried out to analysis of the correlation between the CAD、F5H、COMT、CCoAOMT gene expression with the lignin contains in different cultivates ramie in XiangZhu1, XiangZhu3, Xiangtandayebai and Chengbuqingma. We carried out a visual quantitative of the lignin by tissue stain with phloroglucin analysis of the stems’ cross section with the four ramies. The correlation between the quantity of lignin and the gene expression are analysised of the ramie lignin with the four key enzymes in lignin synthesis pathways. The conlusion is following:1. The ramie lignin key enzymes CAD、F5H gene cDNA are cloned and sequenced and the BnCAD whole cDNA sequence was submitted in to GeneBank and its registry numbers is KF758396,could be translated into a putative protein with359amino acids. BLAST and protein structure analysis showed that this cDNA sequence is in high homologous with other plant CAD gene, and with97%homology compared with Medicago truncatula, which confirmed that this cDNA sequence was the CAD gene.2. The quantitative of the gene expression of the four genes indicates the expressions of BnCAD gene was much higher in phloem than xylem in Xiangzhu1and Xiangzhu3, while Chengbuqingma the opposite, and no obvious difference observed in Xiangtandayebai.Other-waise the BnF5H、BnCOMT、BnCCoAOMT gene expression are mainly in the xylem and their expression quantity are changing among the varieties.3. Histochemical lignin assays showed that the staining degree between different cultivars is directly proportional to the quantitative expression of CAD transcription suggesting an important role of CAD during ramie lignin synthesis.