Cloning And Expression Analysis Of Flax (Linum Usitatissimum) Critical Lignin Metabolism Genes

Flax (Linum usitatissimum) is an important source of cellulose-rich natural fibres. Its fibre is characterized by the strong pulling force,soft,fineness slender and so on. In the textile industry and paper industry, high lignin contents incease the difficulty in degumming process, increasing waste pollution and decreasing the fiber quality. Therefore cloning and functional characterization of the genes involved in lignin metabolism will be helpful to understand the lignin metabolism pathway and make it possible to create lower lignin content flax by genetic engineering method. In this research, using flax (Linum usitatissimum) Variety "Baihua" as materials, RACE technology was used to clone CCoAOMT1,4CL2 and COMT cDNA sequences. Expression patterns of CCoAOMT1,CCoAOMT1,F5H2,F5H3,COMT,4CL1 and 4CL2 were analyzed by semi-quantitative RT-PCR in different flax tissues and in xylem and phloem (bark) of different development stages. The main results were as the following:(1) The COMT 5'end cDNA partial sequence was cloned, which is of 512 bp in length and coding for 135 amino acid residues. The sequence is 97% identical to the reported flax COMT partial sequence.(2) The Cloned 4CL2 3'end cDNA partial sequence was cloned, which is of, 727 bp in length and coding for 199 amino acid residues.(3) The CCoAOMT1 full length cDNA sequence was cloned. The sequence is of 1027 bp in length and coding for 247 amino acid residues. This gene was registered to GenBank with accession number EU926495 and putative protein sequence accession number ACH47151. The CCoAOMT1 cDNA sequence was 85% identical to the reported CCoAOMT full length cDNA.(4)Semi-quantitative PCR analysis revealed that CCoAOMT1,CCoAOMT1,F5H2,F5H3,COMT,4CL1 and 4CL2 were all expressed in seedlings, roots, florescence xylem, florescence phloem, leaves, flowers and young fruits, except for no detection of F5H1 expression in the roots. Expression of CCoAOMT1 was consistently high in all the tissues detected. There existed expression complementary characters between two CCoAOMT,two 4CL genes and among the three F5H genes in various tissues.(5)In the phloem, all genes lowly expressed except for 4CL2 at young stage(30-day-old)and highly expressed after flowering(70-day-old). Expression of 4CL1 was relatively high, and that of F5H3 was relatively low in the phloem at all stages comparing to the other genes.(6)In the xylem, expression of 4CL1 gene was low at all the developing stages. The expression of F5H1 reached the peak before flowering and the expression of the other genes in the xylem reached the highest at 30-day-old xylem and gradually declined at late growing stages, with a sharp decrease to very lower level after flowering.(7)Different from others genes examined in this research, the expression of 4CL1 was significantly higher in phloem than that in xylem.