| Lignin is a major structural compound of cell wall in vascular plants (pteridophytes, gymnosperms and angiosperms), being incorporated into the cell wall together with the process of thickening plant secondary wall. It not only plays a significant role in the growth and development of plants, but also is highlighted by more and more scientists because of its use in the human life. In order to regulate the biosynthesis of lignin in its molecular structure, quantity and composition, we firstly clone and characterize the gene of cinnamoyl-CoA reductase, a key enzyme gene which is considered as a potential control point regulating the carbon flux towards lignins.Two cDNAs encoding CCR are identified from wheat (Triticum aestivum L. cv. H4564), which is titled W-cr6 and W-crl9. Sequence and phylogenetic analysis with CCR protein suggest that W-cr6 and W-crl9 are the members of CCR family. Their proteins contain a motif KNWYCYGK which is extremely well conserved in the CCR sequence. The nucleotide of the W-cr6 and W-crl9 are GC rich over 60%. They share 76% and 69% similarity in the DNA and protein level respectively. RT-PCR and Nothern blot hybridization show that W-cr6 is expressed active in stem and leaf tissue, but W-crl9 is expressed in the root and stem tissue. The results suggest that there should be at least two genes encoding CCR in wheat genome.Using W-cr6 as a probe, a 1317 bp full-length cDNA of CCR gene, named as TaCCRl, is screened and cloned form the cDNA library of wheat. TaCCRl contains 1047 bp open reading fragment encoding a protein composed of 347 amino acids, with 72 bp 5' non-translated sequence and 198 bp 3' non-translated sequence. The molecular weight of the protein is deduced about 37.4 kDa. A high homology and similarity (over 60%) is found between TaCCRl and CCR from other plant in the level of nucleic acid and protein.To confirm the function of CCR gene in the lignin biosynthesis, we construct the anti-sense and sense expression vector pAtCCR, pStCCR harboring TaCCRl for transforming tobacco. 30 transgenic tobaccos with anti-sense TaCCRl gene and 12transgenic lines with sense TaCCRl are gained via Agrobacterium-mediated transformation. All transgenic lines including sense and anti-sense display common features, a decrease in the lignin content, orange brown coloration of the xylem cell wall, the collapsed or contorted vessels, and the dwarf development, but no variation about fecundity of transgenic plants and germinability of transgenic seeds are observed.Another two constructs pBSCl (sense) and pBACl (anti-sense) contain full-length TaCCRl are carried out for transforming the wheat through pollen tube-mediated. About 5000 seeds are conducted on the flower stage of wheat. We gain one transgenic wheat after screening using 0.2ug/ml PPT and identification of PCR, and southern. It required characterized in genetics, physiology and morphology. And other is conducting through PPT screen and molecular analysis.Here we analyze the relation between the lignin quantity and stem maximum bending stress. The results show that the lignin quantity together the number of vascular bundle and the culm area can contribute to the maximum bending stress. And the structure and density of vascular bundle in the culm have no significant effect on stiffness properties of stem. So it builds a foundation in physiology for improving the stem properties via genetic engineering using the genes in the lignin biosynthesis. |
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