| Lignins are composed of three difference kind of hydroxyl coumaryl alcohol—p-coumaryl alcohol, coniferyl alcohol and sinapyl alcohol, forms H, G, and S monolignin separately. Lignin, as one kind of main component in tracheophyta plant, is one important characteristics of plant to adapt terrestrial environment. However, its existence can influence the application of plant in paper-making and animal husbandry. Cinnamoyl-CoA reductase (CCR) is the first key enzyme in the specific pathway of lignin subunit biosynthesis. Its suppress can decrease lignin biosynthesis, so it is benefit to paper industry and feed utilizationThe Main results are as follows:1. CCR gene was cloned into pMD20-T in antisense direction. Using a variety of biological softwares, the ccr sequence was analyzed. Compared with Popolus trichocarpa and Popolus tomentosa, the homologies of nucleotide sequence were97%. Its mature protein was hydrophilic, mainly localized on plasma membrane, had the KNWYCYGK motif conserved in most plant CCR, its secondary structure had12a-helices,20p-strands and11coils.2. pMD20-T-ccr and pBI121plasmid were cut by restriction endonuclease Xba I and Sma I, the recycled CCR gene fragments and pBI121large fragments was ligated, the recombinant plasmid (we named pBI121-ccr) was identified, then transferred into Agrobacterium LBA4404.3. Tissue culture system for soybean was also established. Firstly soybean seed was disinfected, germinated for1-2days, the explants were disinfected in70%alcohol for30s, then sterilized in0.1%mercuric chloride for10s. They were transferred onto MS solid medium. Aseptic Soybean Seedlings were obtained in two weeks.4. Using leaf disc method, the plant tissue was infected by Agrobacterium mediation. Callus was obtained from soybean hypocotyl and tobacco leaf cultured on MS medium with NAA+6-BA; Adventitious buds were gotten from callus in the MS+0.5mg/L6-BA+0.05mg/L NAA; And when medium was MS+0.35mg/L NAA, adventitious roots were obtained. |
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