CN100415882C - Method for regulating lignin in populus tomentosa - Google Patents
Method for regulating lignin in populus tomentosa Download PDFInfo
- Publication number
- CN100415882C CN100415882C CNB2003101017995A CN200310101799A CN100415882C CN 100415882 C CN100415882 C CN 100415882C CN B2003101017995 A CNB2003101017995 A CN B2003101017995A CN 200310101799 A CN200310101799 A CN 200310101799A CN 100415882 C CN100415882 C CN 100415882C
- Authority
- CN
- China
- Prior art keywords
- lignin
- gene
- genes
- substratum
- xylogen
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired - Fee Related
Links
- 229920005610 lignin Polymers 0.000 title claims abstract description 56
- 238000000034 method Methods 0.000 title claims abstract description 20
- 230000001105 regulatory effect Effects 0.000 title claims description 5
- 241000249899 Populus tomentosa Species 0.000 title abstract description 9
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 23
- 230000009261 transgenic effect Effects 0.000 claims abstract description 17
- NGSWKAQJJWESNS-UHFFFAOYSA-N 4-coumaric acid Chemical compound OC(=O)C=CC1=CC=C(O)C=C1 NGSWKAQJJWESNS-UHFFFAOYSA-N 0.000 claims abstract description 12
- 239000013604 expression vector Substances 0.000 claims abstract description 12
- 229940093681 4-coumaric acid Drugs 0.000 claims abstract description 6
- 230000001404 mediated effect Effects 0.000 claims abstract description 5
- 230000006696 biosynthetic metabolic pathway Effects 0.000 claims abstract description 3
- 108060004795 Methyltransferase Proteins 0.000 claims abstract 4
- 241000196324 Embryophyta Species 0.000 claims description 35
- 238000012216 screening Methods 0.000 claims description 15
- 238000012549 training Methods 0.000 claims description 10
- 229930006000 Sucrose Natural products 0.000 claims description 9
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 claims description 9
- 239000001963 growth medium Substances 0.000 claims description 9
- 239000005720 sucrose Substances 0.000 claims description 9
- 108090000364 Ligases Proteins 0.000 claims description 7
- 230000007423 decrease Effects 0.000 claims description 6
- 230000004069 differentiation Effects 0.000 claims description 5
- 101150044508 key gene Proteins 0.000 claims description 5
- 241000193830 Bacillus <bacterium> Species 0.000 claims description 4
- QHRGJMIMHCLHRG-GQCTYLIASA-N s-[2-[3-[[4-[[[5-(6-aminopurin-9-yl)-4-hydroxy-3-phosphonooxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-hydroxyphosphoryl]oxy-2-hydroxy-3,3-dimethylbutanoyl]amino]propanoylamino]ethyl] (e)-3-(3,4-dihydroxyphenyl)prop-2-enethioate Chemical group O1C(N2C3=NC=NC(N)=C3N=C2)C(O)C(OP(O)(O)=O)C1COP(O)(=O)OP(O)(=O)OCC(C)(C)C(O)C(=O)NCCC(=O)NCCSC(=O)\C=C\C1=CC=C(O)C(O)=C1 QHRGJMIMHCLHRG-GQCTYLIASA-N 0.000 claims description 4
- 241000168036 Populus alba Species 0.000 claims description 2
- 230000000692 anti-sense effect Effects 0.000 abstract description 22
- 238000004537 pulping Methods 0.000 abstract description 11
- 108090000790 Enzymes Proteins 0.000 abstract description 10
- 230000008859 change Effects 0.000 abstract description 9
- 239000002994 raw material Substances 0.000 abstract description 4
- 230000009467 reduction Effects 0.000 abstract description 4
- 230000015572 biosynthetic process Effects 0.000 abstract description 3
- 238000010353 genetic engineering Methods 0.000 abstract description 3
- 239000002023 wood Substances 0.000 abstract description 3
- 241000589158 Agrobacterium Species 0.000 abstract description 2
- 230000009466 transformation Effects 0.000 abstract description 2
- 230000001131 transforming effect Effects 0.000 abstract description 2
- 241000894007 species Species 0.000 abstract 2
- QAIPRVGONGVQAS-DUXPYHPUSA-N trans-caffeic acid Chemical compound OC(=O)\C=C\C1=CC=C(O)C(O)=C1 QAIPRVGONGVQAS-DUXPYHPUSA-N 0.000 abstract 2
- ACEAELOMUCBPJP-UHFFFAOYSA-N (E)-3,4,5-trihydroxycinnamic acid Natural products OC(=O)C=CC1=CC(O)=C(O)C(O)=C1 ACEAELOMUCBPJP-UHFFFAOYSA-N 0.000 abstract 1
- 108010011449 Long-chain-fatty-acid-CoA ligase Proteins 0.000 abstract 1
- 229940074360 caffeic acid Drugs 0.000 abstract 1
- 235000004883 caffeic acid Nutrition 0.000 abstract 1
- QAIPRVGONGVQAS-UHFFFAOYSA-N cis-caffeic acid Natural products OC(=O)C=CC1=CC=C(O)C(O)=C1 QAIPRVGONGVQAS-UHFFFAOYSA-N 0.000 abstract 1
- 238000010367 cloning Methods 0.000 abstract 1
- 239000000470 constituent Substances 0.000 abstract 1
- 230000002265 prevention Effects 0.000 abstract 1
- 238000007873 sieving Methods 0.000 abstract 1
- 108010035812 caffeoyl-CoA O-methyltransferase Proteins 0.000 description 22
- 239000000178 monomer Substances 0.000 description 13
- 102100034581 Dihydroorotase Human genes 0.000 description 10
- 241000219000 Populus Species 0.000 description 10
- 230000009182 swimming Effects 0.000 description 10
- 239000002299 complementary DNA Substances 0.000 description 8
- 102000004190 Enzymes Human genes 0.000 description 7
- 108020002739 Catechol O-methyltransferase Proteins 0.000 description 6
- 102100040999 Catechol O-methyltransferase Human genes 0.000 description 6
- 238000009396 hybridization Methods 0.000 description 6
- 238000006116 polymerization reaction Methods 0.000 description 6
- 102000003960 Ligases Human genes 0.000 description 5
- 241000208125 Nicotiana Species 0.000 description 5
- 235000002637 Nicotiana tabacum Nutrition 0.000 description 5
- 108700019146 Transgenes Proteins 0.000 description 5
- 229920002678 cellulose Polymers 0.000 description 5
- 239000001913 cellulose Substances 0.000 description 5
- 230000000694 effects Effects 0.000 description 5
- 239000007788 liquid Substances 0.000 description 5
- 239000013612 plasmid Substances 0.000 description 5
- 230000008569 process Effects 0.000 description 5
- 241000124033 Salix Species 0.000 description 4
- 238000004458 analytical method Methods 0.000 description 4
- 238000006722 reduction reaction Methods 0.000 description 4
- LZFOPEXOUVTGJS-ONEGZZNKSA-N trans-sinapyl alcohol Chemical compound COC1=CC(\C=C\CO)=CC(OC)=C1O LZFOPEXOUVTGJS-ONEGZZNKSA-N 0.000 description 4
- 101150029435 4CL gene Proteins 0.000 description 3
- 241000894006 Bacteria Species 0.000 description 3
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 3
- DYUQAZSOFZSPHD-UHFFFAOYSA-N Phenylpropyl alcohol Natural products CCC(O)C1=CC=CC=C1 DYUQAZSOFZSPHD-UHFFFAOYSA-N 0.000 description 3
- 230000001851 biosynthetic effect Effects 0.000 description 3
- 238000001514 detection method Methods 0.000 description 3
- 239000012634 fragment Substances 0.000 description 3
- 239000002609 medium Substances 0.000 description 3
- 239000000203 mixture Substances 0.000 description 3
- 238000011160 research Methods 0.000 description 3
- 239000000126 substance Substances 0.000 description 3
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 2
- 101000762164 Arabidopsis thaliana Cytochrome P450 84A1 Proteins 0.000 description 2
- 244000025254 Cannabis sativa Species 0.000 description 2
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 2
- 240000004923 Populus tremuloides Species 0.000 description 2
- 235000011263 Populus tremuloides Nutrition 0.000 description 2
- 238000002105 Southern blotting Methods 0.000 description 2
- 108010036937 Trans-cinnamate 4-monooxygenase Proteins 0.000 description 2
- 230000003321 amplification Effects 0.000 description 2
- 230000033228 biological regulation Effects 0.000 description 2
- LZFOPEXOUVTGJS-UHFFFAOYSA-N cis-sinapyl alcohol Natural products COC1=CC(C=CCO)=CC(OC)=C1O LZFOPEXOUVTGJS-UHFFFAOYSA-N 0.000 description 2
- JMFRWRFFLBVWSI-NSCUHMNNSA-N coniferol Chemical compound COC1=CC(\C=C\CO)=CC=C1O JMFRWRFFLBVWSI-NSCUHMNNSA-N 0.000 description 2
- DKZBBWMURDFHNE-NSCUHMNNSA-N coniferyl aldehyde Chemical compound COC1=CC(\C=C\C=O)=CC=C1O DKZBBWMURDFHNE-NSCUHMNNSA-N 0.000 description 2
- 238000005520 cutting process Methods 0.000 description 2
- 230000029087 digestion Effects 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 230000002708 enhancing effect Effects 0.000 description 2
- 239000004459 forage Substances 0.000 description 2
- 230000002068 genetic effect Effects 0.000 description 2
- 238000005286 illumination Methods 0.000 description 2
- 230000005764 inhibitory process Effects 0.000 description 2
- 239000003550 marker Substances 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 108020004999 messenger RNA Proteins 0.000 description 2
- 235000015097 nutrients Nutrition 0.000 description 2
- COLNVLDHVKWLRT-UHFFFAOYSA-N phenylalanine Natural products OC(=O)C(N)CC1=CC=CC=C1 COLNVLDHVKWLRT-UHFFFAOYSA-N 0.000 description 2
- -1 phenylpropyl alcohol alkane Chemical class 0.000 description 2
- 238000011084 recovery Methods 0.000 description 2
- 238000003757 reverse transcription PCR Methods 0.000 description 2
- 239000000523 sample Substances 0.000 description 2
- DKZBBWMURDFHNE-UHFFFAOYSA-N trans-coniferylaldehyde Natural products COC1=CC(C=CC=O)=CC=C1O DKZBBWMURDFHNE-UHFFFAOYSA-N 0.000 description 2
- 239000013598 vector Substances 0.000 description 2
- 239000002699 waste material Substances 0.000 description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 2
- KJPRLNWUNMBNBZ-QPJJXVBHSA-N (E)-cinnamaldehyde Chemical compound O=C\C=C\C1=CC=CC=C1 KJPRLNWUNMBNBZ-QPJJXVBHSA-N 0.000 description 1
- NQBWNECTZUOWID-UHFFFAOYSA-N (E)-cinnamyl (E)-cinnamate Natural products C=1C=CC=CC=1C=CC(=O)OCC=CC1=CC=CC=C1 NQBWNECTZUOWID-UHFFFAOYSA-N 0.000 description 1
- XMIIGOLPHOKFCH-UHFFFAOYSA-N 3-phenylpropionic acid Chemical compound OC(=O)CCC1=CC=CC=C1 XMIIGOLPHOKFCH-UHFFFAOYSA-N 0.000 description 1
- 102000007698 Alcohol dehydrogenase Human genes 0.000 description 1
- 108010021809 Alcohol dehydrogenase Proteins 0.000 description 1
- 108020005544 Antisense RNA Proteins 0.000 description 1
- 101000984031 Aspergillus flavus (strain ATCC 200026 / FGSC A1120 / IAM 13836 / NRRL 3357 / JCM 12722 / SRRC 167) Cytochrome P450 monooxygenase lnaD Proteins 0.000 description 1
- 108010067661 Caffeate O-methyltransferase Proteins 0.000 description 1
- 108010074879 Cinnamoyl-CoA reductase Proteins 0.000 description 1
- 241000218631 Coniferophyta Species 0.000 description 1
- 101710088194 Dehydrogenase Proteins 0.000 description 1
- PXIKRTCSSLJURC-UHFFFAOYSA-N Dihydroeugenol Chemical group CCCC1=CC=C(O)C(OC)=C1 PXIKRTCSSLJURC-UHFFFAOYSA-N 0.000 description 1
- COLNVLDHVKWLRT-QMMMGPOBSA-N L-phenylalanine Chemical compound OC(=O)[C@@H](N)CC1=CC=CC=C1 COLNVLDHVKWLRT-QMMMGPOBSA-N 0.000 description 1
- OUYCCCASQSFEME-QMMMGPOBSA-N L-tyrosine Chemical compound OC(=O)[C@@H](N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-QMMMGPOBSA-N 0.000 description 1
- 241000209510 Liliopsida Species 0.000 description 1
- 108090000856 Lyases Proteins 0.000 description 1
- 102000004317 Lyases Human genes 0.000 description 1
- 108090000854 Oxidoreductases Proteins 0.000 description 1
- 102000004316 Oxidoreductases Human genes 0.000 description 1
- 238000012408 PCR amplification Methods 0.000 description 1
- 102000003992 Peroxidases Human genes 0.000 description 1
- 108700023158 Phenylalanine ammonia-lyases Proteins 0.000 description 1
- 235000008331 Pinus X rigitaeda Nutrition 0.000 description 1
- 241000018646 Pinus brutia Species 0.000 description 1
- 235000011613 Pinus brutia Nutrition 0.000 description 1
- ATUOYWHBWRKTHZ-UHFFFAOYSA-N Propane Chemical group CCC ATUOYWHBWRKTHZ-UHFFFAOYSA-N 0.000 description 1
- 108020004518 RNA Probes Proteins 0.000 description 1
- 239000003391 RNA probe Substances 0.000 description 1
- 229920002684 Sepharose Polymers 0.000 description 1
- 241000775848 Syringa oblata Species 0.000 description 1
- 241000219793 Trifolium Species 0.000 description 1
- 240000008042 Zea mays Species 0.000 description 1
- 235000005824 Zea mays ssp. parviglumis Nutrition 0.000 description 1
- 235000002017 Zea mays subsp mays Nutrition 0.000 description 1
- 230000005856 abnormality Effects 0.000 description 1
- 230000004913 activation Effects 0.000 description 1
- 238000012231 antisense RNA technique Methods 0.000 description 1
- 238000013459 approach Methods 0.000 description 1
- 238000010936 aqueous wash Methods 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000008827 biological function Effects 0.000 description 1
- 230000003570 biosynthesizing effect Effects 0.000 description 1
- UCKZMPLVLCKKMO-LHLIQPBNSA-N cephamycin Chemical compound S1CC(C)=C(C(O)=O)N2C(=O)[C@@H](C)[C@]21OC UCKZMPLVLCKKMO-LHLIQPBNSA-N 0.000 description 1
- NQBWNECTZUOWID-QSYVVUFSSA-N cinnamyl cinnamate Chemical compound C=1C=CC=CC=1\C=C/C(=O)OC\C=C\C1=CC=CC=C1 NQBWNECTZUOWID-QSYVVUFSSA-N 0.000 description 1
- 230000004087 circulation Effects 0.000 description 1
- 229940119526 coniferyl alcohol Drugs 0.000 description 1
- 238000011109 contamination Methods 0.000 description 1
- 235000005822 corn Nutrition 0.000 description 1
- WBCMGDNFDRNGGZ-ACNVUDSMSA-N coumarate Natural products COC(=O)C1=CO[C@H](O[C@H]2O[C@H](CO)[C@@H](O)[C@H](O)[C@H]2O)[C@H]3[C@@H]1C=C[C@]34OC(=O)C(=C4)[C@H](C)OC(=O)C=Cc5ccc(O)cc5 WBCMGDNFDRNGGZ-ACNVUDSMSA-N 0.000 description 1
- 230000001186 cumulative effect Effects 0.000 description 1
- 230000009615 deamination Effects 0.000 description 1
- 238000006481 deamination reaction Methods 0.000 description 1
- 238000006356 dehydrogenation reaction Methods 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 230000018109 developmental process Effects 0.000 description 1
- 238000005265 energy consumption Methods 0.000 description 1
- 238000003912 environmental pollution Methods 0.000 description 1
- 241001233957 eudicotyledons Species 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 238000013467 fragmentation Methods 0.000 description 1
- 238000006062 fragmentation reaction Methods 0.000 description 1
- 239000011521 glass Substances 0.000 description 1
- 230000012010 growth Effects 0.000 description 1
- 230000033444 hydroxylation Effects 0.000 description 1
- 238000005805 hydroxylation reaction Methods 0.000 description 1
- 125000004464 hydroxyphenyl group Chemical group 0.000 description 1
- 230000008676 import Effects 0.000 description 1
- 230000002779 inactivation Effects 0.000 description 1
- 238000002347 injection Methods 0.000 description 1
- 239000007924 injection Substances 0.000 description 1
- 230000009545 invasion Effects 0.000 description 1
- 230000002015 leaf growth Effects 0.000 description 1
- 244000144972 livestock Species 0.000 description 1
- 238000010841 mRNA extraction Methods 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 239000011259 mixed solution Substances 0.000 description 1
- 239000013642 negative control Substances 0.000 description 1
- 238000003199 nucleic acid amplification method Methods 0.000 description 1
- 238000011330 nucleic acid test Methods 0.000 description 1
- 239000002773 nucleotide Substances 0.000 description 1
- 125000003729 nucleotide group Chemical group 0.000 description 1
- 230000010355 oscillation Effects 0.000 description 1
- 230000037361 pathway Effects 0.000 description 1
- 108040007629 peroxidase activity proteins Proteins 0.000 description 1
- 230000002085 persistent effect Effects 0.000 description 1
- 229930015704 phenylpropanoid Natural products 0.000 description 1
- 150000002995 phenylpropanoid derivatives Chemical class 0.000 description 1
- 230000008635 plant growth Effects 0.000 description 1
- 229920000642 polymer Polymers 0.000 description 1
- 239000013641 positive control Substances 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
- 238000009790 rate-determining step (RDS) Methods 0.000 description 1
- 238000004064 recycling Methods 0.000 description 1
- 230000008929 regeneration Effects 0.000 description 1
- 238000011069 regeneration method Methods 0.000 description 1
- 238000010839 reverse transcription Methods 0.000 description 1
- 230000002786 root growth Effects 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 239000000243 solution Substances 0.000 description 1
- 230000005082 stem growth Effects 0.000 description 1
- NGSWKAQJJWESNS-ZZXKWVIFSA-N trans-4-coumaric acid Chemical compound OC(=O)\C=C\C1=CC=C(O)C=C1 NGSWKAQJJWESNS-ZZXKWVIFSA-N 0.000 description 1
- 230000002103 transcriptional effect Effects 0.000 description 1
- OUYCCCASQSFEME-UHFFFAOYSA-N tyrosine Natural products OC(=O)C(N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-UHFFFAOYSA-N 0.000 description 1
Images
Landscapes
- Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
Abstract
The present invention discloses a method for adjusting and controlling the lignin in Chinese white poplars. The present invention comprises the following steps: cloning key enzyme genes from a Chinese white poplar by a biosynthetic pathway; constructing the expression vectors of the antisense genes of the key enzyme genes and connecting a promoter; through agrobacterium-mediated transformation, transforming functional genes into an explant of a Chinese white poplar; then, sieving a transgenic strain which expresses antisense genes with high efficiency and has reduced lignin content. The key enzyme genes are caffeic acid methyltransferase genes, coffee acylcoenzymea A methyltransferase genes or 4-coumaric acid coenzyme A ligase genes. The present invention utilizes a genetic engineering technique to inhibit the lignin biosynthesis of a Chinese white poplar which is a wood species for making paper and reduces the lignin content or change the constituents of the Chinese white poplar so as to provide a raw material wood species which has the advantages of low cost and little pollution and is suitable for making paper with paper pulping. The present invention has important theoretical and practical significance for strongly developing paper-making industry with lumber and paper pulping in China with the compromise of cost reduction and pollution prevention.
Description
Technical field
The present invention relates to utilize transgenic engineering to regulate biosynthetic method in the plant, especially utilize the method for xylogen in the transgenic engineering regulation and control Cortex Populi Tomentosae.
Background technology
Xylogen is that content is only second to cellulosic a kind of larger molecular organics matter in the plant, and the xylogen of terrestrial plant is synthetic to be one of important evolution feature that adapts to terrestrial environment, has important biological function.Xylogen is filled in the Mierocrystalline cellulose framework, can strengthen the physical strength of plant materials, helps organizing the invasion and attack of carrying out the moisture transportation and resisting extraneous severe environment.Yet the key link of pulping and paper-making is with a large amount of chemical the xylogen in the raw material to be separated with Mierocrystalline cellulose, and Mierocrystalline cellulose is used for papermaking, and the xylogen after separating etc. become the main waste of paper industry, and environment is caused severe contamination.And, drop into delignified chemical products and recycling waste liquid, need to consume a large amount of energy, increase the papermaking cost.In addition, because the xylogen in the forage grass also influences livestock digestion and absorbs, the height of content of lignin also is one of index of forage grass quality.Therefore, reduce content of lignin or change its component, can utilize the plant resources of occurring in nature better.
Xylogen is complicated phenylpropyl alcohol alkane monomer-polymer, three kinds of principal monomers is arranged: tonquinol (coumarylalcohol), lubanol (coniferyl alcohol) and sinapyl alcohol (sinapyl alcohol).According to the monomer difference, xylogen can be divided into three types: syringyl lignin (the syringyl lignin that is formed by Syringa oblata Lindl. base propane structure monomer polymerization, the S-xylogen), guaiacyl xylogen (the guaiacyl lignin that forms by guaiacyl propane structure monomer polymerization, the G-xylogen) with by right-right-hydroxy phenyl xylogen (hydroxy-phenyl lignin, H-xylogen) that hydroxy phenyl propane structure monomer polymerization forms.
The content of xylogen is between 15-36% in the plant.Mainly contain guaiacyl xylogen (G) in the gymnosperm, dicotyledons mainly contains guaiacyl syringyl lignin (G-S), and monocotyledons then mainly contains guaiacyl-Syringa oblata Lindl. base-right-hydroxy phenyl xylogen (G-S-H).The big diversity of lignin monomer tool, the about kind more than 20 of the chemical bond that connects between monomer.Also in any plant, do not find the enzyme of degradable xylogen at present.
The synthetic of lignin monomer undertaken by phenylpropionic acid approach (phenylpropanoid pathway), it is initial to form styracin by phenylalanine (or tyrosine) deamination, through a series of hydroxylations, methylate and reduction reaction, finally generate three kinds of principal monomers.The polymerization of lignin monomer, phenylpropyl alcohol alkyl structure unit aggregates into the xylogen macromole and has proved dehydrogenation polymerization.
Mutant [Barriere etc., 1994 that content of lignin and component change in several plants such as corn, pine tree, have been found; Mackay etc., 1997], studies show that be because coffic acid-3-O-methyltransgerase (COMT, caffeicacid-3-O-methyltransferase) or cinnyl ethanol dehydrogenase (CAD, cinnamoyl alcoholdehydrogenase) inactivation [Pravan, 1997] of causing.Being found to be of mutant utilizes modern biotechnology to reduce that content of lignin provides thinking in the plant,, by suppressing the native gene of plant lignin's monomer synthetic enzyme, reaches the purpose that reduces the transgenic plant content of lignin that is.The gene of a plurality of enzymes in the lignin monomer biosynthetic process is cloned from different plants in succession, for the research that utilizes genetic engineering regulation plant lignin is laid a good foundation.
The initial COMT of discovery can cause that with the CAD transgenation content of lignin descends, so COMT and CAD become focus [Tsai, 1998] in utilizing the biosynthetic research of genetically engineered adjusting plant lignin.Atanassva (1995) thinks the COMT activity inhibited up to 80-90%, could the component of xylogen be exerted an influence.Jouanin etc. (2000) just find the content of lignin reduction in the COMT activity is almost all suppressed the transgenic poplar of (activity residual less than 3%).This shows that the degree of COMT activity inhibited influences the content and the component of xylogen in the transgenic plant.
The content of lignin of antisense CAD transgene tobacco, clover and willow does not obviously reduce [Baucher, 1996 yet; Baucher, 1999; Higuchi, 1994, Hibino, 1995], special component such as coniferyl aldehyde (coniferylaldehyde) etc. are significantly increased.Halpin etc. (1994) make the CAD activity reduce to 7% of wild-type in transgene tobacco, but content of lignin does not reduce yet.Therefore, in the biosynthetic process of xylogen, CAD is not a rate-limiting step, the active needs that just can satisfy the phytosynthesis xylogen of extremely low endogenous CAD.Yet, in the repressed transgenic poplar of CAD, special red xylogen is separated easily, the slurrying experiment shows that the kappa value reduces, though illustrate and suppress the content that CAD does not reduce xylogen, but change the composition structure of xylogen, helped delignified pulping chemistry technology [Baucher, 1996].Pilate etc. (2002) detect the transgenic poplar in 4 years of growth, and the result shows that also active reduction of CAD only can make the faint reduction of content of lignin, but changes lignin component, helps pulping and paper-making, therefore, also obviously improves the pulping and paper-making performance of transgenic poplar.
Suppress phenylalanine lyase (PAL, Phenylalanine ammonialyase) [Bate, 1994; Sewalt, 1997; Elkind, 1990], styracin-4-hydroxylase (C4H, cinnamate 4-hydroxylase) [Sewalt, 1997] and cinnhyl CoA reductase (CCR, cinnamoyl-CoA reductase) [Piquemal, 1998; Ralph, 1998] transgenic plant xylogen descends, and follows the improper growth of plant simultaneously, thereby is difficult to practical application.The expression of inhibition or enhancing forulic acid-5-hydroxylase (F5H, ferulate 5-hydroxylase) only influences component [Franke, 2000 of xylogen; Rugger, 1999].Ipelcl etc. suppress the expression of a kind of peroxidase of willow, content of lignin decline 10-20%[Ipelcl, 1999], this is unique inhibition lignin monomer polymerization and reduce the report of content of lignin.
In recent years, utilize the 4-coumaric acid: CoA ligase enzyme (4CL, coumarate:CoA ligase) obtained gratifying result with the research of caffeoyl CoA3-O-methyltransgerase (CCoAOMT, caffeoyl-CoA 3-O-methyltransferase) generegulation plant lignin synthetic.Kajita etc. (1996) obtain 4CL and express repressed transgene tobacco, content of lignin decline 19-35%, and phenylacrolein and S-component reduce, and transgene tobacco and wild-type do not have difference [Kajita, 1996,1997] on form.Hu etc. (1999) suppress quaking aspen 4CL genetic expression with Antisense RNA Technique, and content of lignin descends 45% in the active transgenic line that descends 90% or more, and root, stem and leaf growth enhancing, and content of cellulose has also increased by 15%.Transgenosis quaking aspen Mierocrystalline cellulose is compared with wild-type with the total content of xylogen and is remained unchanged, and shows xylogen and cellulosic synthetic can the compensation mutually in the plant.
Zhong etc. (1998) obtain the transgene tobacco that antisense CCoAOMT expresses, Klason content of lignin decline 36-47%.The amplitude that G-xylogen ratio-S xylogen reduces is big, and S/G ratio increases.Meyermans etc. (2000) find that in the transgenic poplar that suppresses the CCoAOMT expression content of lignin reduces by 12%, and S/G ratio increases by 11%.Zhong etc. (2000) change antisense CCoAOMT over to willow, find that also content of lignin obviously descends, and short texture are beneficial to delignification.
Pulping and paper-making is one of important industry of China's economic, also is the rich and influential family of environmental pollution and energy consumption.Cortex Populi Tomentosae is one of main paper making raw material, utilize genetic engineering technique to suppress the xylogen biosynthesizing of papermaking seeds, reduce content of lignin or change its component, produce the good raw material seeds that are suitable for pulping and paper-making low-cost, that pollution is few, greatly develop pulping wood papermaking for China, reduce cost to take into account and prevent and remedy pollution, have important theory and realistic meaning.
The innovation and creation content
The purpose of this invention is to provide a kind of method of regulating and control xylogen in the Cortex Populi Tomentosae.
The present invention seeks to be achieved through the following technical solutions: a kind of method of regulating and control xylogen in the Cortex Populi Tomentosae, it is a kind of key gene in clone's xylogen biosynthetic pathway from Cortex Populi Tomentosae (Populus tomentosa), make up the expression vector of its inverted defined gene, the type that connects to form is expressed promotor, in being total to culture medium, pass through agriculture bacillus mediated, inverted defined gene is changed in the Cortex Populi Tomentosae explant of cultivating 1-2 days on pre-training substratum in advance, after the differentiation screening and the screening of taking root, transplant, screening was transplanted more than 1 year, efficiently expressed inverted defined gene, content of lignin descends, the normal transgenic line of growing; Described key gene is caffeoyl coenzyme A methyltransgerase (CCoAOMT) gene or 4-coumaric acid CoA ligase (4CL) gene; The prescription of described pre-training substratum is: 1/2MS+6-BA0.25mg/L+ZT0.25mg/L+IBA0.25mg/L+ sucrose 20g/L; The described substratum of training altogether is to cover filter paper on pre-training substratum; The prescription of described differentiation screening culture medium is: 1/2MS+6-BA0.25mg/L+ZT0.25mg/L+IBA0.125mg/L+ kantlex 50mg/L+ sucrose 20g/L; The prescription of the described screening culture medium of taking root is: 1/2MS+IBA0.25mg/L+VB110mg/L+ kantlex 50mg/L+ sucrose 15g/L.
Wherein, the encoding gene of described key enzyme is:
1) caffeoyl coenzyme A methyltransgerase (CCoAOMT, caffeoyl Co-enzyme A 3-O-methyltransferase), its encoding gene is SEQ ID № in the sequence table: 1;
2) 4-coumaric acid CoA ligase (4CL, 4-coumarate Co-enzyme A ligase), its encoding gene is SEQ ID № in the sequence table: 2;
The encoding gene cDNA of above-mentioned two kinds of key enzymes, all registration (AF240466, AF314180) in GenBank.
The present invention adopts composition type expression promoter, as the CaMV35S promotor.This promoter expression efficient height can suppress the expression of target gene comparatively efficiently, reaches the purpose that reduces the transgenic plant content of lignin.
The present invention is an explant with blade, stem apex or the statoblast of setation white poplar then, sets up the Cortex Populi Tomentosae regeneration system rapidly.By agriculture bacillus mediated, functional gene is changed in the plant materials over to the transgenic line of screening content of lignin decline 10-20%.
Breed the Cortex Populi Tomentosae strain system that content of lignin descends in batches, after becoming a useful person, just can be used for pulping and paper-making.
The process of the inventive method as shown in Figure 1.
Cortex Populi Tomentosae is the distinctive seeds of China, also is important papermaking resource plant.The present invention is creatively with the key gene in the xylogen biosynthetic pathway--and the cDNA of CCoAOMT or 4CL gene oppositely imports in the Cortex Populi Tomentosae, content of lignin to transfer-gen plant is measured, the content of lignin of transgenic line all has decline in various degree as a result, most reach 37%, and its no abnormality seen that grows.Therefore, utilize the resulting transgenosis Cortex Populi Tomentosae of the inventive method to contain the xylogen of low levels, and change the composition of xylogen, can improve the performance of papermaking resource plant, help pulping and paper-making, for the development of China's paper-making industry with prevent and treat industrial pollution caused by paper manufacturing, have important theory and practical significance.
Description of drawings
Fig. 1 is a schema of the present invention.
Fig. 2 is the building process of antisense expression vector APCOA
Fig. 3 is the building process of antisense expression vector AP4CL
Fig. 4 is the PCR detected result of antisense CCoAOMT transgenosis Cortex Populi Tomentosae
Fig. 5 is the Southern results of hybridization of transgenosis Cortex Populi Tomentosae
Fig. 6 is the Northern detected result that antisense CCoAOMTcDNA expresses in the transgenosis Cortex Populi Tomentosae
Fig. 7 is the Western results of hybridization
Fig. 8 analyzes histogram for the Klason content of lignin
Fig. 9 is that transgenosis Cortex Populi Tomentosae and wild-type content of lignin compare histogram
Figure 10 is the PCR detected result of antisense 4CL transgenosis Cortex Populi Tomentosae
Figure 11 is the Southern results of hybridization of transgenosis Cortex Populi Tomentosae
Figure 12 is the Western results of hybridization
Figure 13 analyzes histogram for the Klason content of lignin
Embodiment
The clone of embodiment 1, CCoAOMT and 4CL gene, the structure of antisense expression vector.
Adopt the RT-PCR method, the cDNA of separation of C CoAOMT and 4CL gene from Cortex Populi Tomentosae.The degenerated primer sequence of RT-PCR is as follows:
CCoAOMT:5 ' primer 5 '-CAN AGN CAN GCN GGN AGN CAC C-3 '
3 ' primer: 5 '-GGT CCC TCA NTG NAT NCG A-3 '
4CL:5 ' primer 5 '-CGC CAC AAT GAA NCC NCA NG-3 '
3 ' primer: 5 '-GTT ANA TNC CNG GNA ANT TNT CNT TNA G-3 '
Extract total RNA with TRIzol (GIBCOBRL), with PolyAT tract mRNA Isolation System III (promega) purified mRNA.MRNA with purifying is a template, and polyT (12-18) carries out reverse transcription for primer.With the cDNA that obtains is template, carries out pcr amplification with above-mentioned degenerated primer.Amplification program: 94 ℃ of 3min of sex change meet 94 ℃ of 1min of 30 circulations, 57 ℃ of 1min, 72 ℃ of 1.5min, total elongation 7min.Reclaim specific amplification products with purifying from sepharose, CCoAOMT is cloned on the PMD-18-T vector (TAKARA), and 4CL is cloned on the T-easy vector (Promega).
CCoAOMT antisense expression vector building process downcuts the CCoAOMTcDNA fragment with Hinc II (flush end) and Xba I as shown in Figure 2 from PMDCOA, with EcoICRI (flush end) and the GUS expressed sequence excision of Xba I with binary expression vector pBI121.CCoAOMT cDNA fragment is inserted among the pBI121 that has excised the GUS expressed sequence, makes CCoAOMT cDNA be reversely connected to the downstream of CaMV 35S promoter, be built into the antisense expression vector of CCoAOMT, called after APCOA.
4CL antisense expression vector building process as shown in Figure 3,4CL cDNA is downcut from T-4CL and the purifying recovery with Spe I and Sac II, with Spe I and Sac II the SK plasmid is cut simultaneously, purifying reclaims the back and is connected with above-mentioned recovery fragment, is built into shuttle plasmid (shuttle plamid) pSK-4CL.With Xba I and Sac I GUS encoding sequence excision with binary expression vector pBI121.With Spe I pSK-4CL is cut simultaneously, purifying is used partially digested this segment of Sac I after reclaiming again, and purifying reclaims the dna fragmentation that comprises complete 4CL cDNA.It is connected with the pBI121 that has excised the GUS encoding sequence, is built into the 4CL antisense expression vector, called after AP4CL.
Substratum is as follows:
Pre-training substratum: 1/2MS+6-BA0.25mg/L+ZT0.25mg/L+IBA0.25mg/L sucrose 20g/L
Train substratum altogether: train in advance and cover filter paper on the substratum
Differentiation screening culture medium: 1/2MS+6-BA0.25mg/L+ZT0.25mg/L+IBA0.125mg/L+ microbiotic sucrose 20g/L
The screening culture medium of taking root: 1/2MS+IBA0.25mg/L+VB110mg/L+ microbiotic sucrose 15g/L
1, Agrobacterium activation: choose single bacterium colony shaken overnight in YEB (Rif 100mg/l, Str 50mg/l, Kan 50mg/l) liquid nutrient medium and cultivate.Get 1mL bacterium liquid and be added to fresh YEB liquid nutrient medium (containing the 500uM Syringylethanone) relaying persistent oscillation cultivation 3-5 hour, when OD600 is about 0.4, can be used for transforming.
2, get then that healthy and strong aseptic seedling blade or the stem section of Cortex Populi Tomentosae are cut into 0.5-1cm, train pre-the cultivation 1-2 days on the substratum in advance.In activatory bacterium liquid, infected 10-20 minute then.Take out explant, after blotting with aseptic filter paper, change in the common training substratum that is covered with one deck aseptic filter paper, in sterilized water, give a baby a bath on the third day after its birth time in 25 ℃ of dark cultivations 2-3 days, at the aqueous wash medium of the cephamycin of 250mg/L once, put into and contain kantlex 50mg/L screening culture medium and cultivate.
3, wait the budlet of 1.5-2.0cm to occur after, the clip budlet places and contains 50mg/L screening root media and cultivate.
The mensuration of embodiment 3, the Cortex Populi Tomentosae plant detection of antisense CCoAOMT transgenosis and analysis and transfer-gen plant content of lignin
Total genomic dna with extraction in a small amount is a template, and the PCR that carries out the transgenosis Cortex Populi Tomentosae detects.For fear of the interference of native gene, one of PCR primer is a 5 ' primer of target gene, and another primer is one section nucleotide sequence corresponding to the CaMV 35S promoter.The result as shown in Figure 4, swimming lane 1 is DNA marker among the figure, swimming lane 2 is a wild-type, swimming lane 3 is the transgenosis Cortex Populi Tomentosae for plasmid APCOA (positive control) swimming lane 4-9, the result among the figure shows that tentatively target gene is integrated in the transfer-gen plant.Fig. 5 is Southern hybridization detected result, and swimming lane 1 is XbaI+EcoRI digested plasmid APCOA among the figure; Swimming lane 2 is; Swimming lane 3 is the XbaI+EcoRI double digestion genomic dna of wild-type; Swimming lane 4 is the XbaI enzyme cutting genomic dna of transgenosis poplar; Swimming lane 5 is the XbaI enzyme cutting genomic dna of wild-type, and the result shows that antisense CCoAOMT is incorporated in the genome.Fig. 6 is Northern dot blot result, and the upper left corner is wild-type Cortex Populi Tomentosae negative control among the figure; Other 3 is the transgenosis poplar.Use corresponding to the RNA hybridization total with it of the single stranded RNA probe of CCoAOMT antisense strand, the result shows that antisense CCoAOMT expresses at transcriptional level.Fig. 7 is the Western analytical results, and left swimming lane is the wild-type contrast among the figure, and all the other are the transgenosis Cortex Populi Tomentosae, shows that CCoAOMT expression amount comparison illumination shows minimizing in the transfer-gen plant.
Fig. 8 is for transplanting the Klason content of lignin measurement result of the transgenosis Cortex Populi Tomentosae in 2 years, 1-transgenosis poplar among the figure; The contrast of 2-wild-type shows that transgenosis Cortex Populi Tomentosae content of lignin descends 10% than wild-type.
The Klason content of lignin is analyzed: cut the bottom stem of transgenosis Cortex Populi Tomentosae with the contrast willow of transplanting, grind to form uniform powder after the oven dry, (40-60um) sieves.After the methyl alcohol extracting, oven dry.Get about 200mg sample, 72%H
2SO
430 ℃ of extractings 1 hour add 112ml H
2O boiled 1 hour, noted keeping cumulative volume constant.This mixed solution filters through raw glass sieve (40-60um), and uses the 500ml hot water injection, weighs after the oven dry.Content of lignin accounts for the percentage ratio calculating of primary sample weight with the xylogen of last acquisition, the result finds as shown in Figure 9, Cortex Populi Tomentosae strain system (YCOA38) content of lignin descends 17% than the wild-type contrast, and 1 is wild-type among the figure, and 2 is that the strain of transgenosis Cortex Populi Tomentosae is YCOA38.
The mensuration of embodiment 4, the Cortex Populi Tomentosae plant detection of antisense 4CL transgenosis and analysis and transfer-gen plant content of lignin
Detection method is with embodiment 3, and Figure 10 detects for PCR, and a left side 1 is molecular weight marker among the figure, a left side 2 positive contrasts, and remaining is the transgenosis Cortex Populi Tomentosae, shows that tentatively target gene has been integrated in the transfer-gen plant; Figure 11 is Southern hybridization analysis result, and right 1 is digested plasmid among the figure, and right 2 are the wild-type contrast, and all the other are different transfer-gen plants, show that antisense 4CL is incorporated in the genome; Figure 12 is Western hybridization analysis result, and right 1 is the wild-type contrast among the figure, and all the other are different transfer-gen plants, shows apparent minimizing of CCoAOMT expression amount comparison illumination in the transfer-gen plant.
Figure 13 analyzes for the antisense 4CL transgenosis Cortex Populi Tomentosae plant Klason xylogen of transplanting 1 year, and 1 is the wild-type contrast among the figure, and 2 is the transgenosis Cortex Populi Tomentosae, and the result shows that transgenosis Cortex Populi Tomentosae content of lignin descends 26.25%.
Sequence table
<160>1
<210>1
<211>726
<212>DNA
＜213〉Populus Cortex Populi Tomentosae (Populus tomentosa)
<400>2
caaagccagg caggaaggca ccaggaagtt ggccacaaga gccttttgca aagtgatgct 60
ctttaccagt atattcttga gactagtgtg tatccaagag agcctgaatg catgaaggag 120
ctcagggagg tgactgccaa gcatccttgg aacatcatga ccacatctgc tgatgaaggg 180
caattcttga atatgctttt gaagcttgtc aatgccaaga acaccatgga gatcggtgtt 240
tacactggct attctctctt ggccaccgcc ctggctatcc ctgaggatgg caagatcttg 300
gctatggaca tcaacagaga aaactatgaa ttgggtctcc cagtaattca gaaagctggt 360
gttgcgcaca agattgattt caaggaaggc cctgctctac cagttcttga tcaaatgatt 420
gaagatggga agtaccatgg aagttttgat ttcatctttg tggatgctga caaggacaat 480
tatataaatt atcacaagag gttgattgag cttgttaaag ttggtggact gattgggtac 540
gacagcactc tatggaatgg atctgtggtg gcaccacctg atgctccgat gaggaagtat 600
gtgaggtact accgggactt tgttttggag ctcaacaagg cacttgctgc tgaccccagg 660
attgaaattt gcatgcttcc tgttggtgat ggcatcactc tctgccgtcg gatccaatga 720
gggacc 726
<210>2
<211>1619
<212>DNA
＜213〉Populus Cortex Populi Tomentosae (Populus tomentosa)
<400>3
cgccacaatg aatccacaag aagaattcat ctttcgctca aaattaccag acatctacat 60
cccgaaaaac cttcccctgc attcatacgt tcttgaaaac ttgtctaaac attcatcaaa 120
accttgcctg ataaatggcg cgaatggaga tgtctacacc tgtgctgacg ttgagctcac 180
agcaagaaga gttgcttctg gtctcaacaa gattggtatt caacaaggtg acgtgatcat 240
gctcttccta ccaagttcac ctgaattcgt gcttgctttc ctaggcgctt cacacagagg 300
tgccatcacc actgctgcca atcctttctc cacccctgca gagctagcga aacatgccaa 360
ggcctcgaga gcaaagcttc tgataacaca ggcttgttac tacgagaagg ttaaagattt 420
tgccagagaa agtgatgtta aggtcatgtg cgtggactct gccccggatg ggtgcttgca 480
cttctcagag ctaacacagg cagacgaaaa tgaagcgcct caggtcgaca ttagtcctga 540
tgatgtcgtg gcattgcctt attcatcagg gactacaggg ttgccaaaag gggtcatgtt 600
aacgcacaaa gggctaataa ccagtgtggc tcaacaggta gatggagaca atcctaacct 660
gtattttcac agtgaagatg tgattctgtg tgtgctccct atgttccata tctatgctct 720
gaattcaata atgctgtgtg ggctgagagt cggtgcctcg attttgataa tgccaaagtt 780
tgagattggt actttgctgg gattgattga gaagtacaag gtatctatag caccggttgt 840
tccacctgtg atgttggcaa ttgctaggtc acctgatctt gacaagcatg acttgtcttc 900
tctgaggatg ataaaatctg gaggggctcc attgggcaag gaacttgaag atactgtcag 960
agctaagttt cctcaggcta gacttggtca gggatatgga atgaccgagg caggacctgt 1020
tctagcaatg tgcttggcat ttgccaagga accattcgac ataaaaccag gtgcatgtgg 1080
gactgtagtc aggaatgcag agatgaagat tgttgaccca gaaacagggg cctctctacc 1140
gaggaaccag cctggtgaga tctgcatccg gggtgatcag atcatgaaag gatatcttaa 1200
tgaccctgag gcaacctcaa gaacaataga caaagaagga tggttgcaca caggcgatat 1260
cggctacatt gatgatgatg atgagctttt catcgttgac aggttgaagg aattgatcaa 1320
gtataaaggg tttcaggttg ctcctgctga actcgaagct ttgttaatag cccatccaga 1380
gatatccgat gctgctgtag taggaatgaa agatgaggat gcaggagaag ttcctgttgc 1440
atttgctgtg aaatcagaaa agtctcaggc caccgaagat gaaattaagc agtatatttc 1500
aaaacaggtg atattctaca agagaataaa acgagttttc ttcattgaag ctattcccaa 1560
ggcaccatct ggcaagatcc tgaggaagaa tctgaaagag aagttgccag gcatataac 1619
Claims (5)
1. method of regulating and control xylogen in the Cortex Populi Tomentosae, it is a kind of key gene in clone's xylogen biosynthetic pathway from Cortex Populi Tomentosae, make up the expression vector of its inverted defined gene, the type that connects to form is expressed promotor, in being total to culture medium, pass through agriculture bacillus mediated, inverted defined gene is changed in the Cortex Populi Tomentosae explant of cultivating 1-2 days on pre-training substratum in advance, transplant through the differentiation screening and the screening back of taking root, screening was transplanted 1 year, 2 years or 2-3, efficiently express inverted defined gene, content of lignin decline, the normal transgenic line of growing; Described key gene is caffeoyl coenzyme A methyl transferase gene or 4-coumaric acid CoA ligase gene; The prescription of described pre-training substratum is: the IBA of ZT, the 0.25mg/L of 6-BA, the 0.25mg/L of adding 0.25mg/L and the sucrose of 20g/L in the 1/2MS substratum; The described substratum of training altogether is to cover filter paper on pre-training substratum; The prescription of described differentiation screening culture medium is: the kantlex of IBA, the 50mg/L of ZT, the 0.125mg/L of 6-BA, the 0.25mg/L of adding 0.25mg/L and the sucrose of 20g/L in the 1/2MS substratum; The prescription of the described screening culture medium of taking root is: the kantlex of VB, the 50mg/L of IBA, the 110mg/L of adding 0.25mg/L and the sucrose of 15g/L in the 1/2MS substratum.
2. method according to claim 1 is characterized in that: the base sequence of described caffeoyl coenzyme A methyl transferase gene is shown in SEQ ID NO:1.
3. method according to claim 1 is characterized in that: the base sequence of described 4-coumaric acid CoA ligase gene is shown in SEQ ID NO:2.
4. method according to claim 1 is characterized in that: described explant is blade, stem apex or the statoblast of setation white poplar then.
5. the application of the described method of claim 1 in improvement papermaking resource plant.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB2003101017995A CN100415882C (en) | 2003-10-28 | 2003-10-28 | Method for regulating lignin in populus tomentosa |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB2003101017995A CN100415882C (en) | 2003-10-28 | 2003-10-28 | Method for regulating lignin in populus tomentosa |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN1611602A CN1611602A (en) | 2005-05-04 |
| CN100415882C true CN100415882C (en) | 2008-09-03 |
Family
ID=34756254
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CNB2003101017995A Expired - Fee Related CN100415882C (en) | 2003-10-28 | 2003-10-28 | Method for regulating lignin in populus tomentosa |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN100415882C (en) |
Families Citing this family (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2009000046A (en) * | 2007-06-21 | 2009-01-08 | Hitachi Zosen Corp | Gene encoding enzyme involved in mevalonic acid pathway of eucommia ulmoides oliver |
| CN102115759A (en) * | 2009-08-21 | 2011-07-06 | 纳迦竺那能源私人有限公司 | Transgenic sweet sorghum with altered lignin composition and process of preparation thereof |
| CN102465135A (en) * | 2010-11-05 | 2012-05-23 | 中国中医科学院中药研究所 | Scutellaria baicalensis Georgi caffeoyl-CoA-3-O-methyltransferase (SbCCOMT2) gene, and coding product and application thereof |
| CN109694402B (en) * | 2017-10-24 | 2021-05-11 | 中国农业科学院作物科学研究所 | Plant lignin synthesis related protein and coding gene and application thereof |
| CN108753816A (en) * | 2018-06-20 | 2018-11-06 | 闫秋洁 | The transgenic arabidopsis strain and its construction method of SYTA gene overexpressions |
| CN111621520A (en) * | 2020-06-22 | 2020-09-04 | 南京林业大学 | Application of masson pine PmCOMT gene |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN87101264A (en) * | 1987-12-26 | 1988-07-27 | 武汉市纺织科学研究所 | The efficient chemical degumming of hemp method of removing lignin |
-
2003
- 2003-10-28 CN CNB2003101017995A patent/CN100415882C/en not_active Expired - Fee Related
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN87101264A (en) * | 1987-12-26 | 1988-07-27 | 武汉市纺织科学研究所 | The efficient chemical degumming of hemp method of removing lignin |
Non-Patent Citations (1)
| Title |
|---|
| 毛白杨CCoAOMT cDNA片段的克隆与转基因杨木质素含量的调控. 魏建华，赵华燕，张景昱，刘惠荣，宋艳茹.植物学报，第43卷第11期. 2001 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN1611602A (en) | 2005-05-04 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Poovaiah et al. | Altered lignin biosynthesis using biotechnology to improve lignocellulosic biofuel feedstocks | |
| US7514596B2 (en) | Methods for simultaneous control of lignin content and composition, and cellulose content in plants | |
| Pichon et al. | Cloning and characterization of two maize cDNAs encoding cinnamoyl-CoA reductase (CCR) and differential expression of the corresponding genes | |
| CN101258246B (en) | Plant yield improvement by ste20-like gene expression | |
| RU2326527C2 (en) | Potato with increase starch in terms of each plant and method of its cultivation | |
| Selman-Housein et al. | Molecular cloning of cDNAs coding for three sugarcane enzymes involved in lignification | |
| US20060260011A1 (en) | Methods and genetic constructs for modification of lignin composition of corn cobs | |
| CN102264906A (en) | Vegetabile material, plants and a method of producing a plant having altered lignin properties | |
| CN101460611A (en) | Plants having improved growth characteristics and method for making the same | |
| US20080213871A1 (en) | Altering regulation of maize lignin biosynthesis enzymes via RNAi technology | |
| CN100415882C (en) | Method for regulating lignin in populus tomentosa | |
| CN108130334A (en) | Switchgrass S-adenosylmethionine synthase gene SAMS1 regulates and controls the application of lignin synthesis | |
| CN108588098A (en) | Eucalyptus urophylla CAD genes and its application | |
| US9738901B2 (en) | Regulation of galactan synthase expression to modify galactan content in plants | |
| US9238818B2 (en) | Methods and genetic constructs for modification of lignin composition of corn cobs | |
| CA2856244C (en) | Use of fructokinases and sucrose synthases for increasing cell wall polymers | |
| Jia et al. | Obtaining the transgenic poplars with low lignin content through down-regulation of 4CL | |
| Lu et al. | Xylem-specific expression of a GRP1. 8 promoter:: 4CL gene construct in transgenic tobacco | |
| US6479732B1 (en) | cDNA of 4-coumarate: coenzyme a ligase and process for modifying lignin in plants | |
| Zhao et al. | Lignin biosynthesis by suppression of two O-methyl-transferases | |
| WO2009095641A2 (en) | Enhanced plant growth | |
| CN103748225A (en) | Enhancing cell wall properties in plants or trees | |
| CN101809147A (en) | Modified xylan production | |
| Khan et al. | Genetic engineering of phenylpropanoid pathway in Leucaena leucocephala | |
| CN1699574A (en) | Process for culturing brownness resistant sweet potatoes utilizing gene engineering technology |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20080903 Termination date: 20161028 |
|
| CF01 | Termination of patent right due to non-payment of annual fee |