CN1611602A - Method for regulating lignin in populus tomentosa - Google Patents
Method for regulating lignin in populus tomentosa Download PDFInfo
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Abstract
This invention discloses one method of adjusting and controling Chinese white poplar lignin. This invention clons, from the chinese white poplar, one key enzyme gene in the process of compounding lignin creatures, constructs its antogene's expression vector, joins promoter, transmits the functional genes into chinese white poplar's explant by the medium of agricultural bacillus, then sieves highly active expression antogenes and transgenic plant types of lower content of lignin; The above key enzyme gene consists of caffeic acid methyltransferase gene, caffeic acylase A methyltransferase gene or 4- coumaric acid acylase joining enzyme gene. This invention takes advantage of gene project technique to inhibit the composition of lignin creatures of chinese white poplars for paper-making, to reduce the content of lignin or change its components, and thus to produce material tree species suitable for pulping and paper making with low cost and little pollution. This invention has a great theoretical and practical significance in the development of timber pulping and paper making industry with low cost and little pollution.
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, be from Cortex Populi Tomentosae (Populus tomentosa), to clone a kind of key gene in the xylogen biosynthetic pathway, make up the expression vector of its inverted defined gene, connect promotor, by agriculture bacillus mediated, functional gene is changed in the explant of Cortex Populi Tomentosae, screening efficiently expresses the transgenic line of inverted defined gene, content of lignin decline then; Described key gene is caffeoyl coenzyme A methyltransgerase (CCoAOMT) gene or 4-coumaric acid CoA ligase (4CL) gene.
Wherein, the encoding gene of described key enzyme is:
1) caffeoyl coenzyme A methyltransgerase (CCoAOMT, caffeoyl Co-enzyme A3-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 lmin, 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 (6)
1, 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, make up the expression vector of its inverted defined gene, connect promotor, by agriculture bacillus mediated, functional gene is changed in the explant of Cortex Populi Tomentosae, screening efficiently expresses the transgenic line of inverted defined gene, content of lignin decline then; Described key gene is caffeoyl coenzyme A methyl transferase gene or 4-coumaric acid CoA ligase gene.
2, method according to claim 1 is characterized in that: described caffeoyl coenzyme A methyl transferase gene has SEQ ID № in the sequence table: 1 sequence.
3, method according to claim 1 is characterized in that: described 4-coumaric acid CoA ligase gene has SEQ ID № in the sequence table: 2 sequence.
4, method according to claim 1 is characterized in that: described promotor is the promotor of constitutive expression.
5, method according to claim 1 is characterized in that: described explant is blade, stem apex or the statoblast of setation white poplar then.
6, the application of the described method of claim 1 in improvement papermaking resource plant.
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
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| 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 |
| CN102505018B (en) * | 2007-06-21 | 2014-06-04 | 日立造船株式会社 | Gene encoding enzyme involved in mevalonic acid pathway of eucommia ulmoides oliver |
| CN108753816A (en) * | 2018-06-20 | 2018-11-06 | 闫秋洁 | The transgenic arabidopsis strain and its construction method of SYTA gene overexpressions |
| CN109694402A (en) * | 2017-10-24 | 2019-04-30 | 中国农业科学院作物科学研究所 | Plant lignin's synthesis associated protein and its encoding gene and application |
| CN111621520A (en) * | 2020-06-22 | 2020-09-04 | 南京林业大学 | Application of masson pine PmCOMT gene |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1004288B (en) * | 1987-12-26 | 1989-05-24 | 武汉市纺织科学研究所 | The efficient chemical degumming of hemp method of removing lignin |
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| CN102505018B (en) * | 2007-06-21 | 2014-06-04 | 日立造船株式会社 | Gene encoding enzyme involved in mevalonic acid pathway of eucommia ulmoides oliver |
| CN102505019B (en) * | 2007-06-21 | 2015-03-11 | 日立造船株式会社 | 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 |
| CN109694402A (en) * | 2017-10-24 | 2019-04-30 | 中国农业科学院作物科学研究所 | Plant lignin's synthesis associated protein and its encoding gene and application |
| 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 |
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