CN103451169B

CN103451169B - A kind of regulate and control timber grow related gene and application thereof

Info

Publication number: CN103451169B
Application number: CN201210179885.7A
Authority: CN
Inventors: 李来庚; 赵运军
Original assignee: Shanghai Institutes for Biological Sciences SIBS of CAS
Current assignee: Center for excellence and innovation in molecular plant science, Chinese Academy of Sciences
Priority date: 2012-06-01
Filing date: 2012-06-01
Publication date: 2016-05-25
Anticipated expiration: 2032-06-01
Also published as: CN103451169A

Abstract

The present invention relates to a kind of regulate and control timber grow related gene and application thereof. Particularly, the invention provides a kind of new PtMAN6 polypeptide or its coded sequence, is the amino acid sequence of described PtMAN6 polypeptide as SEQ? ID? is described coded sequence as SEQ shown in NO:2? ID? shown in NO:1. The present invention also provides the purposes of the albumen of PtMAN6 gene and coding thereof, and they can reduce the accumulation that xylem secondary cell wall synthesizes and/or reduce crystalline fibers living beings; Overexpression PtMAN6 can be in plant the mannosan hydrolase of enrichment degraded mannans hemicellulose, while is due to the regulating and controlling effect of PtMAN6, in transfer-gen plant, content of lignin reduces, avicel cellulose reduces, and greatly reduces the cost in lignocellulosic alcohol production and paper industry; The promoter of PtMAN6 gene has special vessel cell positioning function, can the expression of accuracy controlling gene in xylem vessel's cell.

Description

A kind of regulate and control timber grow related gene and application thereof

Technical field

The invention belongs to biotechnology and bioenergy field, particularly, the present invention relates to a kind of regulate and control timber growRelated gene and application thereof.

Background technology

Along with the continuous increase of socio-economic development and population, the mankind are more and more to the demand of the energy, due to fossil energySource is limited, and the price of the products such as oil rises steadily, and the stable sustainable development of economy is threatened.

Reproducible biomass energy is one of desirable alternative energy source, and bio-ethanol is to have at present prospect and topmost mostBiomass energy. Since the oil crisis in 20 century 70 mid-terms, start to amass taking the U.S. and Brazil as some main countriesThe utmost point is carried out bio-ethanol development plan, and since this century, global bio-ethanol output increases sharply. Utilize in early days cereal crops moreProduce ethanol as corn etc. as raw material, cause higher food prices, have contradiction with national grain security, can not carry out largeLarge-scale production. In recent years, the American-European a large amount of inputs of state that wait are carried out bio-ethanol production technology and the works taking lignocellulosic as raw materialIndustry practice, actively cultivates and is applicable to national energy crop simultaneously. China is also at the positive lignocellulose ethanol fermentation of carrying outResearch.

Lignocellulosic alcohol production mainly contains four steps: 1 pretreatment, the structure of destruction lignocellulosic, removes and hinder sugarThe materials such as the lignin of changing and ferment; 2 acid or enzyme hydrolysis cellulose become monose with hemicellulose; 3 use zymophytes by hexose andPentose is fermented into ethanol; 4 distillation dehydration purifying ethanols, make it reach the requirement as the energy. Wherein being produced into of the 1st, 2 stepsThis is too high, and efficiency is very low, has seriously limited the development of the fibrous biomass energy, causes lignin bio-ethanol production cost to occupy highNot.

In addition, lignocellulosic is also widely used in paper industry. Mainly contain at present two large class paper-making pulping process:Mechanical feedback and chemical pulping. Mechanical feedback, by mechanical separation papers fiber, is not removed lignin wherein, can obtainTo high paper output, but because the existence of lignin causes it can bleachability low, the paper of production is easily jaundice under light. ChangeLearn legal system slurry and utilize chemical substance to remove the lignin in cell membrane, can obtain long and pliable and tough paper fiber, thereby can produce highThe paper of quality. The bleaching of paper pulp is other one important procedure of pulp and paper industry, is mainly further by chemical substanceRemove the lignin in paper pulp or change lignin chromophoric group, thereby improving whiteness and the retention of whiteness of paper pulp. TraditionalBleaching agent mostly is chlorine bleaches, contains the noxious material with strong carcinogenicity in waste water, and environmental pollution is very large.

Therefore this area does not also have a kind of xylplant that is suitable for producing bio-fuel and papermaking, therefore in the urgent need to researchThe related gene that regulation and control timber is grown, and exploitation is applicable to the timber variety of production regenerative resource and papermaking.

Summary of the invention

Object of the present invention be just to provide a kind of regulate and control timber grow related gene and application thereof.

In a first aspect of the present invention, the coded sequence of a kind of PtMAN6 polypeptide or PtMAN6 polypeptide is provided, described inThe amino acid sequence of PtMAN6 polypeptide is as shown in SEQIDNO.:2, and the coded sequence of described PtMAN6 polypeptide is as SEQIDShown in NO.:1.

In a second aspect of the present invention, the purposes of PtMAN6 polypeptide or its coded sequence is provided, described purposes is certainly optionalOne or more of lower group:

(1) reduce the synthetic of xylem secondary cell wall;

(2) accumulation of reduction crystalline fibers living beings;

(3) content of reduction lignin (preferably reducing plant stem lignin).

In another preference, described PtMAN6 polypeptide is selected from lower group:

(I) there is the polypeptide of amino acid sequence shown in SEQIDNO.:2 or SEQIDNO.:17;

(II) by one or several amino of amino acid sequence process as shown in SEQIDNO.:2 or SEQIDNO.:17Acid residue replacement, disappearance or interpolation and form by (1) derivative polypeptide; Or

(III) homology of amino acid sequence shown in amino acid sequence and SEQIDNO.:2 or SEQIDNO.:17 >=90% (preferably >=95%, more preferably 98%) is by (1) derivative polypeptide.

In another preference, the coded sequence of described PtMAN6 polypeptide is selected from lower group:

(i) coding has the polynucleotides of the polypeptide of amino acid sequence shown in SEQIDNO.:2 or SEQIDNO.:17;

(ii) polynucleotides of sequence as shown in SEQIDNO.:1 or SEQIDNO.:16;

(iii) homology >=95% of sequence shown in nucleotide sequence and SEQIDNO.:1 or SEQIDNO.:16 (Good ground >=98%, more preferably >=99%) polynucleotides;

(iv) as shown in SEQIDNO.:1 or SEQIDNO.:16,5 ' of polynucleotides end and/or 3 ' is held brachymemma or addsAdd the polynucleotides of 1-60 (preferably 1-30, more preferably 1-10) nucleotides; Or

(v) with (i)-(iv) polynucleotides of arbitrary described polynucleotides complementation.

In a third aspect of the present invention, provide a kind of method that improves plant trait, described improvement plant trait choosingFrom lower group: the secondary cell wall that (1) reduces plant xylem synthesizes; (2) accumulation of reduction plant crystalline fibers living beings; (3)Reduce the content of plant lignin (preferably reducing plant stem lignin);

Described method comprises step: the expression or the activity that improve PtMAN6 polypeptide in described plant or its coded sequence.

In another preference, described method comprises step: the coded sequence of PtMAN6 polypeptide is imported in plant cell,Cultivate described plant cell, regeneration plant.

In another preference, described plant is Salicaceous Plants, is preferably willow.

In a fourth aspect of the present invention, a kind of promoter element is provided, described promoter element is plant PtMAN6The promoter of gene, and described promoter element has the function that plant xylem vessel cell-specific starts.

In another preference, described promoter element does not have the merit of the special startup of plant xylem cambial cellEnergy.

In another preference, described promoter element is selected from lower group:

(a) there are the polynucleotides of sequence as shown in SEQIDNO.:3;

(b) homology >=95% of sequence shown in nucleotide sequence and SEQIDNO.:3 (preferably >=98%, more preferably >=99%), and there are the polynucleotides of plant xylem vessel cell-specific start-up performance;

(c) as shown in SEQIDNO.:3 brachymemma 1-60,5 ' of polynucleotides end and/or 3 ' end (preferably 1-30, moreGood ground 1-6) nucleotides, and there are the polynucleotides of plant xylem vessel cell-specific start-up performance.

In a fifth aspect of the present invention, a kind of construction is provided, described construction contains foreign gene and and external sourcePromoter element described in the fourth aspect present invention that gene is operatively connected.

In another preference, described foreign gene is selected from lower group:

Resistant gene, selection markers gene, antigenic protein gene, RNAi gene, microRNA gene, biologic product baseCause or plant quality related gene.

In a sixth aspect of the present invention, a kind of expression cassette is provided, described expression cassette from 5 ' to 3 ' has following unit successivelyPart: promoter element, gene ORF sequence and terminator described in fourth aspect present invention.

In a seventh aspect of the present invention, a kind of carrier is provided, described carrier contains opening described in fourth aspect present inventionExpression cassette described in mover element or sixth aspect present invention.

In a eighth aspect of the present invention, a kind of host cell is provided, described host cell contains seventh aspect present inventionDescribed carrier or its chromosomal integration have promoter element or its chromosome described in the fourth aspect present invention of external source wholeClose the expression cassette described in sixth aspect present invention.

In a ninth aspect of the present invention, the promoter element described in fourth aspect, the structure described in the 5th aspect are providedThe purposes of the expression cassette described in thing or the 6th aspect, for specificity regulation and control foreign gene at plant xylem vessel cellExpress.

In a tenth aspect of the present invention, provide a kind of at plant xylem vessel cell specific expression foreign geneMethod, comprises step:

(a) provide a construction, described construction contains foreign gene and is operably connected with this foreign genePromoter element described in fourth aspect;

(b) construction in step (a) is imported to plant xylem vessel cell, obtain the xylem vessel transforming thinBorn of the same parents.

In should be understood that within the scope of the present invention, above-mentioned each technical characterictic of the present invention and in below (eg embodiment) toolBetween each technical characterictic of volume description, can combine mutually, thereby form new or preferred technical scheme. As space is limited, existThis is tired stating no longer one by one.

Brief description of the drawings

Following accompanying drawing is used for illustrating specific embodiment of the invention scheme, defined by claims and be not used in to limitThe scope of the invention.

Fig. 1 shows 35S:PtMAN6 over-express vector structure.

Fig. 2 shows 35S:PtMAN6-GFP over-express vector structure.

Fig. 3 shows 35S:PtMAN6AS antisense expression vector structure.

Fig. 4 shows MAN6G transient expression carrier structure.

Fig. 5 shows MAN6sG transient expression carrier structure.

Fig. 6 has shown various transfer-gen plant phenotypes. Fig. 6 A shows that WersternBlot detects transfer-gen plant result;Fig. 6 B shows overexpression PtMAN6(OE), wild type (WT), antisense expression PtMAN6(AS) phenotype of plant, scale is 20 lisRice; Fig. 6 C-Fig. 6 E shows transfer-gen plant petiole mechanical strength, and overexpression plant (Fig. 6 D, OE) is with respect to wild type (figure6C, WT) and antisense expression plant (Fig. 6 E, AS) petiole deliquescing, arrow shows petiole, scale is 10 centimetres; Fig. 6 F-Fig. 6 H is aobvious respectivelyThe deposition of lignin in plant cane petiole in diagram 6C-Fig. 6 E, petiole is through free-hand section, phloroglucinol stain, red displayThe deposition of lignin, scale is 500 microns.

Fig. 7 shows the result of variations of lignin deposition and wood components in transgenosis and wild type willow stem. Fig. 7 A)-Tu7C): wild type (Fig. 7 A), PtMAN6OE(Fig. 7 B), and PtMAN6AS(Fig. 7 C) the free-hand section isophthalic three of 14 joint stems in plantPhenol dyeing, red display lignin deposition, arrow indicates the accumulation of lignin in myelocyte; Fig. 7 D-Fig. 7 F: be respectively figureThe enlarged drawing of black surround part in 7A-Fig. 7 C, arrow indicates the accumulation of lignin in vessel cell; Fig. 7 G-Fig. 7 I: ultraviolet detects wildRaw type (Fig. 7 G), PtMAN6OE(Fig. 7 H), and PtMAN6AS(Fig. 7 I) 12 joint sections in plant, lignified cell autofluorescenceStronger, be blue, the cell that lignifying is weak, autofluorescence is purple a little less than, and arrow indicates immature vessel cell; Fig. 7 JFor the comparative result of ABSL lignin in transfer-gen plant timber, cross lignin in expression PtMAN6 plant (OE) and significantly reduce;Fig. 7 K is the comparison of avicel cellulose in transfer-gen plant timber, crosses avicel cellulose in expression PtMAN6 plant (OE) and significantly fallsLow; Fig. 7 L is that the area of transfer-gen plant prematurity vessel cell changes result, and institute's survey conduit is leading that in figure G-I, arrow indicatesPipe; Statistical analysis adopts T inspection,^*Represent p < 0.05,^**Represent p < 0.01, in Fig. 7 A-Fig. 7 C, scale is 500 μ m; Fig. 7 D-figureIn 7I, scale is 100 μ m, and Fig. 7 J-Fig. 7 L error line refers to the standard error of statistics.

Fig. 8 shows height and the girth analysis result of 1.5 years raw transgenic poplar timber, and wherein, Fig. 8 A showed scaleReach PtMAN6 willow (OE), the trunk height of the willow of antisense expression PtMAN6 and wild type (WT) willow does not have notable difference;Fig. 8 B shows that the girth of the butt of OE, AS and WT willow does not have notable difference, and statistical analysis adopts T inspection.

Fig. 9 has shown the enzyme activity assay of PtMAN6; Wherein, the optimal pH measurement result that Fig. 9 A is PtMAN6, substrate isBeing dissolved in pH scope is the 1%AZCL-glactomannan suspension of 0.1M citric acid-phosphate buffer of 3.0-8.0, WT andPtMAN6 refers to respectively from wild type and crosses and express the zymoprotein (lower same) extracting plant mature leaf; Fig. 9 B shows PtMAN6'sOptimal reactive temperature measurement result, substrate is the 1%AZCL-that is dissolved in 0.1M citric acid-phosphate buffer of pH5.0Glactomannan suspension; Fig. 9 C shows PtMAN6 deglycosylation result, in Werstern hybridization analysis plant, extractsPtMAN6(the first swimming lane), and EndoH_fProcess the PtMAN6 of 0.5 hour (the second swimming lane) and 1 hour (the 3rd swimming lane), M:The protein label dying in advance; Fig. 9 D is that glycosylation affects enzymatic activity result, compares PtMAN6 warp with contrasting (control)EndoH_fDeglycosylation 2 hours, enzymatic activity is approximately reduced to original 50%.

Figure 10 shows the quantitative PCR analysis result of PtMAN6 gene in willow. From the xylem of 1 year growing poplar, tough respectivelySkin zone, spire, Lao Ye and stem top tissue extraction RNA carry out quantitative RT-PCR analysis, using Actin gene in willow as interiorGinseng.

Figure 11 shows the immunolocalization analysis result of PtMAN6; Wherein, Figure 11 A and Figure 11 B are 1 year raw wild type willowThe sectional view of six internodes, shows that PtMAN6 is positioned xylem vessel's cell; Figure 11 B is the enlarged drawing at black surround position in Figure 11 A;Figure 11 C is the rip cutting figure of 1 year raw wild type willow the 6th internode, shows that PtMAN6 is positioned xylem vessel's cell; Figure 11 D andFigure 11 E is the sectional view of 1 year raw wild type willow stem top tissue, shows that PtMAN6 is positioned xylem vessel's cell; Figure 11 EIt is the enlarged drawing at black surround position in Figure 11 D; The negative contrast of Figure 11 F, the sectional view of 1 year raw wild type willow the 6th internode, notAny framing signal can be detected.

Figure 12 shows that PtMAN6-GFP protein localization is in plasma membrane. Figure 12 A and Figure 12 B show stably express in arabidopsis rootPtMAN6-GFP albumen, this protein localization is in plasma membrane; Figure 12 C and Figure 12 D, 30% sucrose plasmolysis arabidopsis root cells, showsPtMAN6-GFP protein localization is in plasma membrane; Figure 12 E is the carrier for transient expression; Figure 12 F is subcellular componentsWerstern detects, and M is for indicating protein molecular, and 1 is kytoplasm component, and 2 is membrane component; Figure 12 G and Figure 12 H use particle bombardment in oceanMAN6G carrier in transient expression Figure 12 E in green onion epidermal cell; Figure 12 I and Figure 12 J show with particle bombardment at onion epidermis cellMAN6sG carrier in middle transient expression Figure 12 E; Particle bombardment transient expression figure in onion epidermis cell for Figure 12 K and Figure 12 LThe carrier of CK in 12E; A, C, G, I, K is GFP fluorescence picture; B, D, H, J, L is light field picture, scale is 50 μ m.

Figure 13 shows relative expression's level of secondary wall formation associated transcription factor in the plant of overexpression PtMAN6.RNA extracts from 1 year growing poplar xylem, and the expression of wild type is defined as 1, and willow Actin gene is as internal reference.

Figure 14 has shown in the plant of overexpression PtMAN6 and has changed with the expression of secondary wall component synthetic gene. Figure 14 AShow that in the plant of overexpression PtMAN6, the gene of lignin monomer route of synthesis and lignin polymerization's related gene are subject toSuppress; Figure 14 B shows that in the plant of overexpression PtMAN6, the expression of the cellulose synthetic gene CesA8 that secondary wall is relevant is subject toTo suppressing; Figure 14 C shows in the plant of overexpression PtMAN6, the expression of the xylan synthetic gene GT43B that secondary wall is relevantBe suppressed. RNA extracts from 1 year growing poplar xylem, and the expression of wild type is defined as 1, and willow Actin gene is as interiorGinseng.

Detailed description of the invention

The inventor, through extensive and deep research, has found a kind of new PtMAN6 polypeptide or its code sequence firstRow, the amino acid sequence of described PtMAN6 polypeptide is as shown in SEQIDNO.:2, and described coded sequence is as SEQIDNO.:1 instituteShow. In addition, the present invention also provides the purposes of the albumen of PtMAN6 gene in plant and coding thereof, and they can be for regulation and controlThe secondary cell wall of xylem synthesizes and regulates and controls the accumulation of fibrous biomass; Overexpression PtMAN6 can fall in plant in enrichmentSeparate the mannosan hydrolase of mannans hemicellulose, the while is due to the regulating and controlling effect of PtMAN6, in this transfer-gen plantContent of lignin reduces, and crystalline fibers cellulose content reduces, thereby has reduced the cost of lignocellulosic alcohol production and papermaking; ThisOutward, PtMAN6 albumen has special vessel cell positioning function, and its gene promoter energy accuracy controlling gene is led at xylemExpression in solencyte. Complete on this basis the present invention.

Term

As used herein, term " polypeptide of the present invention ", " albumen of the present invention ", " PtMAN6 polypeptide ", " PtMAN6 albumen " or" hemicellulose mannose hydrolase " can exchange use. In a preference, refer to and there is SEQIDNO.:2 or SEQThe polypeptide of amino acid sequence shown in IDNO.:17, or its active fragment, or derivative.

PtMAN6 polypeptide of the present invention preferably derives from plant, preferably derives from Salicaceae, Populus, Salix or brill daySalix etc.

As used herein, term " gene of the present invention ", " PtMAN6 gene " can exchange use, all refer to and derive from oneNucleotide sequence and the derived sequence thereof of coding hemicellulose mannose hydrolase. In a preference of the present invention, described inNucleotide sequence as shown in SEQIDNO.:1 or SEQIDNO.:16.

PtMAN6 gene of the present invention preferably derives from plant, preferably derives from Salicaceae, Populus, Salix or brill daySalix etc.

As used herein, term " separation " refers to that material separates (if natural thing from its primal environmentMatter, primal environment is natural surroundings). If the polynucleotide under the native state in active somatic cell and polypeptide are not separatePurifying, but same polynucleotide or polypeptide as separated in same other materials that exist, for separating from native statePurifying.

PtMAN6 polypeptide of the present invention can be recombinant polypeptide, natural polypeptides, synthetic polypeptide. Polypeptide of the present invention can beThe product of natural purifying, or the product of chemical synthesis, or use recombinant technique for example, from protokaryon or eucaryon host (, bacterium, fermentMother, higher plant, insect and mammalian cell) middle generation. The host used according to recombinant production scheme, polypeptide of the present inventionCan be glycosylated, can be maybe nonglycosylated. Polypeptide of the present invention also can comprise or not comprise initial methionineResidue.

The present invention also comprises that having regulation and control xylem secondary cell wall synthesizes with fibrous biomass accumulation active and functionPtMAN6 protein fragments and analog. As used herein, term " fragment " and " analog " refer to and substantially keep of the present inventionThe biological function that natural PtMAN6 albumen is identical or active many skins.

Polypeptide fragment of the present invention, derivative or analog can be: (i) have one or more conservative or non-conservation ammoniaThe substituted polypeptide of base acid residue (preferably conservative amino acid residue), and the amino acid residue of such replacement can be also canNot encoded by genetic code; Or (ii) in one or more amino acid residues, there is the polypeptide of substituted radical; Or(iii) mature polypeptide and another compound (for example, such as extending the compound of polypeptide half-life, polyethylene glycol) merge institute's shapeThe polypeptide becoming; Or (iv) additional amino acid sequence be fused to this peptide sequence and the polypeptide that forms (as targeting sequencing or secretionSequence or be used for sequence or the proteinogen sequence of this polypeptide of purifying or fusion). According to these fragments of definition herein, spread outBiological and analog belongs to the known scope of those skilled in the art.

The present invention also comprise with PtMAN6 polypeptide of the present invention or albumen have 50% or above (preferably more than 60%, 70% withUpper, more than 80%, more preferably more than 90%, more preferably more than 95%, most preferably more than 98%, as 99%) homology have identical orThe polypeptide of identity function or albumen. (be generally 1-60, preferably 1-30 is individual, more can to pass through several in protein variantGood ground 1-20,1-10 best) replace, lack or add the derived sequence of at least one amino acid gained, and at C endEnd and/or N end add one or several (being generally in 20, is preferably in 10, is more preferably in 5) ammoniaBase acid. For example, in described albumen, while replacement with the close or similar amino acid of performance, conventionally can not change proteinFunction, C end and/or the end function of adding or several amino acid and conventionally also can not change protein. These guarantorsThe variation of keeping property is best replaces and produces according to table 1.

Table 1

The difference that the present invention includes PtMAN6 protein analogue and natural PtMAN6 albumen can be on amino acid sequenceDifference, can be also the difference not affecting on the modified forms of sequence, or have both at the same time. The analog of these albumen comprisesNatural or induce genetic variant. Induce variation body can obtain by various technology, as by radiation or be exposed to mutagenesisAgent and produce random mutagenesis, also can knownly divide biological technology by direct mutagenesis method or other. Analog also comprises toolHave the analog that is different from the amino acid whose residue of natural L-(as D-amino acid), and have non-natural exist or synthetic ammoniaThe analog of base acid (as β, gamma-amino acid). Should be understood that albumen of the present invention is not limited to the above-mentioned representational egg exemplifyingIn vain.

(conventionally the not changing primary structure) form of modification comprises: in body or the chemically derived form of external albumen as acetoxylationOr carboxylated. Modify and also comprise glycosylation, as those carry out glycosylation modified in protein synthesizes and processes. This modification canTo carry out glycosylated enzyme (as mammiferous glycosylase or deglycosylating enzyme) and complete by albumen is exposed to. ModifyForm also comprises the have phosphorylated amino acid residue sequence of (as phosphotyrosine, phosphoserine, phosphothreonine).

The present invention also provides the polynucleotide sequence of coding PtMAN6 polypeptide, albumen or its variant. Multinuclear of the present inventionThuja acid can be DNA form or rna form. DNA form comprises: DNA, genomic DNA or artificial synthetic DNA, DNA can beStrand or double-stranded. DNA can be coding strand or noncoding strand. The polynucleotides of encoding mature polypeptide comprise: only codingThe coded sequence of mature polypeptide; The coded sequence of mature polypeptide and various additional code sequence; The coded sequence of mature polypeptide (withOptional additional code sequence) and non-coding sequence.

Term " polynucleotides of coded polypeptide " can be the polynucleotides that comprise this polypeptide of encoding, and can be still to compriseThe polynucleotides of additional code and/or non-coding sequence. The invention still further relates to the variant of above-mentioned polynucleotides, its coding and thisInvention has many glycosides of identical amino acid sequence or fragment, analog and the derivative of polypeptide. The variant of these polynucleotides canTo be the allelic variant of natural generation or the variant that non-natural occurs. These nucleotide diversity bodies comprise replace variant,Deletion mutation body and insertion variant. As known in the art, allelic variant is the replacement form of polynucleotides, and it canCan be replacement, disappearance or the insertion of one or more nucleotides, but can be from not changing in fact the function of polypeptide of its coding.

The invention still further relates between above-mentioned sequence hybridization and two sequences and have at least 50%, preferably at least 70%,The more preferably polynucleotides of at least 80% homogeny. The present invention be more particularly directed under stringent condition and polynucleotides of the present inventionInterfertile polynucleotides. In the present invention, " stringent condition " refers to: (1) assorted compared with under LIS and higher temperatureHand over and wash-out, as 0.2 × SSC, 0.1%SDS, 60 DEG C; Or (2) hybridization time is added with denaturant, as 50% (v/v) first phthalein amine, 0.1%Calf serum/0.1%Ficoll, 42 DEG C etc.; Or (3) only at the homogeny between two sequences at least more than 90%, be more preferably95% just hybridizes when above.

Should be understood that although PtMAN6 gene of the present invention is preferably from willow, from other plant and willowOther gene of PtMAN6 gene height homology (as have more than 80%, as 85%, 90`%, 95%, even 98% sequence homogeny)Also within the scope of considering in the present invention. The Method and kit for of aligned sequences homogeny is also that this area is known, for exampleBLAST。

PtMAN6 nucleotides full length sequence of the present invention or its fragment can be used pcr amplification method, recombination method or artificial conventionallySynthetic method obtains. For pcr amplification method, can relevant nucleotide sequence disclosed according to the present invention, especially open readdingFrame sequence designs primer, and with commercially available DNA library or by conventional method well known by persons skilled in the art preparedCDNA storehouse is as template, amplification and must relevant sequence. In the time that sequence is longer, usually need to carry out twice or pcr amplification repeatedly, soAfter again the fragment amplifying for each time is stitched together by proper order. Once obtain relevant sequence, just can be with recombinatingMethod obtains relevant sequence in large quantity. Normally be cloned into carrier, then proceeded to cell, then by conventional method from increaseIn host cell after growing, separate and obtain relevant sequence.

In addition, also can synthesize relevant sequence by artificial synthetic method, especially fragment length more in short-term. Conventionally, logicalAfter first synthetic multiple small fragments, and then connect and can obtain the fragment that sequence is very long. At present, can be completely by having changedSynthesize to obtain the DNA sequence dna of code book invention albumen (or its fragment, or derivatives thereof). Then this DNA sequence dna can be drawnEnter in various existing DNA moleculars as known in the art (or as carrier) and cell. In addition also can will dash forward by chemical synthesis,Become and introduce in protein sequence of the present invention.

The present invention also provides the recombinant vector and the application thereof that comprise gene of the present invention. As the preferred mode of one, heavyThe promoter downstream of group carrier comprises MCS or at least one restriction enzyme site. When needs are expressed the object of the invention geneTime, genes of interest is connected in applicable MCS or restriction enzyme site, thereby genes of interest and promoter can be operatedGround connects. As another kind of optimal way, described recombinant vector comprises (from 5 ' to 3 ' direction): promoter, and genes of interest, andTerminator. If needed, described recombinant vector can also comprise the element that is selected from lower group: 3 ' polymerized nucleoside acidifying signal; Non-Translation nucleotide sequence; Transhipment and target nucleotide sequence; Resistance selective marker (dihyrofolate reductase, neomycin resistance, hygromycinResistance and green fluorescent protein etc.); Enhancer; Or operation.

Well known to those of ordinary skill in the art for the preparation of the method for recombinant vector. Expression vector can be bacteriumPlasmid, bacteriophage, yeast plasmid, plant cell virus, mammalian cell virus or other carriers. In a word, as long as it canIn host, copy and stablize, any plasmid and carrier are all can be adopted.

Those of ordinary skill in the art can use the method for knowing to build the expression that contains gene of the present invention and carryBody. These methods comprise extracorporeal recombinant DNA technology, DNA synthetic technology, the interior recombinant technique of body etc. Use gene structure of the present inventionWhile building recombinant expression carrier, can before its transcription initiation nucleotides, add any enhancement mode, composing type, organizing specific type orInducible promoter.

Comprise gene of the present invention, expression cassette or carrier can be for transforming suitable host cell, so that host expressesProtein. Host cell can be prokaryotic, as Escherichia coli, and streptomyces, Agrobacterium; Or the eukaryotic such as low, asYeast cells; Or higher eucaryotic cells, as plant cell. Persons skilled in the art are all known suitable the carrying of How to chooseBody and host cell. Can carry out with routine techniques well known to those skilled in the art with recombinant DNA transformed host cell. Work as hostDuring for prokaryotes (as Escherichia coli), can use CaCl₂Method processing, also can carry out with electroporation. When host is that eucaryon is rawThing, can select following DNA transfection method: calcium phosphate precipitation, conventional mechanical method is (as microinjection, electroporation, lipidBody packaging etc.). Conversion of plant also can use the method such as Agrobacterium-mediated Transformation or via Particle Bombardment Transformation, for example leaf dish method, rataria conversion method,Bud infusion method etc. Can use conventional method regeneration plant for the plant cell, tissue or the organ that transform, turn thereby obtainThe plant of gene.

Extracellular can be expressed or be secreted into described polypeptide of the present invention in cell or on cell membrane. If needed,Can utilize its physics, the separating by various separation methods with other characteristic and the albumen of purification of Recombinant of chemistry. These methodsWell-known to those skilled in the art. The example of these methods comprises (but being not limited to): conventional renaturation processing, use albumenPrecipitating reagent processing (salt analysis method), centrifugal, the broken bacterium of infiltration, super processing, ultracentrifugation, sieve chromatography (gel filtration), adsorption layerAnalyse, the combination of ion-exchange chromatography, high performance liquid chroma-tography (HPLC) and other various LC technology and these methods.

The invention provides the purposes of PtMAN6 polypeptide or its coded sequence, described purposes is optionally from lower group:

Reduce xylem secondary cell wall synthetic, the accumulation that reduces crystalline fibers living beings, reduce lignin (preferablyReduce plant stem lignin) content.

Described PtMAN6 polypeptide is selected from lower group: (I) have amino acid shown in SEQIDNO.:2 or SEQIDNO.:17The polypeptide of sequence; (II) amino acid sequence as shown in SEQIDNO.:2 or SEQIDNO.:17 is passed through to one or severalReplacement, disappearance or the interpolation of amino acid residue and form by (1) derivative polypeptide; Or (III) amino acid sequence and SEQIDHomology >=90% of amino acid sequence shown in NO.:2 or SEQIDNO.:17 (preferably >=95%, more preferably 98%) is spread out by (1)Raw polypeptide.

The coded sequence of described PtMAN6 polypeptide is selected from lower group: (i) coding has SEQIDNO.:2 or SEQIDThe polynucleotides of the polypeptide of amino acid sequence shown in NO.:17; (ii) sequence is as SEQIDNO.:1 or SEQIDNO.:16 instituteThe polynucleotides that show; (iii) homology >=95% of sequence shown in nucleotide sequence and SEQIDNO.:1 or SEQIDNO.:16The polynucleotides of (preferably >=98%, more preferably >=99%); (iv) multinuclear as shown in SEQIDNO.:1 or SEQIDNO.:16The polynucleotides of 1-60 (preferably 1-30, more preferably 1-10) nucleotides of 5 ' end of thuja acid and/or 3 ' end brachymemma or interpolation;Or (v) and (i)-(iv) polynucleotides of arbitrary described polynucleotides complementation.

The present invention also provides a kind of method of preparing genetically modified plants, comprises step: by the code sequence of PtMAN6 polypeptideRow import in plant cell, cultivate described plant cell, regeneration plant.

The present invention also provides a kind of method that improves plant trait, and described improvement plant trait is selected from lower group:

(1) secondary cell wall that reduces plant xylem synthesizes; (2) accumulation of reduction plant crystalline fibers living beings; (3)Reduce the content of plant lignin; Described method comprises step: improve PtMAN6 polypeptide in described plant or its coded sequenceExpress or activity.

The present invention also provides the plant of a kind improvement, compared with wild-type plant, in described plant lignin and/orThe content of avicel cellulose declines more than 10%, preferably declines more than 20%, more preferably declines 50%, decline best 100% withOn.

Described plant is Salicaceous Plants, is preferably willow.

As used herein, term " promoter of the present invention ", " promoter of xylem vessel's specifically expressing ", " xylem is ledManage the promoter of special location " be used interchangeably, refer to derive from plant, preferably derive from Salicaceae, Populus, Salix or brillThe promoter element of the PtMAN6 of it Salix etc., the typical promoter element sequence of one of the present invention is as SEQIDNO.:3 instituteShow.

As used herein, term " promoter " or " promoter region (territory) " refer to that a kind of accurate and effective initial gene transcribes meritThe nucleotide sequence of energy, guiding gene nucleotide sequence is transcribed into mRNA, and it is present in the upstream (5 ' of genes of interest coded sequence conventionallyEnd), usually, promoter or promoter region provide RNA polymerase and correct initial knowledge of transcribing necessary other factorsOther site.

The invention provides the specific expressed promoter of a kind of xylem vessel specifically expressing, described promoter derives fromPlant, preferably derives from the PtMAN6 gene of Salicaceae. A kind of nucleotide sequence of preferred promoter is as SEQIDNO.:Shown in 3.

Promoter of the present invention can be efficient, single-mindedly at xylem vessel's cells, and in cambial cellWithout any expression, promoter of the present invention can be improved plant xylem vessel cell characteristics for specificity.

In this article, described promoter or promoter region (territory) comprise the variant of promoter, and promoter variants can pass throughInsert or delete regulation and control region, carry out random or rite-directed mutagenesis etc. and obtain.

The present invention also comprises with preferred promoter sequence of the present invention (SEQIDNO.:3) having 50% or above (preferredMore than 60%, more than 70%, more than 80%, more preferably more than 90%, more preferably more than 95%, most preferably more than 98%, as 99%) homologyProperty nucleic acid, described nucleic acid also has specificity regulation and control and starts the function of xylem vessel's cellular expression. " homology " refer to byAccording to the identical percentage in position, the similar level (being sequence similarity or homogeneity) between two or more pieces nucleic acid.

Although should be understood that in example of the present invention the PtMAN6 gene promoter that derives from Salicaceae be provided, comeCome from other similar plant (especially belonging to the plant of a section or genus with willow), have necessarily with promoter of the present inventionThe promoter of homology (conservative), is also included within scope of the present invention, as long as those skilled in the art are reading this ShenPlease after the information that provides according to the application can from other plant, separate and obtain this promoter easily.

As used herein, term " specific expressed " refers to genes of interest specific time and/or specific in plantThe expression of tissue. Described " expression of plant xylem vessel cell-specific " refer under promoter regulation of the present invention,Genes of interest high degree of specificity and in specific manner at plant xylem vessel cells.

As used herein, " external source " or " allos " refers to two or more pieces nucleic acid or the protein sequence of separate sourcesBetween relation. For example, if the combination of promoter and genes of interest sequence is not naturally occurring conventionally, promoter forThis genes of interest is external source. Cell or the organism that particular sequence inserts for it is " external source ".

As used herein, " cis-regulating element " refers to the transcription initiation of gene and transcribes the guarantor that efficiency plays regulatory roleKeeping property base sequence.

Promoter of the present invention can be operationally connected with foreign gene, and this foreign gene is for promoterCan be external source (allos). Foreign gene of the present invention (also referred to as genes of interest) has no particular limits, Ke YiweiRNAi gene or coding have the gene of specific function albumen, for example some in agricultural or plant improvement, have key property orThe albumen of function.

The representative example of described foreign gene includes, but is not limited to: resistant gene, selection markers gene, antigen proteinGene and biologic product gene or plant quality related gene.

Described resistant gene is selected from lower group: anti-herbicide gene, antiviral gene, cold tolerance gene, high temperature resistant gene, anti-Drought gene, waterlogging-resistant gene or anti insect gene. Described selection markers gene is selected from lower group: gus (GUSB) baseCause, hyg (hygromycin) gene, neo (neomycin) gene or gfp (green fluorescent protein) gene. Described antigenic protein geneAnd biologic product gene is selected from lower group: bacterium class antigen protein (as cholera toxin B, tetanus toxin etc.), virus type antigen egg(as canine parvovirus), protozoa antigen protein (amoeba cause of disease LecA), autoantigen albumen are (as type i diabetes in vainCTB-pins) or biologic product (as α 2b interferon, IGF etc.). Described plant quality related geneBe selected from lower group: amino acid improvement related gene, fat improvement related gene, starch improvement related gene or male sterility dependency basisCause.

The present invention also provides a kind of expression casette, and described expression cassette has following elements successively from 5 '-3 ': startSon, gene ORF sequence and terminator. Preferably, described promoter sequence is as shown in SEQIDNO.:3 or and SEQIDHomology >=95% of sequence shown in NO.:1, preferably >=98%, more preferably >=99%.

The present invention also provides a kind of recombinant vector that comprises promoter of the present invention and/or expression casette. As onePlant preferred mode, the promoter downstream of recombinant vector comprises MCS or at least one restriction enzyme site. When needs are expressedWhen genes of interest, genes of interest is connected in applicable MCS or restriction enzyme site, thereby by genes of interest and startupSon is operably connected. As another kind of optimal way, described recombinant vector comprises (from 5 ' to 3 ' direction): promoter, orderGene, and terminator. If needed, described recombinant vector can also comprise the element that is selected from lower group: 3 ' polynucleotideChange signal; Untranslated nucleotide sequence; Transhipment and target nucleotide sequence; (dihyrofolate reductase, neomycin resist resistance selective markerProperty, hygromycin resistance and green fluorescent protein etc.); Enhancer; Or operation.

Those of ordinary skill in the art can use the method structure of knowing to contain promoter of the present invention and/or orderThe expression vector of gene order. These methods comprise extracorporeal recombinant DNA technology, DNA synthetic technology, the interior recombinant technique of body etc.

Promoter of the present invention, expression cassette or carrier, can be for transforming suitable host cell, so that host expresses eggWhite matter. Host cell can be prokaryotic, as Escherichia coli, and streptomyces, Agrobacterium: or the eukaryotic such as low, as fermentMother cell; Or higher eucaryotic cells, as plant cell. Persons skilled in the art are all known the carrier that How to choose is suitableAnd host cell. Can carry out with routine techniques well known to those skilled in the art with recombinant DNA transformed host cell. When host isWhen prokaryotes (as Escherichia coli), can use CaCl₂Method processing, also can carry out with electroporation. When host is eucaryote,Can select following DNA transfection method: calcium phosphate precipitation, conventional mechanical method is (as microinjection, electroporation, liposomePackaging etc.). Conversion of plant also can use the method such as Agrobacterium-mediated Transformation or via Particle Bombardment Transformation, for example leaf dish method, rataria conversion method, flowerBud infusion method etc. Can use conventional method regeneration plant for the plant cell, tissue or the organ that transform, turn base thereby obtainThe plant of cause.

As a kind of optimal way of the present invention, the method for preparing genetically modified plants is: will carry promoter and object baseBecause the carrier of (both are operably connected) proceeds to Agrobacterium, Agrobacterium is again by whole containing the carrier segments of promoter and genes of interestBe incorporated on the chromosome of plant. The transgene receptor plant relating to is for example arabidopsis, tobacco, fruit tree etc.

Major advantage of the present invention is:

(1) inventor finds that PtMAN6 gene in plant and the albumen of coding thereof can reduce the secondary of xylem firstThe accumulation of Cell wall synthesis and reduction crystalline fibers living beings.

(2) promoter of PtMAN6 gene has special vessel cell positioning function, and energy accuracy controlling gene is woodenExpression in portion's vessel cell.

Below in conjunction with specific embodiment, further set forth the present invention. Should be understood that these embodiment are only for illustrating the present inventionLimit the scope of the invention and be not used in. The experimental technique of unreceipted actual conditions in the following example, conventionally according to conventional barPart is as people such as Sambrook, molecular cloning: laboratory manual (NewYork:ColdSpringHarborLaboratoryPress, 1989) condition described in, or the condition of advising according to manufacturer.

Experiment material and method

1. vegetable material and growing environment

Willow Gene cloning is from comospore poplar (Populustrichocarpa), and expression vector all proceeds to commercially available southern woods 895In kind, poplar seedlings is cultivated glasshouse in the controlled environment chamber, 27 DEG C of natural lightings, and within 2 years, raw sapling is transplanted farm natureWeather plantation.

Be Colombia's type for the arabidopsis transforming, plant in 22 DEG C of phjytotrons, 12 hour photoperiod cultivated.

2. Gene cloning

The genome sequence of PtMAN6 is downloaded (USDepartmentofEnergy, Joint from JGI databaseGenomeInstitute(JGI),http://www.phytozome.net/poplar）。

The xylem of growing from comospore poplar by CTAB method extracts RNA, and with the DNaseI removal DNA dirt of RNase-freeDye.

With primer 5 ' TGAATGGCTAATGGTGGCAAAGA3 ' (SEQIDNO.:4) and 5 'TCAGGAGCCGATGGTCCATAAAA3 ' is the part cDNA of the clone of the method by RT-PCR PtMAN6 (SEQIDNO.:5)Sequence, then use the method for 5 ' RACE clone 5 ' end of this gene (Kit, Invitrogen). With primer 5 'CTGACTCAATACTATGGATACCC3 ' (SEQIDNO.:6) and 5 ' CTTCCTTTTCCTGTTGTGACCGA3 ' (SEQIDNO.:7) complete encoding sequence of clone PtMAN6.

3. gene expression analysis

Gene expression analysis adopts the method for quantitative RT-PCR, the primer of the design gene specific specific gene sheet that goes to increaseSection (100-300bp). The total RNA PrimerScript of 1 μ g^TMReverse transcription kit (Dalian is precious biological) reverse transcription becomes the first chainCDNA. quantitative PCR adopts SYBRgreen method at MyiQ^TMReal-TimePCR instrument (Bole, the U.S.) is upper to be detected. In willowPtActin gene is as reference gene.

4. antibody preparation

By the sequence alignment of PtMAN family gene, selected two little peptide sections in the non-conservative district of PtMAN6,EQFKTMVEEVDNH (37 to 49 amino acids, SEQIDNO.14) and ELNDVEEDEWL(61 position to 71 amino acids,SEQIDNO.15),, by artificial synthetic, be injected into and in rabbit, produce polyclonal antibody (entrusting Ai Bimate company to complete). Anti-Body detects and can only detect single band in xylem total protein through Western. Monoclonal antibody Anti-GFP and anti-Actin buys from Ai Bimate (Chinese Shanghai) company.

5. vector construction and Plant Transformation

5.135S:PtMAN6 and 35S:PtMAN6AS vector construction:

Following primer 5 ' CTT for the CDS sequence of total length PtMAN6TCTAGACTGACTCAATACTATGGATACCC3’（XbaI) (SEQIDNO:10) and 5 ' CTGCTCGAGCCGATCAACTACTTTTACAAATC3’（XhoI）（SEQIDNO.:11）Or:

5’CTTCTCGAGCTGACTCAATACTATGGATACCC3 ' is (SEQIDNO.:12) and 5 ' (XhoI)CTGTCTAGACCGATCAACTACTTTTACAAATC3 ' is (SEQIDNO.:13) amplification (XbaI), with corresponding enzyme cuttingAmplified fragments, by its subclone in commercially available pBI121 carrier framework.

5.2 transient expression vector constructions:

The CDS sequence of total length PtMAN6 or its front 31 amino acid sequences by subclone to pA7 carrier and carrierEGFP merges. These two carriers called after MAN6G or MAN6sG respectively.

5.335S:PtMAN6-GFP vector construction:

By upper MAN6G CaMV35S promoter, PtMAN6-GFP and NOS terminator together subclone to pCAMBIA2300Carrier.

5.4 Plant Transformation

Stably express, is first transformed into carrier GV3101 agrobacterium strains, preparation engineering bacterium.

Transformation of Arabidopsis thaliana adopts general agriculture bacillus mediated flower-dipping method.

Willow transforms and adopts general Ye Panfa.

6. the extraction of inscribe-Isosorbide-5-Nitrae-'beta '-mannase and enzyme activity determination in willow

Fresh Tissues of Poplar Clones is ground to form to fine powder fast with liquid nitrogen, then add the zymoprotein of 1.5 times of volume precoolingsExtraction buffer is placed on ice 1 hour, 10000g4 DEG C of centrifugal 30min, and then supernatant filters by Miracloth, thenSuper filter tube by 10KD is concentrated and purified. The albumen of purifying BCA reagent quantitative.

Utilize colorimetric method to carry out enzyme assay. Insoluble substrate A ZCL-galactomannan buys from Megazyme(Ireland), 1%(w/v) substrate be dissolved in citric acid-phosphate buffer (pH3.0-8.0) or the 0.1M sodium acetate buffer of 0.1MLiquid (pH5.0) is made suspension. Get the zymoprotein (or BSA contrast) that the above-mentioned substrate suspension of 100 μ l adds 20 μ g purifying, useCorresponding buffer polishing to volume is 200 μ l, is then placed in specific reaction temperature reaction 2h, keeps during this time shake to make alwaysReaction system is even, 100 DEG C of 5min cessation reactions, the centrifugal 5min of 12000g, and the change of surveying absorbance in 590nm, enzymatic activityWith A590nmmg^-1min^-1Represent, result is at least wanted independent repetition three times.

7.PtMAN6 deglycosylation

Analyze for Western, the PtMAN6 of purifying first, through 100 DEG C of sex change 10min, then adds according to descriptionEndoH_f37 DEG C of processing 30min of enzyme (NewEnglandBiolabs) or 1h, sample, in 100 DEG C of inactivation 10min, is got 20 μ lCarry out SDS-PAGE detection.

Survey for enzyme biopsy, directly add from 60 μ g zymoproteins of overexpression PtMAN6 plant or the extraction of wild type plantEnter the EndoH of 6000 units_f37 DEG C of processing 2h of enzyme, the albumen of getting 20 μ g processing carries out enzyme activity assay, and each sample repeats threeInferior.

8. plant section analysis

Lignin deposition detects: 1 year growing poplar stem or petiole free-hand section, and then use 1% phloroglucin (to be dissolved in 12% saltAcid) processing 5min, then microscopic examination at once.

Lignin autofluorescence and conduit area measurement: FAA(5% formaldehyde, 5% acetic acid, 70% ethanol) fixing stem section, stoneWax embedding, after 10 μ m section dehydrations, direct ultraviolet is observed. With ImageJ software statistics conduit area, Student ' st checks systemMeter result.

9. the log in number of the willow gene of studying in the present invention in NCBI is:

PtMAN6XM_002323644，PtrPAL(XM_002326150)，

PtrC4H1(EU603304)，Ptr4CL3(XM_002297663)，

PtrHCT1(EU603313)，PtrC3H1(XM_002308824)，

PtrCCoAOMT1(XM_002313089，EU603307)，

PtrCCR2/7(EU603310，XM_002303809)，

PtrCAld5H1(EU603312)，PtrCOMT2(XM_002317802,EU603317)，

PtrCAD1(EU603306)，PtrLAC17(XM_002317469)，

PtrCesA8(XM_002316779)，PtrGT43B(JF518935)，

PtrWND1A(HQ215847，XM_002317023)，

PtrWND1B(HQ215848)，PtrWND2A(HQ215849)，

PtrWND3A(XM_002322362)，PtrWND3B(XM_002318252)，

PtrWND4A(XM_002329829)，PtrWND5A(XM_002310261)，

PtrWND6A(XM_002327206)，PtrMYB3(XM_002299908)，

PtrMYB20(XM_002313267)，PtrMYB28(XM_002307154)，

Pt-IAA8(XM_002302315)，PtrXCP1(XM_002328102)，

PtrACT1(XM_002298674)。

The expressed sequence of embodiment 1PtMAN6 and the clone of promoter

1. sequence information

The CDS sequence that PtMAN6 is complete is as follows:

atggatacccacaagagggtatttgggttcaagatcattttcttagtgtccgtttttatt60

cttcttaatgaaagttcaaaatgcagcagctctggtgttatggatgatgaacaatttaaa120

acaatggtggaggaagtagacaatcatctaccatcttctagctcaagtcaaggggtttat180

gagttgaatgatgtggaagaagatgaatggctaatggtggcaaagaaaggaaaccagttt240

gtgatcaatgaccaacctttctatgtcaatggatttaacacatactggctgatggtgttt300

gctgctgatcaatctaccagaggaaaggtcactgaggttttccaaaaagcatcctcagtt360

ggtctatcagtttgcaggacttgggcttttaatgatggtcaatggagagctcttcaaaaa420

tctccaggtgtttatgatgaagatgttttcaaggccctggattttgtggtcagtgaagca480

aataagtacaagatcaggctcatattatcattggctaacaattgggatgcatatggtgga540

aaagcacaatatgttaaatggggaaaagcttctggccttaatttgacatctgatgatgat600

ttcttctctcatccaactctcagaagctactacaaggctcatgtcaaggcggtattgaat660

agagtcaatacgatcacaaacataacctacaaggatgaccctacaatatttgcttgggag720

ctgatgaatgaacctcgatgcacctcagatccctctggcgataaactgcagtcatggata780

acagacatggcagtatatgtgaagagcatggacgcaaagcacttagtagagattggattg840

gagggattttatggaccatcggctcctgatagggctcagttcaatccaaactcgtatgct900

acgcaagttggaaccgactttatcaggaaccatcaggttcttggtgttgattttgcttct960

gttcacatatatgcagactcctggatttcgcaaacaattacggattctcatatccaattc1020

accaagtcatggatggaagctcacatagaggatgctgagaaatatctgggaatgccggtt1080

gtgttcgctgagtttggtgtatcttcaaaagatcctggatacaactcatcattccgcgac1140

acactaattaacacagtgtacaagaccctcttgaactcaaccaagagaggtgggagtgga1200

gctgggagccttctgtggcagcttttccctgacgggacagactacatggatgatggatat1260

gcaattgttctatcaaaatctccttccacaacaaacatcatttccctccattcaacacga1320

gtcgcaatcgtcaattccatgtgttcatggaaatgcaaatggggctgcaagaagaggaat1380

cctttagaggcattcctctaccatgatgatttgtaa1416

（SEQIDNO.:1）

Or

atggatacccacaagagggtatttgggttcaagatcattttcttagtgtccgtttttatt60

cttcttaatgaaagttcaaaatgcagcagctctggtgttatggatgatgaacaatttaaa120

acaatggtggaggaagtagacaatcatctaccatcttctagctcaagtcaaggggtttat180

gagttgaatgatgtggaagaagatgaatggctaatggtggcaaagaaaggaaaccagttt240

gtgatcaatgaccaacctttctatgtcaatggatttaacacatactggctgatggtgttt300

gctgctgatcaatctaccagaggaaaggtcactgaggttttccaaaaagcatcctcagtt360

ggtctatcagtttgcaggacttgggcttttaatgatggtcaatggagagctcttcaaaaa420

tctccaggtgtttatgatgaagatgttttcaaggccctggattttgtggtcagtgaagca480

aataagtacaagatcaggctcatattatcattggctaacaattgggatgcatatggtgga540

aaagcacaatatgttaaatggggaaaagcttctggccttaatttgacatctgatgatgat600

ttcttctctcatccaactctcagaagctactacaaggctcatgcggtattgaatagagtc660

aatacgatcacaaacataacctacaaggatgaccctacaatatttgcttgggagctgatg720

aatgaacctcgatgcacctcagatccctctggcgataaactgcagtcatggataacagac780

atggcagtatatgtgaagagcatggacgcaaagcacttagtagagattggattggaggga840

ttttatggaccatcggctcctgatagggctcagttcaatccaaactcgtatgctacgcaa900

gttggaaccgactttatcaggaaccatcaggttcttggtgttgattttgcttctgttcac960

atatatgcagactcctggatttcgcaaacaattacggattctcatatccaattcaccaag1020

tcatggatggaagctcacatagaggatgctgagaaatatctgggaatgccggttgtgttc1080

gctgagtttggtgtatcttcaaaagatcctggatacaactcatcattccgcgacacacta1140

attaacacagtgtacaagaccctcttgaactcaaccaagagaggtgggagtggagctggg1200

agccttctgtggcagcttttccctgacgggacagactacatggatgatggatatgcaatt1260

gttctatcaaaatctccttccacaacaaacatcatttccctccattcaacacgagtcgca1320

atcgtcaattccatgtgttcatggaaatgcaaatggggctgcaagaagaggaatccttta1380

gaggcattcctctaccatgatgatttgtaa1410

（SEQIDNO.:16）

The amino acid sequence of PtMAN6 albumen is as follows:

MDTHKRVFGFKIIFLVSVFILLNESSKCSSSGVMDDEQFKTMVEEVDNHLPSSSSSQGVY60

ELNDVEEDEWLMVAKKGNQFVINDQPFYVNGFNTYWLMVFAADQSTRGKVTEVFQKASSV120

GLSVCRTWAFNDGQWRALQKSPGVYDEDVFKALDFVVSEANKYKIRLILSLANNWDAYGG180

KAQYVKWGKASGLNLTSDDDFFSHPTLRSYYKAHVKAVLNRVNTITNITYKDDPTIFAWE240

LMNEPRCTSDPSGDKLQSWITDMAVYVKSMDAKHLVEIGLEGFYGPSAPDRAQFNPNSYA300

TQVGTDFIRNHQVLGVDFASVHIYADSWISQTITDSHIQFTKSWMEAHIEDAEKYLGMPV360

VFAEFGVSSKDPGYNSSFRDTLINTVYKTLLNSTKRGGSGAGSLLWQLFPDGTDYMDDGY420

AIVLSKSPSTTNIISLHSTRVAIVNSMCSWKCKWGCKKRNPLEAFLYHDDL471

（SEQIDNO.:2）

Or

MDTHKRVFGFKIIFLVSVFILLNESSKCSSSGVMDDEQFKTMVEEVDNHLPSSSSSQGVY60

ELNDVEEDEWLMVAKKGNQFVINDQPFYVNGFNTYWLMVFAADQSTRGKVTEVFQKASSV120

GLSVCRTWAFNDGQWRALQKSPGVYDEDVFKALDFVVSEANKYKIRLILSLANNWDAYGG180

KAQYVKWGKASGLNLTSDDDFFSHPTLRSYYKAHAVLNRVNTITNITYKDDPTIFAWELM240

NEPRCTSDPSGDKLQSWITDMAVYVKSMDAKHLVEIGLEGFYGPSAPDRAQFNPNSYATQ300

VGTDFIRNHQVLGVDFASVHIYADSWISQTITDSHIQFTKSWMEAHIEDAEKYLGMPVVF360

AEFGVSSKDPGYNSSFRDTLINTVYKTLLNSTKRGGSGAGSLLWQLFPDGTDYMDDGYAI420

VLSKSPSTTNIISLHSTRVAIVNSMCSWKCKWGCKKRNPLEAFLYHDDL469

（SEQIDNO.:17）

Clone's PtMAN6 promoter sequence is as follows:

tgaggaccgaaatgagagaaaaaaacatatgagcgaaaacccttggtcaatagttctgaa60

ttttgttgatcttgaggataaaagtgtatttttactgtccaaataatttgaaaaagacta120

atatacccccaaataattttttaatgaccaataaacatcttgaaaagaccaaactaccct180

tatcataaagccttctattataaaattataatcttattatagcaatccacagtcaattat240

aactagtgttacaatgaaaaaccctctccctagtccctatatttcatgttaattgatatt300

gttaataattggcagaacacaggtgcgcacgagtcttaataataaatctcattcaaggtt360

tgggtaattgttttattaggtcaactttaaaattatattgttttttatttaaaaattact420

caaccatgtgtcacttggatttgactccatcaactagtcaatgggatttgattaaaacaa480

ccttcaagaatggcagtagcctctctaacaacaggggtgcaataattcgataaaaacaag540

aaaaacaagaaaaatcctgaacttttgggtgacaattctagcaaggcccagatagaaata600

gaaaaataccagagccatgcaagatgagtgcttcataatgggccaacaaaaatagttcta660

aaaaaaaaaaactgggctaagatacaacattttgcatggtagacttttttttttttcatt720

ataaaaaaacaagggatcgaaatctaagcttctgatggctgaattcagctcatatcttcc780

ctgaaaattttgaatttcaagtttgattaatccaatgttaggtttttgattcaataaaaa840

caaagcaaaatgatgcaaaaatataaactctcgaagccataccaggatacataatgatat900

caaatacgttgttatgattaaagcaggacctcagattccttgggagctaccttttgctac960

ttcaaagttaaagatagttttttccagttgaatttcaagtttgattaatccaatgttaat1020

gggaactttgagactgcaacagagcacagatatgttaatttgacccaaaaacaggacaga1080

aacaaaaagcatatgatagttgataaatcagacaggttatgtgtttggacaatcaaaggt1140

tgccaggctacttaacaggattttttctaggtctgccatatatatatgaaacattcaagt1200

taagcttgcaacagatcagcttagttctgctcacctgcttaccagctccaatatacttct1260

tcaatctcctactctccagattgctttttgcagagaaatggcatgcatcgatttcgtctt1320

tctttccatggatttatttcttccgtgtttgtctatgtatatgtgtgtgtagagaccaat1380

ctagtttgttggttttaggacatgtcaacatgttcttcaagaaagatgcagtgaccttga1440

agtaatgtcagtgtcatctgaactacaagttatctaggtttaaaagggtctcattttctt1500

gtaggacccttaaaacttaccttaggagtcatacctgaagccttgtttaagtatgcaact1560

agacctaggtctgtagcttttgcttgtgtatatccatctatgtatctgtgtttggaagaa1620

aagaaaatggcagagaaagtataagaattgaaattttcttggatttaaagccatggtgta1680

gatcccctcttcaacactcttcttgcatatcacagtttctgctacagccttagatggcca1740

tgcatataaatatggggggtccagtgaagaggggtgtgcaacccttggtttgctgactca1800

at1802

（SEQIDNO.:3）

2. vector construction

2.1 Overexpression vector

Fig. 1 is shown in by 35S:PtMAN6 carrier collection of illustrative plates; 35S:PtMAN6-GFP carrier collection of illustrative plates Fig. 2.

2.2 antisense expression vector

Fig. 3 is shown in by 35S:PtMAN6AS collection of illustrative plates.

2.3 transient expression carriers

MAN6G transient expression carrier is shown in Fig. 4, and MAN6sG transient expression carrier is shown in Fig. 5.

Embodiment 2 crosses expression PtMAN6, and the content of wood lignin and avicel cellulose is reduced

Western detects proves that PtMAN6 crosses expression (OE) carrier and antisense (AS) expression vector successfully proceeds to willow, andAnd in plant, can express, (Fig. 6 A) further plays a role.

Transfer-gen plant analysis shows, with respect to wild type plant, OE plant petiole and cane deliquescing (Fig. 6 B, 6C and6D), AS plant is contrary (Fig. 6 B, 6C and 6E), illustrates that PtMAN6 participates in the mechanical strength of regulation and control cane. Further section pointAnalyse and show, in OE plant petiole, the content of lignin reduces (Fig. 6 F-H). In same stem, lignin deposition also reduces (Fig. 7 A-I).The lignin of autumn wood and avicel cellulose assay further prove PtMAN6 function, cross and express this gene, cause woodIn material, lignin and crystalline fibers cellulose content reduce (Fig. 7 J, 7K).

The living beings of embodiment 3 transgenic poplars obviously do not change

The present embodiment has been measured the living beings of willow, and result shows, with respect to wild type, and 1.5 years raw transgenic poplar woodThe height of material and girth all do not have marked change (Fig. 8).

Fig. 8 shows height and the girth analysis result of 1.5 years raw transgenic poplar timber, and wherein, Fig. 8 A showed scaleReach PtMAN6 willow (OE), the willow (AS) of antisense expression PtMAN6 is obviously not poor with the trunk height of wild type (WT) willowDifferent; Fig. 8 B shows that the girth of the butt of OE, AS and WT willow does not have notable difference, and statistical analysis adopts T inspection.

Embodiment 4 genetically modified plants timber monosaccharide components change

The present embodiment has been measured the component of various monose in poplar wood, and result shows, timber monose in genetically modified plantsComponent has very large change. Compared with wild type plant (WT), cross Poplar Cultivars (PtMAN6OE) wood of expressing PtMAN6-GFPThe monosaccharide component of secondary cell wall enrichment in material, as wood sugar and mannose content reduction, and corresponding primary cell wall enrichment listSugar content raises; Trend and PtMAN6OE strain timber phase that in antisense expression Poplar Cultivars (PtMAN6AS), monosaccharide component changesInstead, concrete outcome is in table 1.

Monosaccharide component is measured by GC – MS, and unit is μ gmg^-1AIR. In table, PtMAN6OE and PtMAN6AS represented respectivelyExpress the transgenic poplar of PtiMAN6-GFP and PtiMAN6 antisense vector. Statistical analysis adopts T inspection,^*Represent p <0.05，^**Represent p < 0.01 (n=4).

Willow enrichment hemicellulase β-Isosorbide-5-Nitrae-inscribe mannase of embodiment 5 overexpression PtMAN6

The present embodiment has been measured the β-Isosorbide-5-Nitrae-inscribe mannosan enzymatic activity in willow plant, and result shows, with wild typeCompare, cross in expression plant at PtMAN6, a large amount of enrichments β-Isosorbide-5-Nitrae-inscribe mannase (Fig. 9), this enzyme optimal reaction pHValue is 5.0, and optimal reactive temperature is 50 DEG C, and glycosylation is most important to maintaining the hydrolysing activity of PtMAN6.

Fig. 9 has shown the enzyme activity assay of PtMAN6. Wherein, the optimal pH measurement result that Fig. 9 A is PtMAN6, substrate isBeing dissolved in pH scope is the 1%AZCL-glactomannan suspension of 0.1M citric acid-phosphate buffer of 3.0-8.0, WT andPtMAN6 refers to respectively from wild type and crosses and express the zymoprotein (lower same) extracting plant mature leaf; Fig. 9 B shows PtMAN6'sOptimal reactive temperature measurement result, substrate is the 1%AZCL-that is dissolved in 0.1M citric acid-phosphate buffer of pH5.0Glactomannan suspension; Fig. 9 C shows PtMAN6 deglycosylation result, in Werstern hybridization analysis plant, extractsPtMAN6(the first swimming lane), and EndoH_fProcess the PtMAN6 of 0.5 hour (the second swimming lane) and 1 hour (the 3rd swimming lane), M:The protein label dying in advance; Fig. 9 D is that glycosylation affects enzymatic activity result, compares PtMAN6 warp with contrasting (control)EndoH_fDeglycosylation 2 hours, enzyme work is reduced to original 50%.

Embodiment 6PtMAN6 gene can be used as xylem vessel's cell marking gene

The present embodiment uses the methods analyst PtMAN6 gene of quantitative RT-PCR in the expression of different parts, finds that PtMAN6 is excellentGesture is expressed in xylem (Figure 10). Further immunohistochemical analysis, finds the special immature wood of willow that is positioned of PtMAN6Matter portion vessel cell. At cambial cell, framing signal (figure in xylem fibrocyte and parenchyma cell, all can not be detected11). Therefore this gene can be used as the marker gene of xylem vessel's cell.

Figure 10 shows the quantitative PCR analysis result of PtMAN6 gene in willow. From the xylem of 1 year growing poplar, tough respectivelySkin zone, spire, Lao Ye and stem top tissue extraction RNA carry out quantitative RT-PCR analysis, using Actin gene in willow as interiorGinseng. Figure 11 shows the immunolocalization analysis result of PtMAN6. Wherein, Figure 11 A and Figure 11 B are Section six, 1 year raw wild type willowBetween sectional view, show PtMAN6 be positioned xylem vessel's cell; Figure 11 B is the enlarged drawing at black surround position in Figure 11 A; Figure11C is the rip cutting figure of 1 year raw wild type willow the 6th internode, shows that PtMAN6 is positioned xylem vessel's cell; Figure 11 D and figure11E is the sectional view of 1 year raw wild type willow stem top tissue, shows that PtMAN6 is positioned xylem vessel's cell; Figure 11 E isThe enlarged drawing at black surround position in Figure 11 D; The negative contrast of Figure 11 F, the sectional view of 1 year raw wild type willow the 6th internode, failsAny framing signal detected.

The location of embodiment 7PtMAN6-GFP albumen

The present embodiment is by transient expression PtMAN6-GFP in stably express in arabidopsis and onion epidermis cell, knotFruit all shows that PtMAN6 protein localization is in plasma membrane, and the contrast that only turns GFP shows that GFP signal spreads all over whole cell, comprises nucleusAnd kytoplasm.

Embodiment 8PtMAN6 negative regulation timber forms the expression of related gene

Quantitative RT-PCR testing result shows, in PtMAN6 overexpression plant, forms relevant PtrWND to timber(PtrWNDA-PtrWND6A) and some myb transcription factors (PtrMYB3,20,28) all lower (Figure 13), with secondary wallSynthetic relevant biosynthetic pathway of lignin gene, the table of cellulose synthetic gene (CesA8) and xylan synthetic gene (GT43B)Reach equally all decline to some extent (Figure 14). Thereby PtMAN6 has affected the thickening of normal cell membrane by these genes of negative regulation,Further affect Wood Properties Within.

Figure 13 has shown relative expression's level of secondary wall formation associated transcription factor in the plant of overexpression PtMAN6.RNA extracts from 1 year growing poplar xylem, and the expression of wild type is defined as 1, and willow Actin gene is as internal reference.

Figure 14 has shown in the plant of overexpression PtMAN6 and has changed with the expression of secondary cell wall composition synthesis related geneBecome. Figure 14 A shows that, in the plant of overexpression PtMAN6, the gene of lignin monomer route of synthesis and lignin polymerization are relevantGene is suppressed; Figure 14 B shows in the plant of overexpression PtMAN6, the cellulose synthetic gene CesA8 that secondary wall is relevantExpression be suppressed; Figure 14 C shows in the plant of overexpression PtMAN6, the xylan synthetic gene that secondary wall is relevantThe expression of GT43B is suppressed. RNA extracts from 1 year growing poplar xylem, and the expression of wild type is defined as 1, willow ActinGene is as internal reference.

All documents of mentioning in the present invention are all quoted as a reference in this application, just as each section of document quilt separatelyQuote as a reference. In addition should be understood that those skilled in the art can after having read above-mentioned instruction content of the present inventionSo that the present invention is made various changes or modifications, these equivalent form of values fall within the model that the application's appended claims limits equallyEnclose.

Claims

1. a purposes for the coded sequence of PtMAN6 polypeptide or PtMAN6 polypeptide, is characterized in that, described PtMAN6 polypeptideAmino acid sequence is as shown in SEQIDNO:2, or the coded sequence of described PtMAN6 polypeptide is as shown in SEQIDNO:1, described inPurposes is to reduce the content of lignin.

2. purposes as claimed in claim 1, is characterized in that, the content of described reduction lignin is wooden for reducing plant stemThe content of element.

3. the purposes of PtMAN6 polypeptide as claimed in claim 1 or PtMAN6 polypeptid coding sequence, is characterized in that, described useWay also comprises:

Reduce the synthetic of xylem secondary cell wall.

4. the purposes of PtMAN6 polypeptide as claimed in claim 1 or PtMAN6 polypeptid coding sequence, is characterized in that, described useWay also comprises:

Reduce the accumulation of crystalline fibers living beings.

5. improve a method for plant trait, wherein, described improvement plant trait is:

Reduce the content of plant lignin;

It is characterized in that, comprise step: improve the PtMAN6 polypeptide shown in SEQIDNO:2 or its coded sequence in described plantExpression or activity.

6. the method for improvement plant trait as claimed in claim 5, wherein, described improvement plant trait also comprises: reduceThe secondary cell wall of plant xylem is synthetic.

7. the method for improvement plant trait as claimed in claim 5, wherein, described improvement plant trait also comprises: reduceThe accumulation of plant crystalline fibers living beings.

8. method as claimed in claim 5, is characterized in that, described method comprises step: by shown in SEQIDNO:2The coded sequence of PtMAN6 polypeptide imports in plant cell, cultivates described plant cell, regeneration plant; And, described in cloneThe primer of the coded sequence of PtMAN6 polypeptide is as shown in SEQIDNO:10 and SEQIDNO:11.