CN110331149A - The method of the application of PagKNAT2/6b gene and poplar adjusted and controlled plant type - Google Patents
The method of the application of PagKNAT2/6b gene and poplar adjusted and controlled plant type Download PDFInfo
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- CN110331149A CN110331149A CN201910729683.7A CN201910729683A CN110331149A CN 110331149 A CN110331149 A CN 110331149A CN 201910729683 A CN201910729683 A CN 201910729683A CN 110331149 A CN110331149 A CN 110331149A
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Abstract
The present invention provides the methods of a kind of application of PagKNAT2/6b gene and poplar adjusted and controlled plant type, are related to field of biotechnology.PagKNAT2/6b gene is the gene for expressing (a1) or (a2): (a1): the albumen containing the amino acid sequence as shown in SEQ ID NO.1；(a2): containing the amino acid sequence with 90% or more identity of amino acid sequence described in (a1), and albumen with the same function.The application of PagKNAT2/6b gene includes poplar adjusted and controlled number of branches and stem pliability, and expression quantity variation of the gene in poplar will affect the formation of poplar branch and the pliability of poplar stem.The method of the poplar adjusted and controlled plant type, including poplar adjusted and controlled number of branches and poplar stem pliability, this method are realized by the expression quantity of poplar adjusted and controlled middle PagKNAT2/6b gene, and the plant type of poplar can more accurately be transformed, and improve regulation efficiency.
Description
Technical field
The present invention relates to field of biotechnology, application more particularly, to a kind of PagKNAT2/6b gene and poplar adjusted and controlled
The method of plant type.
Background technique
Branch has important biological significance to plant growth and development, and crotch angle and quantity directly affect the intercepting and capturing of light
Ability, photosynthetic efficiency and the planting density of plant, the direct shadow of number of branches in the agriculture herbaceous plant such as rice, wheat
Ring its biological yield.And for fancy horticulture class plant, the number of number of branches directly affects plant entirety plant type, to influence
The visual experience of audience.
Plant physiology research has determined auxin, the basic element of cell division, only angle gold lactone, gibberellin, abscisic acid, indoles
The plant hormones such as acetic acid have important regulating and controlling effect to axillary bud development, and wherein auxin and abscisic acid inhibit the generation of branch,
The basic element of cell division promotes branch to generate；Simultaneously intensity of illumination and optical density be influence that most of species axillary bud occurs it is main because
Element.
Poplar is as a kind of economic tree in world's distribution in extensive range, by its growth is rapid, breeding is easy, timber
Wide, the adaptable feature of purposes, becomes China's fast-growing timber forests the most famous and green tree species, and cultivation breeding process is gone through
To be taken seriously.And there is no a kind of methods of effectively poplar adjusted and controlled plant type in the prior art, therefore one kind can orient and change
The method for making poplar plant type needs at present.
In view of this, the present invention is specifically proposed.
Summary of the invention
The first object of the present invention is to provide a kind of application of PagKNAT2/6b gene, and the application includes regulation poplar
The number of branches and/or stem pliability of tree.
The second object of the present invention is to provide a kind of method of poplar adjusted and controlled plant type, and this method is by changing in poplar
The expression quantity of PagKNAT2/6b gene, to change poplar number of branches and change poplar stem pliability, to realize poplar adjusted and controlled
Plant type.
In order to solve the above technical problems, spy of the present invention adopts the following technical scheme that
According to an aspect of the present invention, the present invention provides a kind of application of PagKNAT2/6b gene, which includes
Poplar adjusted and controlled number of branches and/or stem pliability；The PagKNAT2/6b gene is the gene for expressing (a1) or (a2):
(a1): the albumen containing the amino acid sequence as shown in SEQ ID NO.1；
(a2): containing the amino acid sequence with 90% or more identity of amino acid sequence described in (a1), and having identical
The albumen of function.
According to another aspect of the present invention, the present invention also provides a kind of method of poplar adjusted and controlled plant type, this method packets
The expression quantity for changing PagKNAT2/6b gene in poplar is included, by changing poplar number of branches and changing poplar stem pliability
Carry out poplar adjusted and controlled plant type.
Compared with prior art, the invention has the following beneficial effects:
PagKNAT2/6b gene is the homologous gene of arabidopsis KNAT2 and KNAT6, changes PagKNAT2/6b base in poplar
The expression of cause will affect the formation of poplar branch and the pliability of poplar stem.Therefore, pass through poplar adjusted and controlled middle PagKNAT2/
The expression of 6b gene can influence the plant type of poplar, the different scapes to obtain the poplar for being suitable for different demands, applied to poplar
See moulding.The present invention is found through experiments that, raises the expression of the PagKNAT2/6b gene in poplar, can increase poplar branch
Number improves stem pliability so that poplar is easy moulding to improve the crown type of poplar.
In the method for poplar adjusted and controlled plant type provided by the invention, the transformation of the poplar plant type can be by changing poplar branch
Number and stem pliability are realized.Since PagKNAT2/6b gene is related to poplar number of branches and stem pliability, adjust
The expression quantity of control PagKNAT2/6b gene can be realized the regulation to poplar plant type, grant growth to plant compared to traditional
The expression of the mode of the allogenic materials such as element, the basic element of cell division and abscisic acid, the gene of direct regulation and control poplar can be more precisely and fixed
The plant type poplar adjusted and controlled to ground regulates and controls high-efficient.
Detailed description of the invention
It, below will be to specific in order to illustrate more clearly of the specific embodiment of the invention or technical solution in the prior art
Embodiment or attached drawing needed to be used in the description of the prior art be briefly described, it should be apparent that, it is described below
Attached drawing is some embodiments of the present invention, for those of ordinary skill in the art, before not making the creative labor
It puts, is also possible to obtain other drawings based on these drawings.
Fig. 1 is the structural schematic diagram that expression vector pMDC32 is overexpressed PagKNAT2/6b gene in the present invention；
Fig. 2 is the structural schematic diagram that expression vector pCambia1300 is overexpressed PagKNAT2/6b gene in the present invention；
Fig. 3 A is wild type poplar；
PagKNAT2/6b-OE poplar in Fig. 3 B present invention；
GFP-PagKNAT2/6b poplar in Fig. 3 C present invention；
In Fig. 3 D present invention in PagKNAT2/6b-OE poplar OE5 strain and OE7 strain PagKNAT2/6b gene expression
Amount；
PagKNAT2/ in GFP-PagKNAT2/6b poplar GFP1 strain, GFP6 strain and the strain of GFP8 system in Fig. 3 E present invention
The expression quantity of 6b gene；
The plant type of three months earth cultures of PagKNAT2/6b-OE poplar growth compares in Fig. 4 A present invention；
The plant type of four months earth cultures of PagKNAT2/6b-OE poplar growth compares in Fig. 4 B present invention；
The plant type of five months earth cultures of PagKNAT2/6b-OE poplar growth compares in Fig. 4 C present invention；
The plant type of 1 year earth culture of PagKNAT2/6b-OE poplar growth compares in Fig. 4 D present invention；
The plant type of GFP-PagKNAT2/6b poplar tissue-cultured seedling compares in Fig. 5 A present invention；
The plant type of one month earth culture of GFP-PagKNAT2/6b poplar growth compares in Fig. 5 B present invention；
The plant type of three months earth cultures of GFP-PagKNAT2/6b poplar growth compares in Fig. 5 C present invention；
Pass through cell wall thickness in scanning electron microscope and transmission electron microscope analysis PagKNAT2/6b-OE poplar stem in Fig. 6 A present invention
Degree, Ve, vessel, conduit；Xf, xylem fiber, xvlem fibres；
Fig. 6 B is vessel cell wall thickness in OE5 strain in the present invention and OE7 strain stem；
Fig. 6 C is the cell wall thickness of fibrocyte in OE5 strain and OE7 strain stem in the present invention；
Fig. 7 is the cell wall of GFP-PagKNAT2/6b poplar GFP1 strain, GFP6 strain and GFP8 strain stem in the present invention
Thickness compares figure.
Specific embodiment
Technical solution of the present invention is clearly and completely described below in conjunction with embodiment, it is clear that described reality
Applying example is a part of the embodiment of the present invention, instead of all the embodiments.Based on the embodiments of the present invention, the common skill in this field
Art personnel every other embodiment obtained without making creative work belongs to the model that the present invention protects
It encloses.
According to an aspect of the present invention, the present invention provides the application of PagKNAT2/6b gene, the application includes adjusting
Control the number of branches and/or stem pliability of poplar；The PagKNAT2/6b gene is the gene for expressing (a1) or (a2):
(a1): the albumen containing the amino acid sequence as shown in SEQ ID NO.1；
(a2): containing the amino acid sequence with 90% or more identity of amino acid sequence described in (a1), and having identical
The albumen of function.
Albumen containing the amino acid sequence shown in SEQ ID NO.1 refer to as described in the amino acid sequence of albumen can
Think all amino acid sequences as shown in SEQ ID NO.1, can also as the amino acid sequence as shown in SEQ ID NO.1 and
Other amino acid composition, effect of other amino acid in albumen include but is not limited to label, glimmering of the composition for protein purification
Photoprotein marker and the binding site of DNA etc., for example can be in some specific embodiments but be not limited to HIS, GST,
MyC, FLAG, HSV, V5, HA, GFP, RFP, BFP, CAT, DHFR, MBP, T7 or thioredoxin etc..
Wherein " identity " refers to the similitude with amino acid sequence shown in SEQ ID NO.1.With shown in SEQ ID NO.1
Amino acid sequence difference may by the change of one or several amino acid, missing or insertion cause, these changes make egg
White molecule and amino acid sequence shown in SEQ ID NO.1 are not quite identical, but at least have 90% or more, such as can be but
It is not limited to 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity, meanwhile, these
There is the protein molecular of 90% or more identity with amino acid sequence shown in SEQ ID NO.1 and containing shown in SEQ ID NO.1
Amino acid sequence protein molecular function having the same.Identity can with the naked eye or computer software is evaluated, example
Such as it is compared using the blast software of this field routine.Wherein the PagKNAT2/6b gene is expression containing such as SEQ ID
The effect when gene of the albumen of amino acid sequence shown in NO.1, applied to poplar adjusted and controlled number of branches and poplar stem pliability
Preferably.
In some alternative embodiments, the PagKNAT2/6b gene nucleotide series are (b1) or (b2):
(b1): containing the nucleotide sequence as shown in SEQ ID NO.2；
(b2): containing with 90% or more identity of nucleotide sequence described in (b1), and nucleotide with the same function
Sequence.
" the containing " refers to that the PagKNAT2/6b gene nucleotide series can be containing only just like SEQ ID NO.2
Shown in nucleotide sequence, can also the nucleotide sequence shown in SEQ ID NO.2 and other nucleotide sequences form, such as
The nucleotide sequence of the functional units such as binding site of the coding for the label of protein purification, fluorescence egg marker and DNA, or
Element of the coding to genetic transcription and expression with adjustment effect, including but not limited to promoter, strong promoter, enhancer turn
Record factor binding site etc.；Described " containing " can also refer to that the nucleotides sequence as shown in SEQ ID NO.2 is listed in PagKNAT2/6b
It is discontinuous in gene, but can produce the nucleic acid sequence encoding of the nucleotide sequence as shown in SEQ ID NO.2
(cDNA)。
Wherein " identity " refers to the similitude with nucleotide sequence shown in SEQ ID NO.2.With shown in SEQ ID NO.2
The difference of nucleotide sequence may be by the change of one or several nucleotide or missing or insertion and the letter of codon
And property causes, these changes keep the sequence of gene and nucleotide sequence shown in SEQ ID NO.2 not quite identical, but at least have
Have 90% or more, for example, can be but be not limited to 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or
99% identity.Meanwhile nucleotides sequence shown in these and SEQ ID NO.2 is shown the gene of 90% or more identity and is contained
There is the gene function having the same of nucleotide sequence shown in SEQ ID NO.2.Identity can with the naked eye or computer software
It is evaluated, such as is compared using the blast software of this field routine.Wherein the PagKNAT2/6b gene be containing
The gene of the nucleotide sequence as shown in SEQ ID NO.2 be applied to the effect of poplar adjusted and controlled number of branches and stem pliability compared with
It is good.
KNOX gene unconventionality expression influences normal plant growth and development process in arabidopsis, corn and tobacco.Plant
KNOX gene family is divided into two subclass: I class KNOX gene and II class KNOX gene, and I class KNOX gene includes 4 in arabidopsis
A member is respectively: SHOOTMERISTEMLESS (STM), BREVIPEDICELLUS (BP), KNOTTED-like from
Arabidopsis thaliana 2 (KNAT2) and KNAT6.
PagKNAT2/6b gene is homologous gene with arabidopsis KNAT2 and KNAT6 in poplar, it is poplar adjusted and controlled in
The expression of PagKNAT2/6b gene will affect the formation of poplar branch and the pliability of poplar stem.Therefore, by regulating and controlling poplar
The expression of PagKNAT2/6b gene in tree, can influence the plant type of poplar, to obtain the poplar for being suitable for different demands, application
In the different landscape moulding of poplar.The present invention is found through experiments that, raises the expression of the PagKNAT2/6b gene in poplar, energy
Enough increase poplar number of branches and improve stem pliability, improves stem pliability and refer to that the stem of poplar is softer compared to wild type.
Therefore in some preferred embodiments, PagKNAT2/6b gene is applied to increase poplar number of branches, generates poplar rich
The tree crown being full of；Poplar stem pliability is improved, poplar is made to be easy moulding.
The present invention also provides a kind of method of poplar adjusted and controlled plant type, the method for the poplar adjusted and controlled plant type includes poplar adjusted and controlled
Number of branches, while poplar adjusted and controlled stem pliability.Due to PagKNAT2/6b gene and poplar number of branches and stem pliability
Correlation, thus change PagKNAT2/6b gene expression quantity can poplar adjusted and controlled plant type, grant life to plant compared to traditional
The gene of the mode of the substances such as long element, the basic element of cell division and abscisic acid, direct regulation and control poplar can be more precisely and according to people's
Like the poplar adjusted and controlled plant type of orientation, improves regulation efficiency.
Poplar can be made to be easier moulding due to increasing poplar number of branches and raising stem pliability, some preferred
Embodiment in, be overexpressed poplar described in PagKNAT2/6b gene expression quantity, to increase poplar number of branches and raising
Poplar stem pliability.It is wherein overexpressed and promoter, enhancer and other sequences controls with regulating and controlling effect can be used
The expression of PagKNAT2/6b gene, promoter, enhancer and other sequences with regulating and controlling effect can select a use,
It can also be used in combination, optionally promoter and enhancer be used in combination, it is preferable to use strong promoter is overexpressed PagKNAT2/
6b gene.
In some preferred embodiments, the PagKNAT2/ is overexpressed using the expression vector containing strong promoter
6b gene, the carrier include pMDC32 or pCambia1300；PMDC32 and pCambia1300 contains hygromycin phosphoric acid and turns
Enzyme HPT is moved as the selection markers of transgenic poplar can carry out the screening of transgenic poplar with hygromycin；Load is expressed simultaneously
It include LB and RB sequence on body, the PagKNAT2/6b expression frame and riddled basins HPT for promoting to assemble therebetween are integrated into
In poplar recipient chromosome.Through experiments, it was found that crossing table compared to the PagKNAT2/6b that pMDC32 expression vector conversion poplar obtains
Up to strain (OE5 and OE7), pCambia1300 expression vector converts the overexpression for the up-regulation PagKNAT2/6b gene that poplar obtains
The expression quantity of PagKNAT2/6b gene dramatically increases in transgenic poplar, and plant type better effect, therefore preferably
PCambia1300 expresses PagKNAT2/6b gene.
In some preferred embodiments, it is preferable to use Gataway system is cloned and expressed to target gene,
Gataway system is a kind of clone operations technology, including elder generation gene cloning to entry vector, then without relying in restricted
Enzyme cutting, and specific recombination site and recombinase present on carrier are leaned on, target gene efficiently, is rapidly cloned into other tables
Up on carrier.It is preferred that target gene is first cloned into entry vector PDNOR207, then pass through LR reaction in Gateway system again
Target gene is inserted into expression vector, it is easy to operate quick, and the success rate of expression vector establishment is high.
The technical scheme and beneficial effects of the application are further illustrated below with reference to preferred embodiment.
Embodiment 1
Gene cloning:
(1) using the silver-colored gland poplar of 84K (P.alba × P.glandulosa) as material, RNeasy Plant Mini reagent is used
Box and RNase-free DNase I kit (Qiagen, Hilden, Germany) extract RNA；And by using
SuperScript III first-strand synthesis system (Life Technologies, Carlsbad, CA,
USA cDNA) is synthesized.
(2) using 5 software Design primers of Primer (amplicon includes initiation codon and terminator codon), base is carried out
Because of overall length amplification (GATEWAY connector is added in primer)；Wherein, PagKNAT2/6b ORF primer is shown in Table 1；
1 PagKNAT2/6b primer of table
(3) High fidelity PCR reaction system is as follows: TaKaRa high-fidelity amplification enzyme PrimeSTAR, forward primer reversely draw
Object, template are 84K poplar cDNA, and sterile water is supplied；Response procedures: 95 DEG C of initial denaturation, 5min；95 DEG C, 20s；56 DEG C, 20s；72
DEG C, 60s, 28 circulations；72 DEG C, 10min；The final full length gene cDNA sequence that obtains is 930bp, is named as PagKNAT2/6b
Gene.
Embodiment 2
Construction of expression vector: the PagKNAT2/6b gene that embodiment 1 is expanded is inserted into expression vector.
(1) it utilizes the Overexpression vector of clone technology building PagKNAT2/6b gene: using specific PCR primers
(PagKNAT2/6b ORF primer), the 84K cDNA obtained using embodiment 1 carry out PCR amplification, by PagKNAT2/6b as template
Gene ORF is building up to entry vector, entry vector PDNOR207；Reaction system be fresh PCR product (150ng),
II mixed enzyme of PDNOR207 carrier (75ng) and BP Clonase (0.8 μ l)；Sample-adding, which is placed in 25 DEG C of metal baths, connects 4-5h.
(2) product connected above is converted into Escherichia coli, and is coated on the screening and culturing medium containing gentamicin,
Picking positive colony carries out PCR detection and sequence verification from sifting motion cultivation plate after overnight incubation, finally successfully enters building
Door carrier by LR reaction building in Gateway system construct respectively to expression vector pMDC32 be transformed
GFP sequence (is inserted into 35S strong promoter and is inserted into the Gateway recombination site of PagKNAT2/6b by pCambia1300 carrier
Between), the over-express vector of PagKNAT2/6b over-express vector and PagKNAT2/6b fusion GFP is formulated respectively；LR reactant
System are as follows: cloning vector (150ng), expression vector (75ng) and LR Clonase II mix (0.8 μ L), are placed in 25 DEG C of metal baths
React 4-5h；LR reaction after, PagKNAT2/6b gene be directed respectively into plant expression vector pMDC32 with it is improved
In pCambia1300 carrier, contains strongly expressed promoter 35S at 5 ' ends of PagKNAT2/6b gene, PagKNAT2/6b can be made
Albumen high efficient expression in poplar body.
Embodiment 3
By electric shocking method, construct above two kinds of PagKNAT2/6b over-express vectors are transferred to Agrobacterium GV3101 respectively
In, by mediated by agriculture bacillus, PagKNAT2/6b is transferred to poplar (silver-colored gland poplar, 84K poplar), step of converting is as follows: turned for heredity
Hybrid Poplar clone's 84K tissue-cultured seedling of change is trained under conditions of cultivation temperature is 23-25 DEG C, illumination is 16/8h (day/night)
It supports, the Agrobacterium containing PagKNAT2/6b expression vector infects 84K leaf dish when cultivating to OD600=0.6-0.8, after infecting
Leaf dish adds 0.5mg/L6-benzyl in adventitious bud induction culture base (Murashige-Skoog, MS) minimal medium
Aminopurine (6-BA, 6- benzylaminopurine) and 0.05mg/L Naphthaleneacetic acid (NAA, naphthalene second
Acid), it is co-cultured 3-4 days in the case where temperature is 23 ± 2 DEG C of dark condition；Leaf dish after co-cultivation is transferred to containing 3mg/L
It is 23-25 DEG C, light in cultivation temperature on the MS of Hygromycin B (hygromycin B) and 200mg/L Timentin (Ticarcillin/Clavulanate Acid)
According under conditions of 16/8h (day/night) induce and screen resistance adventitious bud；By about 20 days Fiber differentiations, by resistance
Adventitious bud is transferred in the root media containing 3mg/L Hygromycin B and 200mg/L Timentin that (1/2MS is basic
Culture medium adds 0.05mg/L IBA and 0.02mg/L NAA), until inducing adventitious root.
Contain hygromix phosphotransferase HPT on pMDC32 and improved pCambia1300 expression vector, as turning
The selection markers of gene poplar can carry out the screening of transgenic poplar with hygromycin；It simultaneously include LB and RB on expression vector
Sequence, the PagKNAT2/6b expression frame and riddled basins HPT for promoting to assemble therebetween are integrated into poplar recipient chromosome
In, the blade for extracting the positive plant taken root in screening and culturing medium extracts DNA, and PCR sequence verification confirms PagKNAT2/6b mistake
The overexpression transgenic poplar of express transgenic poplar and PagKNAT2/6b fusion GFP are formulated successfully.Conversion is overexpressed
The poplar of the pMDC32 carrier of PagKNAT2/6b is denoted as PagKNAT2/6b-OE, chooses OE5 strain and OE7 strain poplar carries out
Subsequent detection；The poplar that conversion is overexpressed the pCambia1300 carrier of PagKNAT2/6b is denoted as GFP-PagKNAT2/6b, is selected
GFP1 strain, GFP6 strain and GFP8 strain is taken to carry out subsequent detection.
PagKNAT2/6b as shown in Figure 1 is constructed to pMDC32 expression vector, is converted 84K poplar and is obtained PagKNAT2/6b-
It is as shown in Figure 3B that OE is overexpressed transgenic poplar.The PagKNAT2/6b over-express vector of fusion GFP as shown in Figure 2
PCambia1300 building, it is as shown in Figure 3 C that conversion 84K poplar obtains GFP-PagKNAT2/6b overexpression transgenic poplar.It is wild
Type (CK) is as shown in Figure 3A.
Identify that PagKNAT2/6b expression quantity raises respectively in conversion transgenic line OE5, OE7 by real-time quantitative PCR
50,30 (Fig. 3 D), and PagKNAT2/6b expression quantity raises 120/140/150 times (Fig. 3 E) respectively in GFP1, GFP6 and GFP8.
Observed by the plant type of different batches transgenic poplar and non-transgenic poplar CK, discovery PagKNAT2/6b-OE with
The plant type that branch increases in GFP-PagKNAT2/6b material is significant and stablizes (Fig. 4 A- Fig. 4 D and Fig. 5 A- Fig. 5 C).By Fig. 3 B and
Fig. 3 C compares it is found that the increase plant type with PagKNAT2/6b expression quantity is further significant, at the same time by Fig. 4 A- Fig. 4 D with
Fig. 5 A- Fig. 5 C's it was found that with PagKNAT2/6b expression quantity increase number of branches increase.
At the same time, PagKNAT2/6b-OE and GFP-PagKNAT2/6b transgenic poplar branch softness are easy to moulding, solve
Cut open that credit analysis discovery OE5, OE7 and GFP1, cell wall is significantly thinner than CK in GFP6, GFP8 transgenic poplar stem, it is known that
The flexibility of PagKNAT2/6b-OE and GFP-PagKNAT2/6b transgenic poplar stem is caused by its cell wall is thinning, is such as schemed
Shown in 6A-6C and Fig. 7, wherein in Fig. 6 A, icon Ve refers to that conduit (vessel), icon Xf refer to xvlem fibres
(xylem fiber).CK, OE5 and OE7 vessel cell wall thickness are statisticallyd analyze, discovery OE5 vessel cell cell wall thickness is significant
It is thinner than CK (as shown in Figure 6B), statisticallys analyze the cell wall thickness of CK, OE5 and OE7 fibrocyte, discovery OE5 and OE7 fiber finer
Born of the same parents' cell wall thickness is significantly thinner than CK (as shown in Figure 6 C), and the above feature can be used for the landscape moulding of poplar.
The PagKNAT2/6b-OE and GFP-PagKNAT2/6b of above-described poplar adjusted and controlled plant type and poplar stem pliability
The genetic stability and practicability of transgenic poplar: the present embodiment has cloned PagKNAT2/6b gene using silver-colored gland poplar as material；
Meanwhile by PagKNAT2/6b it is gene constructed to Overexpression vector pMDC32 and merge GFP over-express vector
PCambia1300, the gene is after the insertion point in both the above carrier is respectively positioned on strong promoter 35S, in strong promoter
Under the driving of 35S, PagKNAT2/6b can in poplar body overexpression, thus be conducive to poplar number of branches (i.e. crown type) and
The regulation of the flexibility (i.e. moulding) of stem.
Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention., rather than its limitations；To the greatest extent
Pipe present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that: its according to
So be possible to modify the technical solutions described in the foregoing embodiments, or to some or all of the technical features into
Row equivalent replacement；And these are modified or replaceed, various embodiments of the present invention technology that it does not separate the essence of the corresponding technical solution
The range of scheme.
SEQUENCE LISTING
<110>Zhejiang A & F University
<120>method of the application of PagKNAT2/6b gene and poplar adjusted and controlled plant type
<160> 4
<170> PatentIn version 3.5
<210> 1
<211> 309
<212> PRT
<213>silver-colored gland poplar (P. alba × P. glandulosa)
<400> 1
Met Asp Gly Thr Tyr Gly Leu His Ser Thr Val Ala Asp Tyr Ser Asp
1 5 10 15
Lys Ala Leu Met Ser Pro Glu Asp Phe Ile Leu Gln Ser Glu Tyr Gln
20 25 30
Ser Leu Leu Ser Ser Glu Thr Leu Arg Leu Arg Ile Pro Ile Leu Gly
35 40 45
Ser Glu Glu Leu Leu Ser Glu Ala Ala Ser Ile Arg Thr Glu Glu Asp
50 55 60
Met Ser Ala Leu Ile Lys Ala Lys Ile Ala Ser His Pro Cys Tyr Pro
65 70 75 80
Arg Leu Leu Glu Ala Tyr Ile Asp Cys Gln Lys Val Gly Ala Pro Pro
85 90 95
Gly Ile Ala Cys Phe Leu Asp Glu Ile Arg Arg Glu Asn Asp Leu Phe
100 105 110
Lys Gln Gly Ala Val Ser Thr Tyr Trp Gly Ala Asp Pro Glu Leu Asp
115 120 125
Glu Phe Met Glu Thr Tyr Cys Asp Leu Leu Val Lys Tyr Lys Ser Asp
130 135 140
Leu Glu Arg Pro Leu Asp Glu Ala Thr Thr Phe Leu Asn Lys Ile Glu
145 150 155 160
Met Gln Leu Arg Asn Leu Cys Thr Gly Ala Ser Ile Arg Ser Leu Ser
165 170 175
Asp Glu Gly Ala Ala Ser Ser Asp Glu Glu Leu Ser Val Gly Glu Leu
180 185 190
Asp Met His Glu Ala Gln Pro Ser Gly Glu Asp Arg Glu Leu Lys Asp
195 200 205
Lys Leu Leu Arg Arg Phe Gly Gly His Ile Gly Thr Leu Lys Leu Glu
210 215 220
Phe Ser Lys Lys Lys Lys Lys Gly Lys Leu Pro Lys Glu Ala Arg Gln
225 230 235 240
Thr Leu Leu Gly Trp Trp Asp Ala His Tyr Lys Trp Pro Tyr Pro Thr
245 250 255
Glu Ala Asp Lys Ile Ala Leu Ala Glu Ser Thr Gly Leu Asp Gln Lys
260 265 270
Gln Ile Asn Asn Trp Phe Ile Asn Gln Arg Lys Arg His Trp Lys Pro
275 280 285
Ser Glu Asn Leu Gln Phe Ala Val Met Asp Asn Leu Ser Gly Gln Phe
290 295 300
Phe Thr Glu Asp Asp
305
<210> 2
<211> 930
<212> DNA
<213>silver-colored gland poplar (P. alba × P. glandulosa)
<400> 2
atggacggaa cgtacggtct gcactcaacg gtggcggact actcagacaa ggcgttgatg 60
tcgccggagg attttatttt acaatcggag taccagagct tgctgtcttc agaaactctt 120
cggctgcgga ttccgattct tggatccgaa gagttgcttt cagaggcggc ttcgatcaga 180
acagaagaag atatgtccgc tttgatcaaa gccaaaattg cctcgcatcc ttgctatcct 240
cgtttactcg aggcttatat cgattgccag aaggtagggg cgcctccggg gatagcgtgt 300
ttcttggatg aaatccggcg agaaaacgac cttttcaagc aaggcgctgt ctccacgtac 360
tggggagctg atcccgagct agacgagttc atggaaacct actgtgattt gttggtgaag 420
tataaatctg atcttgaaag gcctttagat gaagcaacaa ccttcttgaa caagattgaa 480
atgcagcttc gaaatctctg caccggtgcc tccatcagaa gcctctctga tgaaggtgcg 540
gcatcttcag atgaggagtt aagtgtaggg gagttggata tgcatgaagc tcaaccaagc 600
ggtgaagacc gagagcttaa agataaactt ctccgcaggt ttggaggtca tattggtacg 660
ctgaagctgg agttctcaaa gaaaaaaaag aaaggaaagc taccgaagga agctaggcaa 720
accctacttg gatggtggga tgctcactat aaatggccat atccaactga agctgataag 780
atagcattgg ccgaatcgac gggactagat cagaagcaaa ttaataattg gtttataaat 840
caacgtaagc gccactggaa accatctgag aacctgcaat ttgctgttat ggataatctt 900
tctgggcagt tctttacaga agacgactga 930
<210> 3
<211> 56
<212> DNA
<213>artificial sequence
<400> 3
ggggacaagt ttgtacaaaa aagcaggctg catggacgga acgtacggtc tgcact 56
<210> 4
<211> 57
<212> DNA
<213>artificial sequence
<400> 4
ggggaccact ttgtacaaga aagctgggta tcagtcgtct tctgtaaaga actgccc 57
Claims (10)
- The application of 1.PagKNAT2/6b gene, the application include poplar adjusted and controlled number of branches and/or stem pliability；It is described PagKNAT2/6b gene is the gene for expressing (a1) or (a2):(a1): the albumen containing the amino acid sequence as shown in SEQ ID NO.1；(a2): containing the amino acid sequence with 90% or more identity of amino acid sequence described in (a1), and there is identical function Albumen.
- 2. application according to claim 1, which is characterized in that the PagKNAT2/6b gene is expression containing such as SEQ The gene of the albumen of amino acid sequence shown in ID NO.1.
- 3. application according to claim 1, which is characterized in that the nucleotides sequence of the PagKNAT2/6b gene is classified as (b1) or (b2):(b1): containing the nucleotide sequence as shown in SEQ ID NO.2；(b2): containing with 90% or more identity of nucleotide sequence described in (b1), and nucleotide sequence with the same function.
- 4. application according to claim 3, which is characterized in that the PagKNAT2/6b gene nucleotide series be containing The nucleotide sequence as shown in SEQ ID NO.2.
- 5. application according to claim 1-4, which is characterized in that PagKNAT2/6b base described in up-regulation poplar The expression of cause, to increase poplar number of branches and improve poplar stem pliability.
- 6. a kind of method of poplar adjusted and controlled plant type, which is characterized in that the method includes changing PagKNAT2/6b gene in poplar Expression quantity, with by change poplar number of branches and change poplar stem pliability come poplar adjusted and controlled plant type.
- 7. according to the method described in claim 6, it is characterized in that, the PagKNAT2/6b gene is overexpressed, to increase poplar Number of branches and raising poplar stem pliability；Preferably, by promoter and/or enhancer so that the PagKNAT2/6b gene overexpression.
- 8. the method according to the description of claim 7 is characterized in that described be overexpressed includes using described in strong promoter driving The expression of PagKNAT2/6b gene；It is preferable to use 35S for the strong promoter.
- 9. the method according to the description of claim 7 is characterized in that using described in the expression vector overexpression containing strong promoter PagKNAT2/6b gene, the carrier include pMDC32 or pCambia1300；Preferably pCambia1300；Preferably, the PagKNAT2/6b gene is cloned and is expressed using Gataway system, wherein entry vector is excellent Select PDNOR207.
- 10. the method according to claim 6, which is characterized in that described method includes following steps:(a) coded sequence of the PagKNAT2/6b gene of the nucleotide sequence as shown in SEQ ID NO.2 is constructed to introduction and is carried Body PDNOR207；(b) the carrier PDNOR207 for obtaining step (a) is by LR reaction in Gateway system, by PagKNAT2/6b gene structure It builds to pCambia1300, the Gateway recombination site of the pCambia1300 is located at 35S strong promoter and GFP coding albumen Later, the coded sequence of PagKNAT2/6b gene is constructed to 35S-GFP；(c) pCambia1300 that step (b) constructs is transferred in Agrobacterium GV3101, by mediated by agriculture bacillus, by 35S-GFP- PagKNAT2/6b segment is inserted into poplar chromosome.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109161553A (en) * | 2018-09-29 | 2019-01-08 | 安徽农业大学 | A kind of pears transcription factor PbBP and its application |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109082429A (en) * | 2018-08-31 | 2018-12-25 | 南京林业大学 | A kind of key gene PeRBR and its expression albumen and the application of poplar adjusted and controlled adventitious root and xylem development |
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2019
- 2019-08-08 CN CN201910729683.7A patent/CN110331149B/en active Active
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109082429A (en) * | 2018-08-31 | 2018-12-25 | 南京林业大学 | A kind of key gene PeRBR and its expression albumen and the application of poplar adjusted and controlled adventitious root and xylem development |
Non-Patent Citations (2)
| Title |
|---|
| ENRIC BELLES-BOIX ET AL.: "KNAT6: An Arabidopsis Homeobox Gene Involved in Meristem Activity and Organ Separation", 《THE PLANT CELL》 * |
| 赵岩秋等: "杨树中I 类KNOX 基因结构、表达与功能分析", 《林业科学研究》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109161553A (en) * | 2018-09-29 | 2019-01-08 | 安徽农业大学 | A kind of pears transcription factor PbBP and its application |
| CN109161553B (en) * | 2018-09-29 | 2022-02-18 | 安徽农业大学 | Pear transcription factor PbBP and application thereof |
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| CN110331149B (en) | 2021-07-06 |
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