CN109384837A - A kind of poplar anti-drought gene and its application - Google Patents
A kind of poplar anti-drought gene and its application Download PDFInfo
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Abstract
The invention discloses a kind of poplar gene, nucleic acid sequence is as shown in SEQ ID NO.5, and protein sequence is as shown in SEQ ID NO.6.The invention also discloses a kind of methods of prepare transgenosis poplar, gene order shown in SEQ ID NO.5 are transferred to poplar by leaf disc transformation method, the transgenic poplar is relative to wild type poplar in (a) drought resistance；(b) carton dioxide assimilation rate；(c) speed of growth；(d) root breath rate；(e) root hydraulic conductivity；(f) growth of root, (g) lignin concentration of root and structure etc. are significantly increased.
Description
Technical field
The invention belongs to technical field of molecular biology, in particular to a kind of poplar anti-drought gene and its application.
Background technique
30% or so of the forest cover world land gross area is the chief component of earth terrestrial ecosystems,
It is important renewable natural resources, the carbon cycle and the maintenance ecological balance to human lives, nature all have irreplaceable
Status and effect.Under field conditions (factors), forest tree is in growth and development process often by many abiotic stresses
Such as arid, water stain, salt marsh, low temperature, high temperature injury, not to generations such as the growth and development, timber yield and material of forest tree
Benefit influences.Wherein arid is the most common adverse circumstance factor in environment-stress.Global Arid Problem is on the rise, arid and half-dried
Non-irrigated land area is continuously increased, and causes adverse effect to worldwide production of forestry and ecological environment.By global gas
The influence warmed is waited, the frequency and intensity of drought event dramatically increase, to the health of forest ecosystem, it be distributed, structure,
Composition and ecological diversity cause tremendous influence.According to incompletely statistics, the forest over nearly more than 50 years more than 70 percent
Tree kind growth after water source supply is insufficient can be particularly susceptible to encroach on.Researcher's prediction, due to global warming and greatly
The felling of area forest, global Arid Problem and its influence to forest can also be more serious, whole world majority Forest ecosystems
System will be faced with the potential risk of " arid is lethal ".
Forest tree has gradually formed a set of impression and conduction moisture stress signal in long-term evolution and evolution process
System, and a series of mechanism for forming physiology and development carrys out the water avoidance stress in response environment, mitigates arid to the maximum extent
Caused by injure.Forest be perennial arbor species have tall and big trunk and powerful root system, for drought stress response and
It adapts to that there is extremely complex regulation process, includes form, physiology, biochemistry and the multifaceted regulatory mechanism of molecular level.Especially
It is that powerful root system is played an important role for improving the drought-resistance ability of poplar.In recent years, plant responding water stress is ground
Study carefully and achieves many new discoveries in terms of gene expression regulation, protein modified and interaction, signal transmitting and dash forward
It is broken.It is concentrated mainly on regulating and controlling effect of the several big transcription factor gene families such as bZIP, NAC, MYB, WRKY under drought stress,
And especially regulate and control the transcription factor of the growth and development of root about drought-resistant research is regulated and controled in other transcription factor families
It is then relatively fewer.
Bibliography
(1)Jia B,Zhou G,Wang F,et al.Partitioning root and microbial
contributions to soil respiration in Leymus chinensis populations[J].Soil
Biology&Biochemistry,2006,38(4):653-660.
(2)Sluiter,A.,Hames,B.,Ruiz,R.,Scarlata,C.,Sluiter,J.,Templeton,D.,
and Crocker,D.(2008)Determination of structural carbohydrates and lignin in
biomass.National Renewable Energy Laboratory,Golden,CO.
(3)Chen T Y,Wang B,Wu Y Y,et al.Structural variations of lignin
macromolecule from different growth years of Triploid of Populus tomentosa,
Carr[J].International Journal of Biological Macromolecules,2017,101.
(4)Wen,J.-L.；Sun,S.-L.；Xue,B.-L.；Sun,R.-C.Recent advances in
characterization of lignin polymer by solution-state nuclear magnetic
resonance(NMR)methodology.Materials 2013,6,359-391.
Summary of the invention
It is special the present invention provides a kind of new poplar anti-drought gene PdNFY-B21 in order to solve deficiency in the prior art
The growth and development of anisotropic regulation root, transgenic poplar can significantly improve the drought tolerance of poplar.
More specifically, first aspect present invention provides a kind of poplar albumen, the amino acid sequence of the albumen such as SEQ
Shown in ID NO.6.
Second aspect of the present invention provides a kind of poplar gene, the gene nucleic acid sequence encoding such as SEQ ID
Amino acid sequence shown in NO.6.
In some embodiments, the nucleic acid sequence of the gene is as shown in SEQ ID NO.5.
Third aspect present invention provides a kind of gene engineering expression carrier, and the expression vector includes such as the present invention second
Poplar gene described in aspect, it is preferable that the carrier is carrier of the gene engineering expression carrier is PBI121 plasmid.
Fourth aspect present invention provides a kind of recombinational agrobacterium, and the recombinational agrobacterium includes such as third aspect present invention
The expression vector, it is preferable that the Agrobacterium is GV3101.
Fifth aspect present invention provides a kind of method of prepare transgenosis poplar, and the method is by present invention four directions
Recombinational agrobacterium described in face is transformed into poplar, forms the transgenic poplar.
In some embodiments, described method includes following steps:
(1) bacterium liquid activation: by the bacterium solution of recombinational agrobacterium described in fourth aspect present invention mould containing rifampin and Ka Na
It is cultivated in the YEB liquid of element, forms the first mixture；First mixture is centrifuged, supernatant is abandoned, forms the first precipitating；With
First precipitating is resuspended in the WPM culture solution for adding acetosyringone, forms the second mixture；
(2) Agrobacterium is infected: the sterile tender blade of poplar tissue-cultured seedling being put into second mixture and is disseminated；
(3) it co-cultures: blotting the liquid component on dip dyeing rear blade, blade merging WPM is co-cultured into base, it is dark to train
It supports,；
(4) selection culture:
The blade of the co-cultivation is transferred on WPM Selective agar medium, is cultivated under light illumination, resistance adventitious bud is formed；
(5) culture of rootage is screened:
The resistance adventitious bud is transferred in WPM root media and is cultivated, poplar seedlings of taking root are formed.
Sixth aspect present invention provides a kind of for detecting the PCR primer pair of transgenic poplar, and the primer pair includes
First upstream primer and the first downstream primer,
First upstream primer sequence are as follows:
5 '-AGTGGATTGATGTGATATCTCCACTGA-3 ', as shown in SEQ ID NO.9；
First downstream primer sequence are as follows:
5 '-TTATGATAGGGTTGCTAAAAGTTTAGCACTGT-3 ', as shown in SEQ ID NO.4.
Seventh aspect present invention provide it is a kind of for detecting or monitoring the fluorescence PCR primer pair of Drought Resistance of Populus, it is described
Primer pair includes the second upstream primer and the second downstream primer,
Second upstream primer sequence are as follows:
5'-GATGATTTGCTTTGGGCTATGGCTAC-3', as shown in SEQ ID NO.1；
Second downstream primer sequence are as follows:
5'-TCCAGTCTTCGCAGATCCCTTGGT-3', as shown in SEQ ID NO.2.
Eighth aspect present invention provides a kind of for detecting or monitoring the kit of Drought Resistance of Populus, the kit packet
Include the PCR primer pair as described in seventh aspect present invention.
Ninth aspect present invention provides albumen described in a kind of first aspect present invention, as described in respect of the second aspect of the invention
Gene, gene engineering expression carrier as described in the third aspect of the present invention, recombinational agrobacterium as described in the fourth aspect of the present invention,
Or the method for the prepare transgenosis poplar as described in fifth aspect present invention is in poplar character as described in improvement poplar character or improvement
Breeding in purposes, the poplar character includes:
(a) drought resistance；
(b) carton dioxide assimilation rate；
(c) speed of growth；
(d) respiratory rate of root；
(e) root hydraulic conductivity；
(f) growth of root；(g) lignin concentration of root and structure；
It is preferred that the poplar is 84K poplar or black poplar NE19.
Compared with prior art, the invention has the following advantages that
The present invention is to have found the drought resisting function of poplar gene PdNFY-B21 sequence for the first time both at home and abroad, it is to be widely present
Nuclear factor, forefathers researches show that NF-YB transcription factor for improve plant drought resistance important role, exist at present
The function that forest tree drought resistance can be improved in overexpression in forest tree does not have relevant report.The present invention is the experimental results showed that turn
The expression quantity of gene poplar PdNFY-B21 has significant improvement compared with receptor compares (nontransgenic plants), resists
Therefore drought is also enhanced.Therefore the present invention propose can be enhanced using the overexpression of PdNFY-B21 plant drought resistance with
Just it is used for the drought-resistant breeding of forestry, thus slows down Drought Stress injury caused by forest production and quality.
Detailed description of the invention
Fig. 1 is PdNFY-B21 gene tissue specific expression histogram in black poplar NE19, wherein with the expression in leaf
On the basis of amount, numerical value 1；
Fig. 2 is that PdNFY-B21 gene expresses histogram under drought stress in root tissue in black poplar NE19, wherein with 0d
On the basis of expression quantity, numerical value 1；
Fig. 3 is the nucleic acid base sequence schematic diagram of PdNFY-B21 gene；
Fig. 4 is the protein amino acid sequence schematic diagram of PdNFY-B21 gene；
Fig. 5 is PBI121-35S:PdNFYB21:GUS vector construction process intention；
Fig. 6 is that PdNFY-B21 gene transgenic plant screens photo；
Fig. 7 is PdNFY-B21 gene transgenic plant DNA fragmentation electrophoretogram；
Fig. 8 is that PdNFY-B21 gene transgenic plant compares with wild-type leaves GUS stained photographs；
Fig. 9 compares histogram with wild type PdNFY-B21 gene expression amount for PdNFY-B21 gene transgenic plant,
In, on the basis of the expression quantity of TW, numerical value 1；
Figure 10 compares photograph with PdNFY-B21 transgenic plant oxPdNFY-B21#6, #8 drought resisting effect for WT lines
Piece；
Figure 11 is WT lines and PdNFY-B21 transgenic plant oxPdNFY-B21#6, #8 under drought stress environment
Net carbon dioxide Assimilation rate control curve figure；
Figure 12 is WT lines and PdNFY-B21 transgenic plant oxPdNFY-B21#6, #8 under drought stress environment
The high control curve figure of stem；
Figure 13 is WT lines and PdNFY-B21 transgenic plant oxPdNFY-B21#6, #8 under drought stress environment
The size (volume of root long, root diameter and root) of root compares histogram (every cluster is from left to right followed successively by wild type, #6, #8), wherein
Respectively on the basis of the amount of WT, numerical value 1；
Figure 14 is WT lines and PdNFY-B21 transgenic plant oxPdNFY-B21#6, #8 under drought stress environment
Root density compares histogram；
Figure 15 is WT lines and PdNFY-B21 transgenic plant oxPdNFY-B21#6, #8 under drought stress environment
Root/shoot ratio compares histogram (every cluster is from left to right followed successively by wild type, #6, #8)；
Figure 16 is WT lines and PdNFY-B21 transgenic plant oxPdNFY-B21#6, #8 under drought stress environment
Root conductivity compares histogram；
Figure 17 is WT lines and PdNFY-B21 transgenic plant oxPdNFY-B21#6, #8 under drought stress environment
Biomass compares histogram (every cluster is from left to right followed successively by wild type, #6, #8)；
Figure 18 is WT lines and PdNFY-B21 transgenic plant oxPdNFY-B21#6, #8 under drought stress environment
Root system respiration rate control curve figure；
Figure 19 is WT lines and PdNFY-B21 transgenic plant oxPdNFY-B21#6, #8 under drought stress environment
Root hydraulic conductivity control curve figure；
Figure 20 is WT lines and PdNFY-B21 transgenic plant oxPdNFY-B21#6, #8 under drought stress environment
Root content of lignin compares histogram；
Figure 21 is gene WT lines and PdNFY-B21 transgenic plant oxPdNFY-B21#6, #8 in the flow of water
The concentration and structure chart of root lignin under 0.12MPa environment.
Specific embodiment
To make the object, technical solutions and advantages of the present invention clearer, below in conjunction with attached drawing to embodiment party of the present invention
Formula is described in further detail.
Term used in the present invention " transgenic poplar ", which refers to the gene containing importing and can steadily enhance, is led
The gene expression and generation that enter have the poplar of specific biological character.
Usually according to normal condition such as Sambrook et al., molecular cloning: laboratory manual (New York:Cold
Spring Harbor Laboratory Press, 1989) or Draper et al. (Blackwell Science Press, 1988) institute
The condition stated, or the condition with reference to proposed by agents useful for same specification.
The growth of specifically poplar adjusted and controlled of the poplar drought resisting transcription factor protein PdNFY-B21 found in the present invention and
Development is so that improving the drought-resistance ability of poplar including all not there is the report of any correlation function also in poplar, is so far
The albumen and gene of one unknown function.
The purpose of the invention is to provide a kind of poplar (Populus) anti-drought gene PdNFY-B21, the cores of the gene
Nucleotide sequence and protein sequence are shown in Fig. 3 and Fig. 4.Pass through the super table by poplar anti-drought gene PdNFY-B21 in 84K poplar
It reaches, it is found that this gene is remarkably improved the drought resistance of poplar, transgenic poplar energy normal growth after drought stress 40 days, and wood
Material yield is substantially unaffected, and the yield and material for raising timber under Drought Stress provide guarantee.
Another object of the present invention is to be the provision of a kind of poplar anti-drought gene PdNFY-B21 to educate in Tree Drought Resistance
Application in kind improves forest to the tolerance of arid, ensure that forest timber yield and material from or it is few by dry
The adverse effect of drought stress.
In order to achieve the above-mentioned object of the invention, the present invention uses following technical measures:
A kind of poplar anti-drought gene PdNFY-B21, preparation step is:
A kind of black poplar NE19 [P.nigra × (P.deltoides × P.nigra)] anti-drought gene PdNFY-B21 is obtained:
In order to disclose whether NFY-B family gene is able to respond drought stress in poplar, present inventor is according to comospore poplar
PtNFY-B21 (Potri.016G085000.1) gene order devises a pair as shown in SEQ ID NO.1, SEQ ID NO.2
For the fluorescence quantification PCR primer for being named as PdNFY-B21 gene of black poplar NE19, PdNFY-B21 gene is demonstrated in poplar
In root-specific expression and drought stress height expression, further design gene coded sequence such as SEQ ID NO.3, SEQ
Two sides primer shown in ID NO.4.The purpose base is obtained using pcr clone using the first chain of cDNA of black poplar NE19 root as template
The CDS sequence of cause is simultaneously sequenced, and poplar PdNFY-B21 gene order is obtained.
A kind of application of poplar anti-drought gene PdNFY-B21 in poplar Drought-resistant Breeding, application process is:
(1) double digestion is carried out to empty carrier PBI121 plasmid using Xba I and Sma I enzyme, in addition design added with Xba I and
The primer (shown in SEQ ID NO.7, SEQ ID NO.8) of Sma I enzyme restriction enzyme site is reacted by PCR by poplar anti-drought gene
PdNFY-B21 is building up on PBI121 expression vector, is named as PBI121-PdNF-YB21.
(2) the carrier PBI121-PdNF-YB21 prepared in step (1) is transferred to Agrobacterium tumefaciems GV3101
(invitrogen), then by the leaf disc transformation method of mediated by agriculture bacillus by PdNFY-B21 genetic transformation into 84K poplar.
(3) positive plant is screened.
We are infected by blade of the leaf disc transformation method to 84K poplar, and obtaining in the case where card receives mycin screening pressure can
It can be 16 plants of positive plant for turning PdNFY-B21 gene, carry out PCR identification after further extracting to the leaf DNA of positive plant
It was found that there are 9 strains the specific band of about 600bp size occur, rather than do not have then in transgenic line.GUS dyeing discovery
The blade of 9 strains of PCR identification is all blue.Finally confirm that 9 plant are the positive plant that gene has entered.Pass through fluorescence
Quantitative PCR detects the expression quantity of the PdNFY-B21 gene of insertion, it is found that 9 positive strains are not identical, wherein
The expression quantity highest of oxPdNFY-B21#6, oxPdNFY-B21#8, therefore in subsequent experimental, the two strains are chosen as reality
Test material.
The method for cloning heretofore described drought resisting protein gene PdNFY-B21 be in this field frequently with side
Method.Extracting poplar leaf DNA is common Protocols in Molecular Biology, and extracting the method for mRNA, also there are many mature technology, examinations
Agent box (TRIzol Reagent) commercially obtains (Invitrogen company), and construction cDNA library is also common
Protocols in Molecular Biology.Construct heretofore described vector construction and by gene be transfected into digestion used in poplar, connection,
The methods of leaf disc transformation method is also technology commonly used in the art.Media (such as Agrobacterium tumefaciems is used in plasmid involved in it, transfection
GV3101 and agents useful for same ingredient such as sucrose, plant hormone etc.) commercially obtain.
Embodiment 1: the screening of poplar anti-drought gene PdNFY-B21:
In recent years, the research of plant responding drought stress is passed in gene expression regulation, protein modified and interaction, signal
It passs network etc. and achieves many new discoveries and breakthrough.It is concentrated mainly on several big transcriptions such as bZIP, NAC, MYB, WRKY
Regulating and controlling effect of the factor gene family under drought stress, and research drought-resistant about regulation in other transcription factor families,
The transcription factor for especially regulating and controlling the growth and development of root is then relatively fewer.The poplar drought resisting transcription factor egg found in the present invention
Growth and development that white PdNFY-B21 is specifically poplar adjusted and controlled are to improve the drought-resistance ability of poplar, so far, including
All there is not the report of any correlation function also in poplar, is the albumen and gene of a unknown function.
Transcription factor plays an important role in terms of plant responding and regulation water stress.In order to disclose NFY-B house
Whether race's gene is able to respond drought stress in poplar, and present inventor is according to the PtNFY-B21 of comospore poplar
(Potri.016G085000.1) gene order devises a pair as follows and is named as PdNFY-B21 base for black poplar NE19
The fluorescence quantification PCR primer of cause.Carry out following two tests.
Upstream primer sequence are as follows:
5'-GATGATTTGCTTTGGGCTATGGCTAC-3', as shown in SEQ ID NO.1；
Downstream primer sequence are as follows:
5'-TCCAGTCTTCGCAGATCCCTTGGT-3', as shown in SEQ ID NO.2.
1. tissue specificity is tested
Take the consistent annual black poplar NE19 stem section (~15cm) cuttage of growing way in basin, every 10 days every basin watering 1L,
After growth 3 months, it is extracted from basin, gently the soil of root is cleaned up with water, takes its root, stem, leaf is immediately placed in liquid nitrogen
In, it is spare then to put the quantitative fluorescence analysis that -80 DEG C of refrigerators are PdNFY-21 gene.
Then it is carried out in root, stem, leaf using primer pair PdNFY-B21 shown in SEQ ID NO.1 and SEQ ID NO.2
Fluorescent quantitation expression analysis, discovery PdNFY-B21 specificity is expressed in root, as a result referring to Fig. 1.
2. temporal is tested
In order to exclude the influence of growth period and growing environment factor to drought stress effect, five groups of poplar seedlings difference are taken successively
It adds after Huo Gelan (Hoagland's) nutrient solution (no PEG6000) culture is added containing 30%PEG6000 (w/v)
(Sigma) Huo Gelan (Hoagland's) nutrient solution is cultivated, and 20 days in total, is all tested at the 20th day.
The specific scheme is that taking the consistent annual black poplar NE19 stem section (~15cm) cuttage of growing way in basin, 3 are grown
Start to test after month, test be divided into 5 groups with poplar seedlings, poplar seedlings are extracted from basin, gently the soil of root is cleaned up with water,
Being placed on addition, Huo Gelan (Hoagland's) nutrient solution (no PEG6000) is inner successively cultivates 20,15,10,5,0 days, is then placed on
It is added in Huo Gelan (Hoagland's) nutrient solution of 30%PEG6000 (w/v) (Sigma) and successively cultivates 0,5,10,15,20
It.The root of five groups of poplar seedlings quickly removed to (PEG6000 Drought stress simulation, first group is equivalent to and does not do respectively after 20 days
The control group of drought stress), it is immediately placed in liquid nitrogen, then putting -80 DEG C of refrigerators is that quantitative fluorescence analysis is spare.
Using primer shown in SEQ ID NO.1 and SEQ ID NO.2, pass through fluorescent quantitation expression analysis PdNFY-B21
Relative expression quantity, as a result referring to fig. 2.From figure 2 it can be seen that for the control group for not doing drought stress,
PdNFY-B21 is gradually increased in the expression that drought stress handles PdNFY-B21 in 5-15 days, after processing 20 days, PdNFY-B21
Expression quantity fall after rise, still slightly above aforementioned control.
It is highly expressed that result shown in Fig. 2 can illustrate PdNFY-B21 to a certain extent and be that drought stress induction mentions, this
Mechanism encounters self-protection under drought stress for poplar.It expresses and is declined after coercing more than 20 days, this may be because originally
What experiment detected is the expression of RNA, and the expression of albumen has accumulated time, and the accumulation of the albumen of PdNFY-B21 at this time is
The signal path and/or metabolic regulation network for being achieved the effect that be effective against the albumen participation of arid or PdNFY-B21 are
Through having certain adaptation to drought stress by the mechanism such as compensatory.Be also likely to be PdNFY-B21 gene expression it is repaired
The damage of drought stress.
Based on above-mentioned discovery, next the present invention is directed to PdNFY-B21 gene and has carried out various researchs.
Embodiment 2: the clone of poplar anti-drought gene PdNFY-B21:
The comospore Yankee with the PdNFY-B21 sequence homology of black poplar NE19 is found according to the comospore poplar genomic data of announcement
Because of sequence PtNFY-B21 (Potri.016G085000.1), the two sides primer of gene coded sequence, forward primer: 5 '-are designed
ATGGCGGCAGAGGCACCGGC-3′(SEQ ID NO.3)；Reverse primer: 5 '-TTATGATAGGGTTGCTAAAAGTTTAGC
ACTGT-3′(SEQ ID NO.4)。
1, poplar mRNA is extracted
The extraction (extracting RNA using TRIZOL TM Kit) of RNA.
Liquid nitrogen grinding 100mg black poplar NE19 root, stem, leaf (weight ratio 1:1:1) mixing sample.
Plus 1ml TRIZOL, (22-25 DEG C, similarly hereinafter) placement 5min of room temperature A..
B. 200 μ l chloroforms are added, acutely vibrates 30s, is placed at room temperature for 2min.
C.13000rpm, it is centrifuged 15min, 4 DEG C, supernatant is taken to be transferred in new pipe, 500 μ l isopropanols are added, mixes rear chamber
Temperature places 15min.
D.13000rpm, it is centrifuged 15min, 4 DEG C, supernatant is removed, 1ml70% (absolute alcohol and H is added2The volume ratio of O) second
Alcohol.
E.7500rpm, it is centrifuged 7min, 4 DEG C, removes supernatant, air drying.
F. the processed deionized water dissolving RNA of DEPC is used, is put into -80 DEG C of ultra low temperature freezers and saves backup.
2, the reverse transcription of the first chain of cDNA operates reference using the TIANScript II cDNA kit of Tiangeng company
Used kit illustrates to be operated.
3, PCR amplification is carried out by template of cDNA, pcr amplification product has obtained a kind of poplar anti-drought gene through sequencing
PdNFY-B21 (is sequenced) through Sangon Biotech (Shanghai) Co., Ltd., and nucleotide coding sequence is (SEQ shown in Fig. 3
ID NO.5), shown in the nucleotide sequence coded protein sequence such as Fig. 4 (SEQ ID NO.6).
The 50 μ l reaction systems of PCR amplification poplar gene PdNFY-B21 are as follows:
The time of reaction and temperature are as follows:
94℃3min
94℃30s
59℃30s
72 DEG C of 1min, 34cycles
72℃10min
The building of embodiment 3:PdNFY-B21 expression vector and the conversion of Agrobacterium:
Other PCR steps are same as Example 2, and difference is that PCR primer includes Xba I and Sma I enzyme restriction enzyme site, tool
Body are as follows:
Forward primer:
5′-TCTAGAATGGCGGCAGAGGCACCGGC(SEQ ID NO.7)；
Reverse primer:
5′-CCCGGGTTATGATAGGGTTGCTAAAAGTTTAGCACTGT-3′(SEQ ID NO.8)。
Using sequence shown in SEQ ID NO.7 and SEQ ID NO.8 as primer, the poplar drought resisting base that is obtained by PCR amplification
Because of PdNFY-B21 amplified production, by using Xba I and Sma I enzyme to above-mentioned pcr amplification product and empty carrier PBI121 plasmid
(referring to Fig. 5) carries out double digestion respectively；Poplar anti-drought gene PdNFY-B21 is building up to by DNA ligase connection reaction
On PBI121 expression vector, recombinant vector pBI121-PdNF-YB21 is obtained, and recombinant vector is transformed into competence Agrobacterium
Cell GV3101 obtains the recombinational agrobacterium containing gene PdNFY-B21 (with reference to Sambrook et al., molecular cloning: laboratory
Handbook (New York:Cold Spring Harbor Laboratory Press, 1989), kanamycin screening are inserted into piece
Section is after vector primer (35S promoter aligning primer) and the identification of poplar PdNFY-B21 downstream of gene primer PCR, with correct weight
The Agrobacterium-mediated Transformation poplar of PdNFY-B21 is organized.
The anti-drought gene PdNFY-B21 of embodiment 4:84k poplar is converted
One, poplar culture medium is prepared
WPM re-suspension liquid: WPM+30g/L sucrose+100mol/LAs；
WPM co-cultures base :+100 μm of ol/LAs+2.0mg/L6-BA+0.3mg/L NAA+5.5- of WPM+30g/L sucrose
6.0g/L agar；
WPM Selective agar medium: WPM+30g/L sucrose+100mg/L Kana+2.0mg/L6-BA+0.3mg/L NAA+
400mg/L Cef+5.5-6.0g/L agar；
WPM screens root media: WPM+30g/L sucrose+150mg/L Kana+0.05mg/L IBA+0.05mg/L
NAA+400mg/L Cef+5.5-6.0g/L agar；After the ingredient in addition to agar mixes, it is by acidometer tune pH
5.80~5.85, agar, 121 DEG C of high temperature and high pressure steams sterilizing 20min are added.
Wherein, WPM are as follows: (woody plant medium)；As are as follows: acetosyringone；6-BA are as follows: 6- benzyl amino gland is fast
Purine；NAA are as follows: methyl α-naphthyl acetate；Kana are as follows: kanamycins；Cef is cephalosporin；IBA is indolebutyric acid.
Two, step of converting
(1) bacterium liquid activation: the recombinational agrobacterium containing gene PdNFY-B21 is taken out from -80 DEG C of refrigerators, takes 200 μ L bacterium
Liquid is inoculated in 20mL YEB fluid nutrient medium (+the 100mg/L of rifampin containing 100mg/L kanamycins), is cultivated in 28 DEG C of shaking tables
48h.When bacterium solution in it is golden yellow when, take 5ml bacterium solution be inoculated in 500mlYEB fluid nutrient medium (rifampin containing 100mg/L+
100mg/L kanamycins), it is placed in 28 DEG C of shaking tables and cultivates to bacterium solution OD600=0.6~0.8.By bacterium solution 12 at 18 DEG C,
000r/min is centrifuged 2min, abandons supernatant.With the WPM fluid nutrient medium of addition acetosyringone (100 μm of ol/L), (i.e. WPM is resuspended
Liquid) be resuspended after can be used to convert.
(2) Agrobacterium is infected
It chooses the sterile tender blade of 84K poplar tissue-cultured seedling and is aseptically cut into 0.5 × 0.5cm2Fritter,
It puts it into the bacterium solution being resuspended through WPM re-suspension liquid and disseminates 10-15min, and slowly rocked in soaking process.
(3) it co-cultures
The bacterium solution for the blade surface disseminated is blotted with sterile filter paper, blade is tiled back on WPM co-cultivation base, 25
DEG C ± 2 DEG C, dark culture 2d, form the explant by co-culturing.
(4) selection culture
It will be transferred on WPM Selective agar medium by the explant co-cultured, in 25 DEG C, illumination 2000-10000Lux
Under the conditions of cultivate 2-3 weeks therebetween every 10d replace a subculture.
(5) culture of rootage is screened
When resistance adventitious bud it is long to about 2cm when, cut and be transferred in WPM root media, 10d or so is taken root.
Referring to Fig. 6.
(6) transplanting of transgenosis 84K poplar
Seedling is taken out when under growth root seedling long (30 days or so) is to about 8cm, gently cleans root agar, transplanting to greenhouse soil
It is cultivated in earth, and on preservative film cover, manual simulation's natural conditions illumination (illumination 16 hours, dark 8 hours) took off film after 10 days.
Greenhouse experiment: relative humidity~45%, 20-24 DEG C of constant temperature, periodicity of illumination is 8h dark 16h illumination cultivation.According to the method described above
9 transgenic lines are prepared for, respectively marked as #1, #2, #3, #4, #5, #6, #8, #12, #16.
Embodiment 5: the identification of transgenic poplar
(1) PCR is identified
I. for the wild type 84K poplar of PCR and the extraction for turning PdNFY-B21 gene 84K poplar total DNA
Poplar DNA (Saghaimaroof et al.1984) is extracted using the CTAB method of improvement, the method is as follows:
A. take~the fresh blade of 0.6g in 10ml centrifuge tube, pulverize in liquid nitrogen；3ml 1%CTAB is added immediately
It (is preheated in advance) with 90 μ l beta -mercaptoethanols, 65 DEG C of water-bath 45min (the preceding every 10min of 30min mixes primary), taking-up is put to room
Temperature；
B. be added and CTAB isometric chloroform: isoamyl alcohol (V/V 24:1) acutely lays flat 10min after concussion；16 DEG C,
10000r/min is centrifuged 10min；Take supernatant in separately to be repeated 8 times in centrifuge tube；
C. it takes in supernatant 2ml to 10ml centrifuge tube, the isopropanol being pre-chilled in equal volume is added, it is cotton-shaped heavy to occurring to shake gently
It forms sediment；Flocculent deposit is carefully sucked out with pipette tips in 1.5ml centrifuge tube, twice with the rinsing of 75% ethanol water；The drift of 100% ethyl alcohol
It washes once, is dried in 37 DEG C of baking ovens；50 μ l ddH are added2O, 1 μ l Rnase, 37 DEG C of processing 1.5h；- 20 DEG C of preservations.
II. to turn the genomic DNA of PdNFY-B21 gene 84K Yang Yuwei transgenic plant as template, in 35S promoter
Design primer in sequence and PdNFY-B21 gene order carries out PCR reaction, and target fragment size is about 600bp.Design is drawn
Object is as follows:
Primer name Primer Sequence
5 ' AGTGGATTGATGTGATATCTCCACTGA3 ' of 35S upstream primer (SEQ ID NO.9)
5 '-TTATGATAGGGTTGCTAAAAGTTTAGCACTGT-3 ' of PdNFY-B21 downstream primer (SEQ ID NO.4)
Testing result shows that conversion poplar can amplify the electrophoretic band of expected size, with the recombination as the positive
Plasmid electrophoretic band it is in the same size, and the DNA of negative control nontransgenic plants does not have then, shows transgenic poplar genome
In contained foreign gene DNA fragmentation result such as Fig. 7 and shown.
Wherein, M representation DNA markers；PC represents the positive control (embodiment for having pBI121-PdNF-YB21 plasmid
PBI121 recombinant plasmid in 3)；WT represents wild type 84K poplar sample；#1, #2, #3, #4, #5, #6, #8, #12, #16 difference
Represent corresponding transgenic plant.
(2) GUS dyeing detection
X-Gluc is the substrate of gus gene detection, and X-Gluc can be hydrolyzed to blue material by GUS (beta-glucosidase),
This substance does not dissolve in plant cell tissue, therefore GUS active site will be displayed in blue, it can be achieved that plant GUS fusion protein
Quickly detection.
PdNFY-B21 gene is designed as fusion egg by the expression vector of this experimental construction together with the GUS sequence on carrier
It is white, therefore the expression of the indirect reflection genetically modified plants PdNFY-B21 gene of GUS dyeing can be used.The concrete operations of experiment
It is as follows:
The preparation of A.GUS dye liquor:
50mM sodium phosphate buffer+10mM Na2EDTA,0.5mM K4[Fe(CN)6]·3H2O+0.5mM K3[Fe
(CN)6], 0.1%Triton X-100 and 1mg/mLX-Gluc, (concentration refers in the corresponding entire solution of ingredient pH 7.0
Concentration).
B. clip poplar leaf is put into clean pipe, and adds GUS dye liquor, is totally submerged blade in GUS dyeing liquor, in
It is dyed under 37 DEG C of dark conditions 12 hours, then with 75% alcohol decoloration 20min.Be finally transferred to fixer (chloraldurate:
Water: glycerol；8:3:1；W/v/v it is observed and takes pictures after 2h is fixed in).
GUS coloration result shows, the leaf of transgenic poplar has a blue appearance, and the wild type poplar material after alcohol decoloration
Be then it is colorless and transparent, as a result as Fig. 8 shows.
Wherein, WT represents wild type；#1, #2, #3, #4, #5, #6, #8, #12, #16 respectively represent corresponding transgenic plant.
(3) fluorogenic quantitative detection
The blade RNA for turning PdNFY-B21 gene 84K Yang Yuwei transgenic plant is extracted, NFY-B21 base between each strain is detected
The expression difference of cause.
A. the fluorogenic quantitative detection of target gene
According to the coded sequence (SEQ ID NO.5) for the PdNFY-B21 gene that embodiment 2 is sequenced, use
5 software design fluorescent quantitation primer of primer.
The cDNA that the reversion of each group sample records is diluted to 100ng/ μ l, as template.Use Tiangeng company
FastFire qPCRPreMix (SYBR Green) kit carries out fluorescent quantitation reaction.20 μ l reaction systems include:
Quantitative fluorescent PCR reaction condition is 95 DEG C of 15min, 95 DEG C of 10s, 60 DEG C of 30s, 40 circulations.It is with Actin gene
Reference gene, each sample technology is repeated 4 times, with 2-ΔΔCTThe result of method calculating fluorescent quantitation.
Fluorescent quantitative PCR result shows that the expression quantity of the PdNFY-B21 of 9 positive strains is not identical, wherein
The expression quantity highest of oxPdNFY-B21#6, oxPdNFY-B21#8, therefore in subsequent experimental, the two strains are chosen as reality
Test material.As a result as Fig. 9 shows.WT represents wild type；#1, #2, #3, #4, #5, #6, #8, #12, #16, which are respectively represented, accordingly turns base
Because of plant.
6 transgenic poplar performance test of embodiment
One, poplar plant drought resistance is tested
Prolonged drought treatment conditions
After tissue-cultured seedling is moved into soil and cultivated 1 month, take wild type (parent), oxPdNFY-B21#6, #8 strain more respectively
Strain, control soil water-containing gesture are -1.82MPa as drought environment, carry out prolonged drought processing in 40 days.Use PSYPRO Water
Potential System (WESCOR, Utah, USA) measures the flow of water of the soil in soil basin.Every afternoon, 4:00-5:00 was poured
Water maintains soil water potential -1.82MPa (simulation water deficit conditions, drought status), keeps each basin under the conditions of corresponding soil water potential
40 days.(wherein three plants of poplar seedlings from left to right successively represent wild type, #6, #8 respectively, retain the one of plastic tub as seen from Figure 10
Part is relative size in order to indicate photo), wild type 84K poplar blade starts to turn yellow mostly after arid 40 days, and converts and plant
Strain oxPdNFY-B21#6, #8 have flourishing root system to have no significant effect.Therefore illustrate that transgenic poplar drought resistance is remarkably reinforced.
Two, poplar carton dioxide assimilation is tested
After tissue-cultured seedling is moved into soil and cultivated 30 days, take wild type (parent), oxPdNFY-B21#6, #8 strain more respectively
Strain controls soil water potential -1.82MPa 40 days, and soil water potential uses PSYPRO Water Potential System
(WESCOR, Utah, USA) measurement, every progress carton dioxide assimilation test in 5 days, using photosynthetic instrument LI-COR 6400
(Lincoln, NE, USA) instrument measures carton dioxide assimilation rate.With result referring to Figure 11.
As seen from Figure 11, two strain carton dioxide assimilation speed of oxPdNFY-B21#6, #8 is almost without difference, and the two is all
It is significantly faster than that wild-type parent adjoining tree.
After this illustrates that poplar is transferred to PdNFY-B21 gene, carton dioxide assimilation can be increased significantly and hastened, this will make this
Transgenic poplar is grown faster, and poplar seedlings can faster become a useful person.Transgenic poplar growth is accelerated to be conducive to quickly reduce big
The greenhouse effects of carbon dioxide in gas.
Three, poplar stem height is tested
After tissue-cultured seedling moves into soil and cultivates 30 days, wild type (parent), oxPdNFY-B21#6, #8 strain are taken respectively
It more plants, controls soil water potential -1.82MPa 40 days, every progress stem height test in 10 days.As a result referring to Figure 12.
As seen from Figure 12, the two high difference of strain stem of oxPdNFY-B21#6, #8 is unobvious, and the two is all apparently higher than wild type
Parent control plant.It is being grown always in 40 days under the conditions of transgenic poplar is under non-irrigated stressful environmental, and wild type is the 10th
It is with regard to growthing lag.This explanation, under the drought stress environment, transgenic poplar being capable of continued propagation.This also with aforementioned dioxy
It is corresponding for changing carbon assimilation effect.With advantage.
Four, poplar root size is tested
Root is frequently located in earth's surface in the following, absorbing the moisture and dissolution inorganic salts therein inside soil, and have and support, is numerous
Grow, store the effect of synthesis of organic substance matter.Under drought stress, the absorption and transport of root moisture are played an important role.
After tissue-cultured seedling is moved into soil and cultivated 30 days, take wild type (parent), oxPdNFY-B21#6, #8 strain more respectively
Strain, the size (root long, root diameter, root volume) of progress root and each strain root after control soil water potential -1.82MPa 40 days, 40 days
Number measure test.With result referring to Figure 13 (on the basis of wild type, recording the multiple of #6, #8) and 14.
By Figure 13 and 14 as it can be seen that the size of two strain root of oxPdNFY-B21#6, #8 and the number of each strain root almost do not have
There is difference, the two is all apparently higher than wild-type parent adjoining tree, illustrates that transgenic line has under conditions of arid and more sends out
Big root system, therefore under drought condition, transgenic line being capable of normal growth.
Five, poplar root/shoot ratio is tested
Root/shoot ratio refers to the fresh weight of foot end and aerial part or the ratio of dry weight.Its size reflects plant
The correlation of under ground portion and aerial part, and measure an important indicator of plant drought resistance.
After tissue-cultured seedling is moved into soil and cultivated 30 days, take wild type (parent), oxPdNFY-B21#6, #8 strain more respectively
Strain controls soil water potential -1.82MPa 40 days, every progress root/shoot ratio test in 10 days.As a result referring to Figure 15.
As seen from Figure 15, for two strain root/shoot ratio of oxPdNFY-B21#6, #8 almost without difference, the two is all apparently higher than open country
Raw type parent control plant illustrates that transgenic line is with the root system and root/shoot ratio for more sending out big under conditions of arid, therefore
Under drought condition, transgenic line being capable of normal growth.
Six, poplar root conductivity is tested
The plasma membrane of plant is the poor environments such as interface and barrier arid between living cells and environment, can all make plasma membrane by
Different degrees of damage.The most common method of measurement membrane permeability variation is to measure the variation of the outer sepage conductivity of tissue, is
Measure one of the important indicator of Genes For Plant Tolerance stress ability power.
After tissue-cultured seedling is moved into soil and cultivated 30 days, take wild type (parent), oxPdNFY-B21#6, #8 strain more respectively
Strain controls soil water potential -1.82MPa 40 days, uses DDS-307 (thunder magnetic-DDS-307A, Shanghai) conductivity meter every 10 days
Measure the conductivity meter of root.With result referring to Figure 16.
As seen from Figure 16, for the conductivity of two strain root of oxPdNFY-B21#6, #8 almost without difference, the two is all obvious low
In wild-type parent adjoining tree, illustrate under conditions of arid transgenic line by the stress of arid influence will lower than pair
According to plant.
Seven, Biomass of Poplar is tested
After tissue-cultured seedling moves into soil and cultivates 30 days, wild type (parent), oxPdNFY-B21#6, #8 strain are taken respectively
More plants, control soil water-containing gesture is -1.82MPa, carries out biomass test.As a result referring to Figure 17.
As seen from Figure 17, two strain biomass of oxPdNFY-B21#6, #8 is all significantly more than open country almost without difference, the two
Raw type parent control plant.
Eight, Poplar Roots respiratory rate is tested
The breathing of root and it grows to the absorption of mineral element and with it and differentiation has a substantial connection, respiratory rate with
Anabolism, the speed of growth are all positively correlated.
After tissue-cultured seedling is moved into soil and cultivated 30 days, take wild type (parent), oxPdNFY-B21#6, #8 strain more respectively
Strain controls soil water potential -1.82MPa 40 days, every progress root breath speed trial in 5 days, using photosynthetic instrument LI-8100A
(Lincoln, NE, USA) instrument measures the respiratory rate of soil, and the respiratory rate (document 1) of root system is calculated by exclusive method.With knot
Fruit is referring to Figure 18.
As seen from Figure 18, for the respiratory rate of two strain root of oxPdNFY-B21#6, #8 almost without difference, the two is all obvious
Higher than wild-type parent adjoining tree.
After this illustrates that poplar is transferred to PdNFY-B21 gene, the respiratory rate of root can be improved significantly, this will make this turn base
Faster because of growth of poplar, there is more flourishing root system, to improve drought tolerance.
Nine, poplar root hydraulic conductivity is tested
The hydraulic conductivity of root system of plant determines biology and the development of plant to a certain extent, especially in moisture scarcity
Under the conditions of, the hydraulic conductivity of root system directly determines the growth conditions of plant.
After tissue-cultured seedling is moved into soil and cultivated 30 days, take wild type (parent), oxPdNFY-B21#6, #8 strain more respectively
Strain controls soil water potential -1.82MPa 40 days, every progress root hydraulic conductivity measurement test in 5 days, using high pressure hydraulic conductivity instrument
HPFM-Gen3 (Dynamax, USA) measures the hydraulic conductivity of root, with result referring to Figure 19.
As seen from Figure 19, for the hydraulic conductivity of two strain root of oxPdNFY-B21#6, #8 almost without difference, the two is all obvious high
In wild-type parent adjoining tree.After this illustrates that poplar is transferred to PdNFY-B21 gene, the hydraulic conductivity of root can be improved significantly,
This will make the transgenic poplar have more flourishing root system, to improve drought tolerance.
Ten, poplar root lignin concentration and structured testing
The survey experiment of the lignin concentration of root is carried out according to bibliography (1), and lignin structure is tested according to bibliography
(2,3,4) it carries out, with result referring to fig. 20 and 21.
The growth and development of root and the concentration of lignin and structure are closely related.
As seen from Figure 20, after tissue-cultured seedling is moved into soil and cultivated 30 days, wild type (parent), oxPdNFY-B21# are taken respectively
6, more plants of #8 strain, control soil water potential -0.12MPa (moisture is sufficient) 40 days, take the root of its poplar to the concentration of its lignin
It is analyzed with structure.As seen from Figure 21, the lignin concentration of two strain root of oxPdNFY-B21#6, #8 and structure (S/G) be almost
There is no difference, the lignin concentration of the two and the ratio of lignin S/G are all apparently higher than wild-type parent adjoining tree.
By above-mentioned check experiment data it is found that PdNFY-B21 gene can enhance the drought resistance of poplar significantly, improve
Growth of poplar speed.
Aforementioned various contrasting datas complement each other, it may be said that and it is bright, it, can after poplar is transferred to PdNFY-B21 gene
Drought stress is resisted significantly, it is normal or close to normally growing.This is beneficial to the plantation for improving arid area poplar, improves
Environment harvests trees material, slows down greenhouse effects.
This gene overexpression is remarkably improved the drought resistance of poplar, under conditions of normal moisture is sufficient, and does not turn base
The plant of cause is compared, and the root for turning PdNFY-B21 poplar has higher lignin concentration and lignin S/G ratio.It is coerced in arid
Compel 40 days, compared with the plant of non-transgenosis, turns PdNFY-B21 poplar energy normal growth, and timber yield and material are basic
It is unaffected, photosynthesis with higher, the respiratory rate of root, the hydraulic conductivity of root, the growth rate of stem, and the root system of hair,
Root/shoot ratio and biomass provide guarantee to improve the yield of poplar under Drought Stress.
Sequence table
<110>Beijing Forestry University
<120>a kind of poplar anti-drought gene and its application
<130> M1CNCN181008
<160> 11
<170> SIPOSequenceListing 1.0
<210> 1
<211> 26
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 1
gatgatttgc tttgggctat ggctac 26
<210> 2
<211> 24
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 2
tccagtcttc gcagatccct tggt 24
<210> 3
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 3
atggcggcag aggcaccggc 20
<210> 4
<211> 32
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 4
ttatgatagg gttgctaaaa gtttagcact gt 32
<210> 5
<211> 546
<212> DNA
<213>poplar (Populus)
<400> 5
atggcggcag aggcaccggc gagtccaggg ggaggaagcc atgagagcgg agaccaaagt 60
cctcgctcta attctaatgt acgtgaacaa gacaggttct taccgatcgc aaatatcagt 120
aggattatga agaaagcgct acctgctaat ggaaagattg ctaaggatgc taaagagact 180
gttcaagaat gtgtttctga gttcattagc tttatcacta gcgaagcaag tgataagtgt 240
cagcgagaaa agaggaagac gataaatggt gatgatttgc tttgggctat ggctacgtta 300
gggtttgagg attatattga tcctcttaag atttacctgt ctcgatacag agagatggag 360
ggtgatacca agggatctgc gaagactgga gatacatctg ctaaaaagga tattcaccct 420
ggtccaaatg cgcaggtgaa atggagcttt atgttgctga gttactgcca tttcttattt 480
ttagtcaatc aacgtacctc cttgtcttta ttttacagtg ctaaactttt agcaacccta 540
tcataa 546
<210> 6
<211> 181
<212> PRT
<213>poplar (Populus)
<400> 6
Met Ala Ala Glu Ala Pro Ala Ser Pro Gly Gly Gly Ser His Glu Ser
1 5 10 15
Gly Asp Gln Ser Pro Arg Ser Asn Ser Asn Val Arg Glu Gln Asp Arg
20 25 30
Phe Leu Pro Ile Ala Asn Ile Ser Arg Ile Met Lys Lys Ala Leu Pro
35 40 45
Ala Asn Gly Lys Ile Ala Lys Asp Ala Lys Glu Thr Val Gln Glu Cys
50 55 60
Val Ser Glu Phe Ile Ser Phe Ile Thr Ser Glu Ala Ser Asp Lys Cys
65 70 75 80
Gln Arg Glu Lys Arg Lys Thr Ile Asn Gly Asp Asp Leu Leu Trp Ala
85 90 95
Met Ala Thr Leu Gly Phe Glu Asp Tyr Ile Asp Pro Leu Lys Ile Tyr
100 105 110
Leu Ser Arg Tyr Arg Glu Met Glu Gly Asp Thr Lys Gly Ser Ala Lys
115 120 125
Thr Gly Asp Thr Ser Ala Lys Lys Asp Ile His Pro Gly Pro Asn Ala
130 135 140
Gln Val Lys Trp Ser Phe Met Leu Leu Ser Tyr Cys His Phe Leu Phe
145 150 155 160
Leu Val Asn Gln Arg Thr Ser Leu Ser Leu Phe Tyr Ser Ala Lys Leu
165 170 175
Leu Ala Thr Leu Ser
180
<210> 7
<211> 26
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tctagaatgg cggcagaggc accggc 26
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<400> 8
cccgggttat gatagggttg ctaaaagttt agcactgt 38
<210> 9
<211> 27
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 9
agtggattga tgtgatatct ccactga 27
<210> 10
<211> 19
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 10
gtcctcttcc agccatctc 19
<210> 11
<211> 19
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 11
ttcggtcagc aataccagg 19
Claims (10)
1. a kind of poplar albumen, the amino acid sequence of the albumen is as shown in SEQ ID NO.6.
2. a kind of poplar gene, nucleic acid sequence encoding amino acid sequence as shown in SEQ ID NO.6 of the gene；
It is preferred that the nucleic acid sequence of the gene is as shown in SEQ ID NO.5.
3. a kind of gene engineering expression carrier, the expression vector includes poplar gene as claimed in claim 2, it is preferable that institute
The carrier is carrier for stating gene engineering expression carrier is PBI121 plasmid.
4. a kind of recombinational agrobacterium, the recombinational agrobacterium includes expression vector as claimed in claim 3, it is preferable that the agriculture
Bacillus is GV3101.
5. a kind of method of prepare transgenosis poplar, the method is that recombinational agrobacterium as claimed in claim 4 is transformed into poplar
In tree, the transgenic poplar is formed.
6. method as claimed in claim 5, described method includes following steps:
(1) bacterium liquid activation: the bacterium solution of recombinational agrobacterium as claimed in claim 4 is trained in the YEB containing rifampin and kanamycins
It is cultivated in nutrient solution, forms the first mixture；First mixture is centrifuged, supernatant is abandoned, forms the first precipitating；With addition acetyl
First precipitating is resuspended in the WPM culture solution of syringone, forms the second mixture；
(2) Agrobacterium is infected: the sterile tender blade of poplar tissue-cultured seedling being put into second mixture and is disseminated；(3) training altogether
It supports: blotting the liquid component on dip dyeing rear blade, blade merging WPM is co-cultured into base, dark culture；
(4) selection culture:
The blade of the co-cultivation is transferred on WPM Selective agar medium, is cultivated under light illumination, resistance adventitious bud is formed；
(5) culture of rootage is screened:
The resistance adventitious bud is transferred in WPM root media and is cultivated, poplar seedlings of taking root are formed.
7. a kind of for detecting the PCR primer pair of transgenic poplar, the primer pair includes the first upstream primer and the first downstream
Primer,
First upstream primer sequence are as follows:
5 '-AGTGGATTGATGTGATATCTCCACTGA-3 ', as shown in SEQ ID NO.9；
First downstream primer sequence are as follows:
5 '-TTATGATAGGGTTGCTAAAAGTTTAGCACTGT-3 ', as shown in SEQ ID NO.4.
8. a kind of for detecting or monitoring the fluorescence PCR primer pair of Drought Resistance of Populus, the primer pair includes the second upstream primer
With the second downstream primer,
Second upstream primer sequence are as follows:
5'-GATGATTTGCTTTGGGCTATGGCTAC-3', as shown in SEQ ID NO.1；
Second downstream primer sequence are as follows:
5'-TCCAGTCTTCGCAGATCCCTTGGT-3', as shown in SEQ ID NO.2.
9. a kind of for detecting or monitoring the kit of Drought Resistance of Populus, the kit includes as claimed in claim 8 glimmering
Fluorescent Quantitative PCR primer pair.
10. a kind of albumen as described in claim 1, gene as claimed in claim 2, genetic engineering as claimed in claim 3
Expression vector, recombinational agrobacterium as claimed in claim 4, or the side such as prepare transgenosis poplar described in claim 5 or 6
Purposes of the method in the breeding of improvement poplar character or the improvement poplar character, the poplar character include:
(a) drought resistance；
(b) carton dioxide assimilation rate；
(c) speed of growth；
(d) root breath rate；
(e) root hydraulic conductivity；
(f) growth of root；
(g) lignin concentration of root and structure；
It is preferred that the poplar is 84K poplar or black poplar NE19.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201811523555.9A CN109384837B (en) | 2018-12-13 | 2018-12-13 | Poplar drought-resistant gene and application thereof |
Applications Claiming Priority (1)
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| CN115838739A (en) * | 2022-12-27 | 2023-03-24 | 沈阳农业大学 | Application of NAC17 gene in improvement of new aspen character |
| CN116574740A (en) * | 2023-05-16 | 2023-08-11 | 沈阳农业大学 | PuNAC47 gene related to drought resistance of populus euphratica and application thereof |
| CN116731139A (en) * | 2023-06-20 | 2023-09-12 | 西南大学 | Application of PtoERF15 gene of populus tomentosa in regulation and control of drought resistance of poplar |
| CN116790627A (en) * | 2023-08-03 | 2023-09-22 | 贵州大学 | Poplar MYBS2 gene and application thereof |
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| CN116731139A (en) * | 2023-06-20 | 2023-09-12 | 西南大学 | Application of PtoERF15 gene of populus tomentosa in regulation and control of drought resistance of poplar |
| CN116731139B (en) * | 2023-06-20 | 2024-03-22 | 西南大学 | Application of PtoERF15 gene of populus tomentosa in regulation and control of drought resistance of poplar |
| CN116790627A (en) * | 2023-08-03 | 2023-09-22 | 贵州大学 | Poplar MYBS2 gene and application thereof |
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