CN110257401A - Comospore poplar PtrMYB119 gene is improving the application in tobacco drought tolerance - Google Patents
Comospore poplar PtrMYB119 gene is improving the application in tobacco drought tolerance Download PDFInfo
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Abstract
The present invention provides comospore poplar PtrMYB119 genes to improve the application in tobacco drought tolerance, belongs to field of plant genetic project technology.The research of the invention finds that comospore poplar PtrMYB119 gene is transferred to tobacco leaf, the anthocyanidin content in the transgene tobacco of acquisition is obviously improved, and can effectively improve the drought resistance of tobacco.
Description
Technical field
The present invention relates to field of plant genetic project technology, and in particular to comospore poplar PtrMYB119 gene is improving tobacco
Application in drought tolerance.
Background technique
Arid will lead to the decline of plant cell water content, and active oxygen radical outburst destroys plant cell membranous system, causes
Plant growth and development is slow, or even dead to cause the plant underproduction.Currently, there is the soil of one third or more to belong in the world
Arid and semi-arid lands, arid be always influence agricultural development the main reason for one of, nowadays Droughts range, degree and
The frequency is in increase trend, keeps the growth and development of crops seriously hampered, and crop yield reduction causes huge economic damage
It loses.The drought-resistant ability for improving plant has become one of the critical issue in modern plants research work, the research of drought resistance mechanism
It is one of basis and the key factor of breeding of Drought-resistant Breeding.Therefore, excavating has the gene of drought resistance function anti-for cultivating
Drought plant has great importance.
Anthocyanidin is a kind of phytochrome with natural bioactive, and the synthesis and accumulation in nutrition organs are to plant
Object adapts to and resistance rugged environment condition is most important, and plant can be enhanced and support to different biotics and abiotic stress
Anti- ability, for example, arid, high temperature, low temperature salt stress, pest and disease damage etc..Colouration mechanism, anthocyanidin of the anthocyanidin in plant tissue
Biosynthetic controlling in terms of research it is more, and its research in terms of plant resistance to environment stress is less, more and more research shows that flower
Green element is coped in abiotic stress and biotic in plant to be played an important role.
Summary of the invention
In view of technical problem present in background technique, the purpose of the present invention is to provide comospore poplar PtrMYB119 bases
Because improving the application in tobacco drought tolerance.
In order to achieve the above-mentioned object of the invention, the present invention the following technical schemes are provided:
The present invention provides comospore poplar PtrMYB119 genes to improve the application in tobacco drought tolerance.
Preferably, the application includes the following steps:
(1) comospore poplar total serum IgE is extracted, reverse transcription obtains cDNA, and using the cDNA as template, PCR amplification is obtained
PtrMYB119 gene；
(2) the PtrMYB119 gene is inserted into initial carrier, obtains recombinant plant expression vector；
(3) recombinant plant expression vector is transformed into agrobacterium tumefaciens, obtains recombination agrobacterium tumefaciens transformant；
(4) the recombination agrobacterium tumefaciens transformant is infected into tobacco leaf, obtains T0 for genetically modified plants.
Preferably, step (1) described PCR amplification is PtrMYB119-F and PtrMYB119-R with primer pair；It is described
The nucleotide sequence of PtrMYB119-F is as shown in SEQ ID No.1；The nucleotide sequence of the PtrMYB119-R such as SEQ ID
Shown in No.2.
Preferably, the reaction system of step (1) described PCR amplification is 20 μ L:cDNA template, 1 μ L, upstream and downstream primer each 0.4
μ L (10 μM), 10 × Buffer, 2 μ L, 1.6 dNTPs μ L, rTaq enzyme 0.1 μ L, ddH2O14.5μL。
Preferably, the program of step (1) described PCR amplification are as follows: 94 DEG C, 3min；30 circulations, each 94 DEG C of circulation,
30s, 60 DEG C, 30s, 72 DEG C, 1min；72 DEG C extend 10min eventually.
Preferably, step (2) initial carrier be pCAMBIA2300, pCAMBIA1300, pCAMBIA1301 or
pCAMBIA3300。
Preferably, step (3) it is described conversion and/or step (4) described transfection after, further include the steps that positive identification.
It preferably, further include that the T0 is transplanted to temperature for genetically modified plants after step (4) obtains T0 for genetically modified plants
The step of room.
Further, the present invention also provides the recombinant plant expression vectors of the PtrMYB119 gene of poplar containing comospore to improve
Application in tobacco drought tolerance.
Further, the present invention also provides the recombination agrobacterium tumefaciens transformants of the PtrMYB119 gene of poplar containing comospore to exist
Improve the application in tobacco drought tolerance.
The utility model has the advantages that the present invention provides comospore poplar PtrMYB119 genes to improve the application in tobacco drought tolerance.This hair
It is bright the study found that comospore poplar PtrMYB119 gene is transferred to tobacco leaf, the anthocyanidin content in the transgene tobacco of acquisition is aobvious
It writes and is promoted, the drought resistance of tobacco can be effectively improved.
Detailed description of the invention:
Fig. 1 is transgenic plant qualification result described in the embodiment of the present invention 1, wherein Fig. 1-A is dyeing qualification result；Figure
1-B is PCR qualification result.
Fig. 2 is the character mutation under transgene tobacco Osmotic treatment, wherein phenotype when Fig. 2-A is Osmotic treatment 0 day；
Phenotype when Fig. 2-B is Osmotic treatment 10 days；Phenotype when Fig. 2-C is Osmotic treatment 30 days；When Fig. 2-D is Osmotic treatment 30 days
Root long statistical result；Root long phenotype when Fig. 2-E is Osmotic treatment 30 days；The phenotype of stem when Fig. 2-F is Osmotic treatment 30 days；
The phenotype of stem cross section when Fig. 2-G is Osmotic treatment 30 days.
Fig. 3 is the variation of anthocyanidin and chlorophyll content under transgene tobacco Osmotic treatment, wherein Fig. 3-A is anthocyanidin
The variation of content；Fig. 3-B is the variation of chlorophyll content.
Fig. 4 is the variation of ABA content and MDA content under transgene tobacco Osmotic treatment, wherein Fig. 4-A is ABA content
Variation；Fig. 4-B is the variation of MDA content.
Fig. 5 is the active variation of POD, SOD and CAT under transgene tobacco Osmotic treatment, wherein Fig. 5-A is that POD is active
Variation；Fig. 5-A is the active variation of SOD；Fig. 5-C is the active variation of CAT.
Fig. 6 is antioxidant genes under transgene tobacco Osmotic treatment, polyamines biosynthesis gene and responses of drought stress gene
Relative expression quantity, wherein Fig. 6-A is the relative expression quantity of antioxidant genes；Fig. 6-B is the opposite table of polyamines biosynthesis gene
Up to amount；Fig. 6-C is the relative expression quantity of responses of drought stress gene.
Specific embodiment
The present invention provides comospore poplar PtrMYB119 genes (Serial No.: Potri.017G125600) to improve tobacco
Application in drought tolerance.The research of the invention finds that comospore poplar PtrMYB119 gene is transferred to tobacco leaf, the transgenosis of acquisition
Anthocyanidin content in tobacco is obviously improved, and can effectively improve the drought resistance of tobacco.
In the present invention, the application preferably includes following steps:
(1) comospore poplar total serum IgE is extracted, reverse transcription obtains cDNA, and using the cDNA as template, PCR amplification is obtained
PtrMYB119 gene；
(2) the PtrMYB119 gene is inserted into initial carrier, obtains recombinant plant expression vector；
(3) recombinant plant expression vector is transformed into agrobacterium tumefaciens, obtains recombination agrobacterium tumefaciens transformant；
(4) the recombination agrobacterium tumefaciens transformant is infected into tobacco leaf, obtains T0 for genetically modified plants.
The present invention first extracts comospore poplar total serum IgE, and reverse transcription obtains cDNA, and using the cDNA as template, PCR amplification is obtained
PtrMYB119 gene.In the present invention, the PCR amplification is preferably PtrMYB119-F and PtrMYB119-R with primer pair；Institute
The nucleotide sequence of PtrMYB119-F is stated as shown in SEQ ID No.1；The nucleotide sequence of the PtrMYB119-R such as SEQ
Shown in ID No.2.In the present invention, the reaction system of step (1) described PCR amplification be preferably 20 μ L:cDNA template, 1 μ L, on
Each 0.4 μ L of downstream primer (10 μM), 10 × Buffer, 2 μ L, 1.6 dNTPs μ L, rTaq enzyme 0.1 μ L, ddH2O14.5μL.Step
(1) program of the PCR amplification is preferred are as follows: and 94 DEG C, 3min；30 circulations, each 94 DEG C, 30s, 60 DEG C, 30s, 72 DEG C of circulation,
1min；72 DEG C extend 10min eventually.
After obtaining the PtrMYB119 gene, the PtrMYB119 gene is inserted into initial carrier by the present invention, is obtained
Recombinant plant expression vector.In the present invention, the initial carrier be preferably pCAMBIA2300, pCAMBIA1300,
PCAMBIA1301 or pCAMBIA3300；More preferably pCAMBIA2300.
The recombinant plant expression vector is transformed into agrobacterium tumefaciens by the present invention, obtains recombination agrobacterium tumefaciens conversion
Son.In the present invention, the agrobacterium tumefaciens are preferably agrobacterium tumefaciens EHA105.The present invention is preferably to the recombination root nodule agriculture
Agrobacterium-transformation carries out positive identification.In the present invention, the preferred picking white single colonie of the positive identification (recombination root nodule agriculture bar
Bacterium transformant), it is seeded in the LB liquid medium containing 100mg/L kanamycins, 250rpm, 28 DEG C are shaken bacterium and cultivated 4 hours,
PCR detection positive colony is carried out with primer PtrMYB119-F and PtrMYB119-R.
After obtaining recombination agrobacterium tumefaciens transformant, the recombination agrobacterium tumefaciens transformant is infected Tobacco Leaf by the present invention
Piece obtains T0 for genetically modified plants.In the present invention, described to infect that it is preferable to use infusion methods: the tobacco leaf that cut into pieces
It is immersed in 1~3min in the LB liquid medium containing the recombination agrobacterium tumefaciens transformant.The present invention is preferably to the T0 generation
Genetically modified plants carry out positive identification.In the present invention, the positive identification preferably uses decoration method or PCR method.
After T0 is obtained for genetically modified plants, it is also preferable to include the T0 is transplanted to greenhouse for genetically modified plants by the present invention
Step.
The present invention clones anthocyanidin biosynthesis related genes PtrMYB119 from comospore poplar, constructs over-express vector, so
Tobacco leaf is transferred to Agrobacterium-mediated Transformation method afterwards.The present invention in tobacco line by being overexpressed the gene, and to its function
It is analyzed.The result shows that: the overexpression of PtrMYB119 gene can promote anthocyanidin and ABA content, antioxidase in tobacco
The expression quantity of activity and related gene, and MDA concentration can be reduced, to improve the drought tolerance of transgene tobacco.
The present invention also provides the recombinant plant expression vector of the PtrMYB119 gene of poplar containing comospore and poplars containing comospore
The recombination agrobacterium tumefaciens transformant of PtrMYB119 gene is improving the application in tobacco drought tolerance.The present invention is to poplar containing comospore
The conversion of the recombination agrobacterium tumefaciens of the recombinant plant expression vector of PtrMYB119 gene and the PtrMYB119 gene of poplar containing comospore
The concrete application mode of son is not particularly limited.
Technical solution provided by the invention is described in detail below with reference to embodiment, but they cannot be understood
For limiting the scope of the present invention.
Embodiment 1:
(1) comospore poplar total serum IgE is extracted, cDNA is obtained after reverse transcription, and amplification is amplified from poplar cDNA with round pcr
PtrMYB119 gene coded sequence out.Detailed process are as follows: arranged with following nucleotides sequence as primer: PtrMYB119-F:5 '-
CTAAGGAAGTGCGTTGAGAA-3 ', PtrMYB119-R:5 '-GCCAAGCAACTTGTGTAGTC-3 ' carries out PCR amplification.Expand
Increasing condition is 94 DEG C, 3min；30 circulations, each 94 DEG C, 30s, 60 DEG C, 30s, 72 DEG C of circulation, 1min；72 DEG C extend eventually
10min；Reaction system is 20 μ L:cDNA template, 1 μ L, each 0.4 μ L of upstream and downstream primer (10 μM), 10 × Buffer, 2 μ L, dNTPs
1.6 μ L, rTaq enzyme 0.1 μ L, ddH2O14.5μL.PCR product carries out agarose gel electrophoresis, is carried out with gel extraction kit
It is tapped and recovered, is stored in -20 DEG C after recovery product measurement concentration, or directly carry out digestion, the reaction such as connection.
(2) plant expression vector will be obtained in obtained PtrMYB119 gene insertion vector pCAMBIA2300
pCAMBIA2300-PtrMYB119.Detailed process are as follows: expanded gene PtrMYB19 obtained by step 1 as gateway recombination system
Volume increase object, which is cloned into over-express vector pCAMBIA2300 (kalamycin resistance), obtains plant expression vector pCAMBIA2300-
PtrMYB119 (bibliography: Karimi, M., Inze, D., Depicker, A., Gateway vectors for
Agrobacterium-mediated planttransformation.Trends Plant Sci.2002 May:7(5):
193-195)。
(3) gained plant expression vector is transformed into agrobacterium tumefaciens EHA105.Detailed process are as follows:
1. the Agrobacterium competent cell of -80 DEG C of preservations is taken to receive its partial melting in palm of the hand piece, it is in ice water mixing shape
It is inserted into ice when state；
2. take 100 μ L competent cells be added 0.01~1.0 μ g plasmid, finger touch tube bottom mix, successively on ice, liquid
5min is respectively stood in nitrogen, in 37 DEG C of water-baths and ice bath；
3. sterile LB liquid medium is added, in 28 DEG C degree shake culture 2~3 hours；
4. 6000rpm be centrifuged 1min receive bacterium, take 50 μ L supernatants gently blow and beat resuspension fungus block be coated on card containing 100mg/L that
On the LB plate of mycin, it is inverted on 28 DEG C of shaking tables and cultivates 2~3 days.
5. positive colony detects: picking white single colonie is seeded in the LB liquid medium containing 100mg/L kanamycins
In, 250rpm, 28 DEG C are shaken bacterium and cultivated 4 hours, carry out PCR detection positive colony with primer, identify it is errorless after, shake bacterium and be ready to use in turn
Change K326 Nicotiana tabacum.
(4) gained Agrobacterium-mediated Transformation is infected into flue-cured tobacco class K326 tobacco leaf, obtains T0 for genetically modified plants.Specific mistake
Journey are as follows: will identify that the sub- pCAMBIA2300-PtrMYB119 of errorless Agrobacterium-mediated Transformation is inoculated in 30mL LB liquid medium
(kanamycins containing 50mg/ml) is cultivated in shaking table of the temperature setting at 28 DEG C until OD600It is 0.5~0.8, bacterium is collected by centrifugation
Body is resuspended Agrobacterium with 30ml conversion culture medium MSo and is taken fresh and tender blade using infusion method transformation of tobacco blade, cut into pieces
Shape is immersed in Agrobacterium culture medium two minutes or so.Blade is taken out, is placed on not antibiotic MS solid medium 28 DEG C
It dark culture 2~3 days, moves back within 2~3 days and waits differentiation on MS differential medium containing kanamycin, change primary culture within one week
Base is detected after waiting blade differentiation to grow resistant buds.
Transgenic plant identification:
1. dyeing identification: the above-mentioned test tube seedling leaf 0.2cm of clip is placed in GUS dye liquor, 37 DEG C of dyeing 12h or more.Later
It is decolourized using dehydrated alcohol.There is blue and then shows that reporter gene is successfully integrated into Plant Genome in blade, the plant
Object is transgenic plant (Figure 1A).
2. PCR is identified: using the PCR kit of precious day biotechnology (Beijing) Co., Ltd production, to dyeing au bleu
Plant carry out transgenic plant PCR verify (Figure 1B).It is arranged with following nucleotides sequence as primer: PtrMYB119-F:5 '-
CTAAGGAAGTGCGTTGAGAA-3 ', PtrMYB119-R:5 '-GCCAAGCAACTTGTGTAGTC-3 ' carries out PCR amplification, expands
Increasing condition is 94 DEG C, 3min；30 circulations, each 94 DEG C, 30s, 60 DEG C, 30s, 72 DEG C of circulation, 1min；72 DEG C extend eventually
10min；Reaction system is 20 μ L:DNA template, 1 μ L, each 0.4 μ L of upstream and downstream primer (10 μM), 10 × Buffer, 2 μ L, dNTPs
1.6 μ L, rTaq enzyme 0.1 μ L, ddH2O14.5μL。
(5) transgenic plant is transplanted in greenhouse, obtains the transgenosis that anthocyanidin content improves and drought resistance improves
Tobacco.Detailed process are as follows: choose three overexpression transgenic lines (A, B and C) and WT lines carry out Osmotic treatment, often
A strain has 5 plant.After these plant finely cultivate 20 days in tissue culture bottle, hardening 7 days in illumination box are placed on,
Transplanting is cultivated in the plastic flowerpot into greenhouse after hardening.Greenhouse experiment: 28 DEG C of temperature, light dark period 16h/8h.It fills
Divide watering that plant growing way is kept unanimously to start Osmotic treatment afterwards, at interval of progress primary sample in 10 days and takes pictures, continuously take 3 times.
(6) anthocyanidin content of transgene tobacco and drought resistance are measured, the specific method is as follows:
To be overexpressed PtrMYB119 transgene tobacco and wild-type tobacco as control, is sent out and surveyed by spectrophotometer
It is colonized the internal anthocyanidin content of strain, the results showed that under Osmotic treatment, be overexpressed the anthocyanidin of PtrMYB119 transgene tobacco
Content is significantly higher than wild-type tobacco.
Under Osmotic treatment, by observing phenotype, measurement root long, blade wilting ratio and stem cross section measure ABA and MDA
Content measures activities of antioxidant enzymes and measures the opposite of antioxidant genes, polyamines biosynthesis gene and responses of drought stress gene
The drought resistance of the methods of expression verifying transgene tobacco, is as a result shown in Fig. 2~Fig. 6.Fig. 2~Fig. 6 shows: and wild-type tobacco phase
Than the drought resistance of transgene tobacco can be significantly improved by being overexpressed PtrMYB119.
The above is only a preferred embodiment of the present invention, it is noted that for the ordinary skill people of the art
For member, various improvements and modifications may be made without departing from the principle of the present invention, these improvements and modifications are also answered
It is considered as protection scope of the present invention.
Sequence table
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<120>comospore poplar PtrMYB119 gene is improving the application in tobacco drought tolerance
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
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ctaaggaagt gcgttgagaa 20
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<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 2
gccaagcaac ttgtgtagtc 20
Claims (10)
1. comospore poplar PtrMYB119 gene is improving the application in tobacco drought tolerance.
2. application according to claim 1, which comprises the steps of:
(1) comospore poplar total serum IgE is extracted, reverse transcription obtains cDNA, and using the cDNA as template, PCR amplification obtains PtrMYB119 base
Cause；
(2) the PtrMYB119 gene is inserted into initial carrier, obtains recombinant plant expression vector；
(3) recombinant plant expression vector is transformed into agrobacterium tumefaciens, obtains recombination agrobacterium tumefaciens transformant；
(4) the recombination agrobacterium tumefaciens transformant is infected into tobacco leaf, obtains T0 for genetically modified plants.
3. application according to claim 2, which is characterized in that step (1) described PCR amplification is with primer pair
PtrMYB119-F and PtrMYB119-R；The nucleotide sequence of the PtrMYB119-F is as shown in SEQ ID No.1；It is described
The nucleotide sequence of PtrMYB119-R is as shown in SEQ ID No.2.
4. application according to claim 3, which is characterized in that the reaction system of step (1) described PCR amplification is 20 μ L:
Each 0.4 μ L of 1 μ L of cDNA template, 10 μM of upstream and downstream primer, 10 × Buffer, 2 μ L, 1.6 dNTPs μ L, 0.1 μ L of rTaq enzyme,
ddH2O 14.5μL。
5. application according to claim 4, which is characterized in that the program of step (1) described PCR amplification are as follows: 94 DEG C,
3min；30 circulations, each 94 DEG C, 30s, 60 DEG C, 30s, 72 DEG C of circulation, 1min；72 DEG C extend 10min eventually.
6. application according to claim 2, which is characterized in that step (2) initial carrier be pCAMBIA2300,
PCAMBIA1300, pCAMBIA1301 or pCAMBIA3300.
7. application according to claim 2, which is characterized in that step (3) conversion and/or step (4) described transfection
Afterwards, further include the steps that positive identification.
8. application according to claim 2, which is characterized in that after step (4) obtains T0 for genetically modified plants, further include by
The step of T0 is transplanted to greenhouse for genetically modified plants.
9. the recombinant plant expression vector of the PtrMYB119 gene of poplar containing comospore is improving the application in tobacco drought tolerance.
10. the recombination agrobacterium tumefaciens transformant of the PtrMYB119 gene of poplar containing comospore is improving the application in tobacco drought tolerance.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111172172A (en) * | 2020-02-18 | 2020-05-19 | 南京林业大学 | Regulatory gene PdeMIXTA02 for initial development of populus deltoides and application thereof |
| CN111172172B (en) * | 2020-02-18 | 2021-02-12 | 南京林业大学 | Regulatory gene PdeMIXTA02 for initial development of populus deltoides and application thereof |
| CN114231541A (en) * | 2022-01-11 | 2022-03-25 | 沈阳农业大学 | MYB transcription factor for improving drought resistance of populus deltoids and application thereof |
| CN114231541B (en) * | 2022-01-11 | 2022-12-06 | 沈阳农业大学 | MYB transcription factor for improving drought resistance of populus deltoids and application thereof |
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