CN115851785A - Grape strigolactone gene VvD14 and application thereof in drought resistance - Google Patents
Grape strigolactone gene VvD14 and application thereof in drought resistance Download PDFInfo
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Abstract
The invention discloses a grape strigolactone gene VvD14, wherein the grape strigolactone gene VvD14 is a nucleotide sequence shown as SEQ ID NO. 1. By adopting the grape strigolactone gene VvD14 and the application thereof in drought resistance, the VvD14 gene has the function of improving the drought resistance of plants, and has important significance for breeding grape drought-resistant varieties and improving the drought resistance of crops.
Description
Technical Field
The invention relates to the technical field of molecular biology and biotechnology, in particular to a grape strigolactone gene VvD14 and application thereof in drought resistance.
Background
Grapes (Vitis vinifera l.) are one of the major commercial crops and are widely grown in various parts of the world. The grapes comprise protein, carbohydrate, crude fiber, calcium, phosphorus, iron, potassium and other trace elements, and have rich nutritional values, so the grapes are known as fruit queen.
Drought stress can cause slow growth and development of plants and shorten the service life of the plants, and is a limiting factor influencing the production of most grape producing areas in China. In recent years, researchers find that novel phytohormone SL plays an important role in resisting drought stress besides the functions of inhibiting plant branches, regulating root morphology, promoting leaf senescence and the like.
D14 encodes a member of the alpha/beta sheet hydrolase superfamily, participates in SL signal transduction network as a substrate receptor, and plays an important role in the metabolism or signal transduction of plant hormones. It has been reported that D14 plays an important role under stresses such as drought, salinization, and low temperature in apple and Arabidopsis thaliana.
Disclosure of Invention
The VvD14 gene can improve the drought resistance of plants and is used for breeding drought-resistant varieties of grapes, so that the drought resistance of crops is improved, the high and stable yield of the crops is ensured, and the application of the grape strigolactone gene VvD14 in drought resistance is of great significance in improving the drought resistance of the crops.
In order to realize the purpose, the invention provides a grape strigolactone gene VvD14, wherein the grape strigolactone gene VvD14 is a nucleotide sequence shown in SEQ ID NO. 1.
Preferably, the VvD14 protein encoded by the gluconolactone gene VvD14 is an amino acid sequence shown in SEQ ID No. 2.
An application of a strigolactone gene VvD14 of a grape in preparing a medicine for improving the drought resistance of plants.
A recombinant vector with a grape strigolactone gene VvD14 comprises a nucleotide sequence shown in SEQ ID NO. 1.
Preferably, the recombinant vector is a cloning vector or a plant overexpression vector.
Preferably, the recombinant vector is a PRI101-an-VvD14 vector constructed from a PRI101-an vector.
An application of the recombinant vector with the strigolactone gene VvD14 of the grape in preparing the drugs for improving the drought resistance of plants.
A transformant of the recombinant vector with the grape strigolactone gene VvD 14.
Preferably, the transformant is an agrobacterium tumefaciens and/or a plant cell (or organism); the organism is a transgenic drought-resistant plant, and is one of rice, corn, wheat, barley, tobacco, soybean, sorghum, cotton, hemp, peanut, rape, sesame, sugarcane, grape or beet, wherein the preferred is grape.
The application of the transformant in preparing a medicament for improving the drought resistance of a plant is disclosed, wherein the plant is arabidopsis thaliana.
Therefore, the invention adopts the grape strigolactone gene VvD14 and the application thereof in drought resistance, the VvD14 gene is constructed to an expression vector PRI101-an, and the VvD14 gene carried by the PRI101-an is transferred into arabidopsis thaliana by an agrobacterium-mediated transformation method to obtain the transgenic arabidopsis thaliana.
After 21 days of simultaneous drought stress of the transgenic arabidopsis thaliana and the non-transgenic arabidopsis thaliana (wild type), leaves of the wild type arabidopsis thaliana are severely shrunk, withered and yellow, while the VvD14 transgenic arabidopsis thaliana is slightly wilted and part of leaves are yellow, after 3 days of rehydration, the wild type arabidopsis thaliana dies, and the VvD14 transgenic arabidopsis thaliana partially restores the phenotype, which shows that the drought resistance of the arabidopsis thaliana can be improved by over-expressing the VvD 14.
The technical solution of the present invention is further described in detail by the accompanying drawings and embodiments.
Drawings
FIG. 1 is a schematic view of an expression vector PRI101-an-VvD14 gel containing the VvD14 gene;
FIG. 2 is a schematic diagram of an Arabidopsis T3 generation plant successfully transformed with the VvD14 gene;
FIG. 3 is a schematic diagram showing the result of PCR identification of the T3 generation of Arabidopsis thaliana successfully transferred into the VvD14 gene;
FIG. 4 is a diagram showing the result of the verification of quantitative expression of T3 generation plants of Arabidopsis thaliana transformed with VvD14 gene;
FIG. 5 is a schematic diagram of drought resistance of VvD14 transgenic Arabidopsis thaliana T3 plants determined by soil drying method.
Detailed Description
The technical solution of the present invention is further illustrated by the accompanying drawings and examples.
Unless defined otherwise, technical or scientific terms used herein shall have the ordinary meaning as understood by one of ordinary skill in the art to which this invention belongs.
It will be evident to those skilled in the art that the invention is not limited to the details of the foregoing illustrative embodiments, and that the present invention may be embodied in other specific forms without departing from the spirit or essential attributes thereof. The present embodiments are therefore to be considered in all respects as illustrative and not restrictive, the scope of the invention being indicated by the appended claims rather than by the foregoing description, and all changes which come within the meaning and range of equivalency of the claims are therefore intended to be embraced therein, and any reference signs in the claims are not intended to be construed as limiting the claim concerned.
Furthermore, it should be understood that although the present description refers to embodiments, not every embodiment may contain only a single embodiment, and such description is for clarity only, and those skilled in the art should integrate the description, and the embodiments may be combined as appropriate to form other embodiments understood by those skilled in the art. These other embodiments are also covered by the scope of the present invention.
It should be understood that the above-mentioned embodiments are only for explaining the present invention, and the protection scope of the present invention is not limited thereto, and any person skilled in the art should be able to cover the technical scope of the present invention and the equivalent replacement or change of the technical solution and the inventive concept thereof in the technical scope of the present invention.
Example one
Obtaining the strigolactone VvD14 gene of the grape
The leaf RNA extraction was performed according to the procedure of the plant RNA extraction kit of the company Baitach, and the cDNA synthesis was performed according to the procedure of the reverse transcription synthesis kit of the company Takara.
First, primers for amplifying the entire CDS sequence of VvD14 gene were designed using Primer Premier 5, based on the CDS sequence, and the Primer sequences (including modified bases) and names were as follows:
VvD14-F:5'-TTGATACATATGCCCGTCGACATGGGTAACACCCTCTTGGAAGC-3' (shown in SEQ ID NO. 3)
VvD14-R:5'-GTTGATTCAGAATTCGGATCCTCACCGTGAGAGGGCACG-3' (shown in SEQ ID NO. 4);
secondly, taking leaves of the Cabernet Sauvignon, extracting total RNA of the leaves, carrying out reverse transcription to generate cDNA, carrying out RT-PCR amplification on VvD14-F/R by using the reversed cDNA as a template, and sequencing the amplified fragment to obtain a VvD14 gene sequence, wherein the sequence is shown in figure 1.
Example two
Construction and genetic transformation of VvD14 gene overexpression vector
1. Construction of overexpression vectors
The drought-resistant related grape gene VvD14 cloned in the first embodiment is connected with a PRI101-an linearized vector by using a homologous recombination method to construct a plant expression vector, which is named as PRI101-an-VvD14 (figure 1), and the specific operation is as follows:
(1) Firstly, a linearized vector is obtained by a double-enzyme digestion Sal1 and BamH1 (Takara) method, and then the linearized vector is obtained by agarose gel electrophoresis and a gel recovery kit (Tiangen Biochemical technology Co., ltd.). After the concentration and purity of the recovered product were checked by a nucleic acid protein analyzer, the next experiment was performed.
(2) Adding the target fragment DNA and the linearized vector into a 1.5mL centrifuge tube according to the molar ratio of 3:1 for recombination reaction, placing the mixture at 37 ℃ for 30min after mixing uniformly, adding 10 mu L of reaction liquid into 50 mu L of DH5 alpha competent cells, mixing the mixture gently by a pipette, incubating the mixture on ice for 30min, performing heat shock in 42 ℃ water bath for 45 seconds, and then quickly placing the mixture on ice for cooling for 2min.
(3) Add 700. Mu.L LB liquid medium and incubate at 37 ℃ for 60min. Centrifuging at 5000rpm for 2min, collecting thallus, discarding part of supernatant, re-suspending thallus with the rest culture medium, lightly spreading on LB solid culture medium containing Kan resistance with sterile spreading rod, and culturing at 37 deg.C for 16-24 hr by inversion in incubator.
(4) Selecting a plurality of clones on the recombinant reaction conversion plate to carry out colony PCR identification, identifying as positive colonies, selecting corresponding single colonies to be cultured in a liquid LB culture medium containing Kan antibiotics at 37 ℃ and 200rpm for overnight, extracting plasmids or directly sequencing bacterial liquid, and identifying the carrier accuracy by enzyme digestion electrophoresis.
2. Genetic transformation
The prepared PRI101-an-VvD14 vector is transferred into Agrobacterium tumefaciens GV3101 (Shanghai Biotechnology, inc.), and then introduced into an Arabidopsis plant, and the specific operations are as follows:
(1) Recombinant vector transferred into agrobacterium GV3101
a. Add 1. Mu.g plasmid DNA to each 50. Mu.L GV3101 Agrobacterium tumefaciens competent cell, mix by gently tapping the tube bottom with a hand, stand on ice for 5min, liquid nitrogen for 5min, water bath at 37 ℃ for 5min, ice bath for 5min.
b. 700. Mu.L of YEB broth without antibiotics was added and shake-cultured at 28 ℃ for 5h. Centrifuging at 6000rpm for 1min, collecting thallus, collecting supernatant of about 100 μ L, gently blowing and beating resuspended thallus, uniformly coating on YEB solid culture medium containing Kan, rif and Gent, inverting, culturing in 28 deg.C incubator for 2d, picking several positive clones, and simply verifying the result by colony PCR.
(2) Plating of Arabidopsis thaliana
a. A number of Arabidopsis seeds were placed in sterile 1.5mL centrifuge tubes. Adding 1mL of 75% ethanol, sterilizing for 5min, removing supernatant,ddH 2 and washing once.
b. Discarding 75% ethanol, adding 1mL of 1% NaClO, sterilizing for 10min, mixing by turning upside down, and discarding the supernatant. ddH 2 And washing twice with water. Adding 1mL of NaCl 1% to sterilize for 1min 2 And washing for 4-5 times with O.
c. Arabidopsis seeds were spotted on prepared plates using a tip. Sealing the plate, vernalizing at 4 ℃ for 48h under the dark condition, culturing the plate in a light incubator after vernalization is finished, and transplanting after one week.
d. Transferring the transgenic plant to a substrate by using tweezers for growing, preserving moisture for 24 hours by using a preservative film, and placing the plant in a plant growing room for culturing until the arabidopsis grows and shoots (about one month) for a transformation experiment.
(3) Genetic transformation:
a. activating agrobacterium: agrobacterium was inoculated in 10mL YEB liquid medium containing Rif, gent and Kan antibiotics, at 28 ℃ and shaking at 180rpm for 2d.
b. And (3) agrobacterium tumefaciens enlarged culture: and sucking a proper amount of the bacterial liquid, inoculating the bacterial liquid into a new 50mL liquid culture medium containing antibiotics, and shaking the bacteria at the speed of 180rpm for 16-24h at the temperature of 28 ℃ until the OD value is between 1.5 and 2.0. Centrifuge at 5000rpm for 10min at room temperature, and discard the supernatant.
c. Preparing a resuspension solution by using a 5% sucrose solution and 0.05% Silwet L-77, and uniformly mixing after resuspending the bacteria solution.
d. Soaking the arabidopsis inflorescence in the infection liquid, slightly rotating for 15s, fully infecting, wrapping the arabidopsis inflorescence with a preservative film, and culturing for 24h under the dark condition. The transformation was repeated once more after one week.
3. Screening and identification of over-expressed plants
(1) Screening positive plants of T3 generation
Seeds harvested from T2 generation of Arabidopsis thaliana are planted, the seeds of T2 generation are disinfected, inoculated on MS screening culture medium containing 30mg/L kanamycin, and cultured in an incubator for 7-10 days, wherein green plants growing healthily are transgenic plants, and yellow plants dying gradually are non-transgenic plants (figure 2).
Among the 12 obtained positive strains, the positive seedlings are transplanted into soil, covered with preservative film for 2-3 days, then the film is uncovered, and then the seedlings grow normally. After the DNA of the leaves of the screened positive plants is extracted, the VvD14 gene is identified by a PCR method, the molecular verification of the target gene of the transgenic plants is carried out (figure 3), and finally the gene is confirmed to be transferred into T3 generation positive plants. In FIG. 3, M is Marker and 1-12 are transgenic T3 plants.
(2) Quantitative expression verification of transgenic T3 generation positive plants
A positive plant of a transgenic plant T3 generation and young leaves of a wild type Arabidopsis plant in the growth period are taken, total RNA of the leaves is extracted by an RNA extraction kit (Beijing Baitak biotechnology Co., ltd.), then cDNA is obtained by a reverse transcription kit (Nanjing NuoZan biotechnology Co., ltd.), and qRT-PCR expression verification (qRT-PCR Mix: nanjing NuoZan biotechnology Co., ltd.; instrument: roche 480) is carried out by taking respective cDNA as templates and Arabidopsis actin2 as an internal reference.
The qRT-PCR primer pair of the arabidopsis actin2 internal reference comprises the following components:
aF:5'-TATGAATTACCCGATGGGCAAG-3' (shown in SEQ ID NO. 5);
aR:5'-TGGAACAAGACTTCTGGGCAT-3' (shown in SEQ ID NO. 6);
the target gene quantitative qRT-PCR primer pair is as follows:
VvD14-F:5'-CTCGGCGTTGATCGTTG-3' (shown in SEQ ID NO. 7);
VvD14-R:5'-TTAGGAATCTGGGAGAAGCA-3' (shown in SEQ ID NO. 8).
The results show that the expression level of VvD14 gene in the leaves of 8 Arabidopsis transgenic lines tested is significantly improved by using non-transgenic wild type Arabidopsis as a control, and the expression of VvD14 gene is not tested in the leaves of non-transgenic control Arabidopsis (FIG. 4). The VvD14 genes were shown to be transformed and inserted into and expressed in the corresponding Arabidopsis genome. Three strains with the highest VvD14 gene expression level are selected, namely L1, L2 and L6.
EXAMPLE III
Determination of drought resistance of VvD14 overexpression transgenic T3 generation positive plants
And selecting positive plants of VvD14 gene overexpression transgenic T3 generation in 3 plants of example II to perform drought stress experiments. The method comprises the following specific steps: 4 transgenic arabidopsis thaliana plants or wild arabidopsis thaliana plants are planted in each pot of the nutrition pot, drought stress is carried out for 21 days after the plants normally grow for 3 weeks, and then the growth condition of the transgenic plants is observed.
The results show that the growth of 3 transgenic plants over-expressed by VvD14 gene cloned by the invention is not very different from the growth conditions of the control plants under normal conditions, but after 21 days of drought stress, the leaves of wild arabidopsis thaliana are yellowed and withered, and the leaves of transgenic arabidopsis thaliana are slightly shrunken. That is, the drought resistance of VvD14 transgenic Arabidopsis line is obviously higher than that of wild Arabidopsis control (figure 5), and the over-expression of VvD14 gene can improve the drought resistance of plants.
Therefore, the invention adopts the grape strigolactone gene VvD14 and the application thereof in drought resistance, the VvD14 gene has the function of improving the drought resistance of plants, is used for breeding grape drought-resistant varieties, and has important significance in improving the drought resistance of crops.
Finally, it should be noted that: the above embodiments are only for illustrating the technical solutions of the present invention and not for limiting the same, and although the present invention is described in detail with reference to the preferred embodiments, those of ordinary skill in the art should understand that: modifications and equivalents may be made to the disclosed embodiments without departing from the spirit and scope of the present invention.
Claims (9)
1. A grape strigolactone gene VvD14, which is characterized in that: the strigolactone gene VvD14 of the grape is a nucleotide sequence shown as SEQ ID NO. 1.
2. The Vitacobalide gene VvD14 of claim 1, wherein: the VvD14 protein coded by the grape strigolactone gene VvD14 is an amino acid sequence shown in SEQ ID NO. 2.
3. An application of a strigolactone gene VvD14 of a grape in preparing a medicine for improving the drought resistance of plants.
4. A recombinant vector having a strigolactone gene VvD14 of a grape, characterized in that: the recombinant vector contains a nucleotide sequence shown as SEQ ID NO. 1.
5. The recombinant vector having the strigolactone gene VvD14 of claim 4, wherein: the recombinant vector is a cloning vector or a plant overexpression vector.
6. The recombinant vector having the strigolactone gene VvD14 of claim 5, wherein: the recombinant vector is a PRI101-an-VvD14 vector constructed from a PRI101-an vector.
7. Use of a recombinant vector having the strigolactone gene VvD14 of any one of claims 4 to 6 in the preparation of a medicament for increasing drought resistance in a plant.
8. A transformant comprising the recombinant vector having the gluconolactone gene VvD14 according to any one of claims 4 to 6.
9. The use of the transformant of claim 8 in the preparation of a medicament for increasing drought resistance in a plant, the plant being Arabidopsis thaliana.
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Non-Patent Citations (3)
| Title |
|---|
| WAN-NI WANG ET AL.: "Physiological and transcriptomic analysis of Cabernet Sauvginon (Vitis vinifera L.) reveals the alleviating effect of exogenous strigolactones on the response of grapevine to drought stress", PLANT PHYSIOLOGY AND BIOCHEMISTRY, pages 400 - 409 * |
| XM_010666213.2: "PREDICTED: Vitis vinifera probable strigolactone esterase DAD2 (LOC100257558), mRNA", GENBANK * |
| 宋今丹 主编: "医学细胞生物学", 人民卫生出版社, pages: 53 * |
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