CN116836992A

CN116836992A - Application of drought-resistant nanlin895 poplar PdMYB2R089 gene

Info

Publication number: CN116836992A
Application number: CN202310677976.1A
Authority: CN
Inventors: 陈英; 张雪梨; 朱嵊; 王浩然; 黄敏仁
Original assignee: Nanjing Forestry University
Current assignee: Nanjing Forestry University
Priority date: 2023-06-08
Filing date: 2023-06-08
Publication date: 2023-10-03

Abstract

The invention discloses application of drought-resistant nanlin895 poplar PdMYB2R089 genes, and belongs to the field of plant molecular biology. The nucleotide sequence of the drought-resistant nanlin895 poplar PdMYB2R089 gene is shown as SEQ ID NO. 1; according to the invention, the tissue culture seedlings of the populus euphratica 895 are taken as materials, the gene PdMYB2R089 of the populus euphratica 895 is obtained through cloning, an overexpression vector pBI121-3HA-PdMYB2R089 is constructed on the basis, and the transgenic plants are obtained after being transferred into arabidopsis, and the gene function identification result shows that under three drought treatment gradients, the transgenic plants have higher total fresh weight and root fresh weight compared with wild type plants; leaf stomata close rapidly under drought stress; and flowering and fruiting earlier; the content of harmful substance MDA in the body under drought stress is obviously lower than that of wild type Arabidopsis thaliana, and the POD content is obviously higher than that of the wild type Arabidopsis thaliana.

Description

Application of drought-resistant nanlin895 poplar PdMYB2R089 gene

Technical Field

The invention belongs to the field of plant molecular biology, and particularly relates to application of drought-resistant nanlin895 poplar PdMYB2R089 genes.

Background

The populus 895 poplar (Puplus. Deltaides. P. Euramericana cv. 'Nanlin 895') is a cultivated variety of poplar veneer and pulp lumber afforestation selected from the filial generation group of populus americana and populus europaea. The method has the advantages of light preference, water preference and fertilizer preference, and very rapid growth under the condition of sufficient water and fertilizer illumination, and is suitable for popularization and forestation in the south of yellow river. Drought and water shortage are serious problems facing global planting industry production and are also important factors for restricting the economic development of agriculture and forestry. The arid and semiarid area of China accounts for 75% of the cultivated land area of the whole country, and the arid seriously affects the development of agriculture and forestry.

MYB (v-MYB avian myeloblastosis viral oncogene homolog) is one of the largest families of transcription factors in plants, involved in cell differentiation, cell cycle regulation, hormonal and stress responses, plant development and metabolic processes. The MYB family can be divided into four subfamilies of 1R-, R2R3-, 3R-, and 4R-MYB, depending on the number of repeats of the N-terminal MYB domain. Wherein R2R3-MYB is not only a subfamily with the most members of the plant MYB family, but also comprises most of the drought stress response related genes. The R2R3-MYB subfamily transcription factors involved in drought stress response regulate drought responses through regulation of downstream genes in a variety of biological pathways, such as ABA-mediated stomatal closure, growth and development of lateral roots, formation of stratum corneum and wood plugs, and phenylpropane metabolic pathways. Most of the functional studies of the R2R3-MYB gene (about 70%) have focused on plant-specific metabolites, however, less research has been conducted on the involvement of the R2R3-MYB gene in abiotic stresses such as drought. At present, an Arabidopsis R2R3-MYB gene family has been identified, and a large number of gene members involved in drought stress and their regulatory mechanisms have been studied and characterized. While poplar is a woody model species, only a few drought-related functions of the R2R3-MYB gene have been reported.

Disclosure of Invention

Aiming at the problems in the prior art, the invention aims to provide an application of drought-resistant nanlin895 poplar PdMYB2R089 gene for plant molecular improvement breeding.

In order to solve the technical problems, the technical scheme adopted by the invention is as follows:

the application of drought-resistant nanlin895 poplar PdMYB2R089 gene, wherein the nucleotide sequence of the PdMYB2R089 gene is shown as SEQ ID NO. 1.

The application of the drought-resistant nanlin895 poplar PdMYB2R089 gene comprises the following steps:

(1) Constructing a vector of a PdMYB2R089 gene;

(2) Transforming the constructed vector of the PdMYB2R089 gene into a plant;

(3) And culturing and screening to obtain transgenic plants with enhanced drought resistance.

The plant is Arabidopsis thaliana.

The vector is a plant expression vector.

The plant expression vector is pBI121-3HA-PdMYB2R089.

The drought resistance southern forest 895 poplar PdMYB2R089 gene is applied to regulating the pore opening of Arabidopsis leaves.

The drought-resistant southern forest 895 poplar PdMYB2R089 gene is applied to improving the POD enzyme activity in Arabidopsis.

The drought-resistant southern forest 895 poplar PdMYB2R089 gene is applied to reducing the MDA content in Arabidopsis thaliana.

The drought-resistant southern forest 895 poplar PdMYB2R089 gene is applied to promoting the early flowering of arabidopsis.

The drought-resistant southern forest 895 poplar PdMYB2R089 gene is applied to shortening of the growth cycle of arabidopsis.

Compared with the prior art, the invention has the beneficial effects that:

according to the invention, the tissue culture seedlings of the populus euphratica 895 are taken as materials, the gene PdMYB2R089 of the populus euphratica 895 is obtained through cloning, an overexpression vector pBI121-3HA-PdMYB2R089 is constructed on the basis, the transgenic plants are transferred into arabidopsis thaliana, homozygous plant lines are obtained through iterative screening, phenotypic observation is carried out, and the gene function identification result shows that under three drought treatment gradients, compared with a wild type, the transgenic plants have higher total fresh weight and root fresh weight; leaf stomata of the transgenic plant are closed under drought stress; the transgenic plant can bloom and fruit earlier, and the growth period of the arabidopsis is shortened; the content of harmful substance MDA in the transgenic plant under drought stress is obviously lower than that of wild type Arabidopsis thaliana, and the POD content is obviously higher than that of the wild type Arabidopsis thaliana.

Drawings

FIG. 1 is a graph of the detection results of PdMYB2R089 transgenic plants;

FIG. 2 is a graph of iterative resistance screening results of progeny of the PdMYB2R089 Arabidopsis transgenic homozygote;

FIG. 3 is a statistical graph of various indexes of wild type plants and transgenic plants under drought stress (a is a statistical graph of the total Fresh Weight (FW) of wild type plants and PdMYB2R089-OE transgenic plants under different drought concentrations; b is a statistical graph of the fresh weight (root-FW) of root systems of plants; c is a statistical graph of pore diameter measurement of WT and PdMYB2R089-OE transgenic arabidopsis thaliana after drought and rehydration treatment; d is a statistical graph of MDA content of plant leaves after 7 days of water deficiency; e is a graph of soluble protein content of plant leaves after 7 days of water deficiency; f is a graph of POD enzyme activity in plant leaves after 7 days of water deficiency, P is less than 0.05; P is less than 0.01; P is less than 0.001.);

FIG. 4 is a diagram of stomatal movement after drought and rehydration of wild type plants and PdMYB2R032-OE transgenic plants;

FIG. 5 phenotype diagrams of flowering and fruiting cycles of wild-type plants and transgenic plants.

Detailed Description

The invention is further described below in connection with specific embodiments. In the following examples, the procedures not described in detail are all routine biological experimental procedures, and can be performed with reference to molecular biology laboratory manuals, journal literature published in the prior art, and the like, or according to the kit and product instructions. Materials, reagents and the like used in the examples described below are commercially available unless otherwise specified.

The plant materials adopted by the invention are Arabidopsis thaliana (Arabidopsis. Thaliana) and Nanlin895 poplar tissue culture seedlings, the culture condition is 25 ℃, and the illumination is 16 hours.

The vector adopted by the invention is pTOPO001 Blunt Simple Cloning Kit purchased from Beijing gold sand (Genesand) biotechnology Co., ltd;

the expression vector used in the invention is pBI121-3HA-des which is stored by the university of Nanjing forestry, forest college;

the invention adopts the escherichia coli strain Trelief ^TM 5α Chemically Competent Cell and agrobacterium GV3101 were purchased from the department of biotechnology limited.

Example 1: nanlin895 poplar PdMYB2R089 gene clone

1. Primer design

The PtrMYB2R089 (Potri.0070134500) gene sequence screened by the populus tomentosa genome database is used as a reference, and an online sequence Primer3 is used for Primer design, wherein the Primer sequence is as follows:

PdMYB2R089-F：5’-AGATTTTCACCTCATCGCCAT-3’；

PdMYB2R089-R：5’-CTAGCGAAACTGAAGGTTGCAT-3’。

2. extraction of Nanlin895 poplar RNA and cDNA synthesis

The total RNA of the 'nan lin895' poplar is extracted by using an RNAprep Pure polysaccharide polyphenol plant total RNA extraction kit (Tiangen), and the specific method is referred to in the specification.

Reverse transcription into eDNA: preparing a reverse transcription reaction solution 1: total RNA 1. Mu.g (x. Mu.L), random 6mer 1.5. Mu. L, oligo (dT) 1.5. Mu. L, RNase-Free dH ₂ O2-xμL, total volume 5 μL; the reverse transcription reaction solution 1 was subjected to a medium temperature bath at 70℃for 5min, followed by a rapid ice bath for 1min. Preparing a reverse transcription reaction solution 2:10 xRT buffer 4. Mu.L, dNTP mix (10 mM) 1. Mu. L, mgCl ₂ 3. Mu.L, imProm II RT (200U/. Mu.L) 4. Mu.L, RNase Inhibitor (400U/. Mu.L) 1. Mu.L, and a total volume of 10. Mu.L. The reverse transcription reaction solution 1 and the reverse transcription reaction solution 2 were mixed and subjected to reverse transcription reaction using a PCR instrument. The reaction procedure is: 25 ℃ for 5min; 60min at 42 ℃; 15min at 70 ℃; preserving at 4 ℃.

Cloning of the gene was performed using KOD high-fidelity enzyme using cDNA synthesized by reverse transcription as a template.

The PCR reaction system is as follows: cDNA 2 mu L, pdMYB2R089-F Primer (10. Mu.M) 1.5 mu L, pdMYB R089-R Primer (10. Mu.M) 1.5. Mu.L, 10 XKOD Buffer 5. Mu. L, mgSO ₄ 3μL、dNTPMixture(10mM each)5μL、KOD-Plus 1μL、ddH ₂ O31. Mu.L, total volume 50. Mu.L.

The PCR reaction procedure was: 94 ℃ for 3min;94 ℃ for 30s, 58-62 ℃ for 30s and 68 ℃ for 2min, and 38 cycles are carried out; 20min at 68 ℃; preserving at 4 ℃.

After the reaction is completed, the amplified products are separated by electrophoresis with 1% agarose gel; the correct size band was excised and recovered for purification.

3. Construction of cloning vector and transformation of E.coli

The reaction system was prepared at room temperature (20-30 ℃ C.) and the details are shown in pTOPO001 Blunt Simple Cloning Kit. The connection system is placed in a PCR instrument or a metal bath (20-30 ℃) to react for 5min, and the escherichia coli is immediately transformed after the reaction is completed. Adding 10 μl of the ligation product into 100 μl of Escherichia coli, flicking the tube wall to mix, and standing on ice for 30min; placing in water bath at 42deg.C for 1min, rapidly placing on ice, and standing for 2-3min; adding 800 mu L of liquid LB culture medium, mixing uniformly, shaking at 37 ℃ with 150rpm for 1h; centrifuging at 3000rpm for 4min, sucking most of the supernatant, keeping about 100 μl, blowing to obtain bacterial precipitate, and mixing the bacterial liquid; the bacterial liquid is gently smeared on LB solid medium containing Amp, and is cultured in an inversion way in a 37 ℃ incubator for overnight; the next day, the bacteria are picked up into LB liquid medium containing Amp, shaking table 37 ℃,250rpm, and shaking for 4-8 hours. The detection of bacterial liquid PCR is carried out, and the reaction system and the reaction procedure are shown in the specification of 2X Rapid Taq Master Mix (vazyme). The reaction product is detected by 1% agarose gel electrophoresis, and the bacterial solution of positive clone with correct band is sent to the manufacturer for sequencing, and the nucleotide sequence of PdMYB2R089 gene is shown as SEQ ID NO. 1.

Example 2: construction and function verification of Nanlin895 poplar PdMYB2R089 gene expression vector

1. Construction of a plant expression vector:

according to the specification, restriction enzymes KpnI and SacI are used for carrying out double enzyme digestion treatment on a PBI121-3HA-des vector, a section of 18bp sequence homologous to the vector is added at two ends of a CDS sequence of a target gene, and the target gene is constructed into the vector pBI121-3HA-des by utilizing a homologous recombination method to obtain a plant expression vector pBI121-3HA-PdMYB2R089.

pBI121-3HA-PdMYB2R089-F：

5’-CCAGATTATGCTAGTCTTATGTCTAGCAGAGGCCATTGG-3’，

pBI121-3HA-PdMYB2R089-R：

5’-GAACGATCGGGGAAATTCCTATAATCCACAAACTTGGTT-3’。

The recombination reaction system is as follows: linearization vector Xμl, target fragment Y μl, 5 XCEII buffer4 μ L, exnase μl, supplemented with ddH ₂ O to a total volume of 20. Mu.L, wherein the linearized support and the fragment of interest volume are dependent on their concentration, but the molar ratio of the two is 1:2. the reaction procedure is: and 0.5h at 37 ℃. And (3) converting the reaction product into an escherichia coli strain, picking and propagating a monoclonal on the next day, and detecting bacterial liquid.

2. Agrobacterium-mediated inflorescence infection method for transforming Arabidopsis thaliana

150-200ng of plasmid to be transformed is taken, 100 mu L of Agrobacterium GV3101 competent cells are added, and the mixture is placed on ice for 20-30min after uniform mixing. Then quick-freezing for 60s by liquid nitrogen, heat-shock for 4min at 37 ℃, and rapidly ice-bathing for 2min. Adding 800 μLLB liquid culture medium, shaking at 28deg.C for 3 hr at 100rpm, centrifuging at 4000rpm for 3min, collecting a small amount of supernatant, blowing, mixing, spreading on a plate, and culturing at 28deg.C for 48 hr. And selecting a monoclonal on the third day, performing bacterial liquid PCR detection, and placing the correct bacteria in a refrigerator at 4 ℃ for standby.

The positive agrobacterium that was correctly detected was activated, after which the single clone was picked up in 5mLLB liquid medium (containing Km and Cef) and incubated for 16-24h at 28 ℃. 1:100, culturing at 28deg.C for 16-24 hr, and standing for OD ₆₀₀ 1.6-2.0. The cells were collected at the bottom of the tube by centrifugation at 4000rpm for 10min at room temperature, resuspended in an equal amount of 5% (w/v) sucrose solution, added with Silwet L-77 at a ratio of 20. Mu.L/100 mL, and immediately mixed. Soaking all inflorescences in the bacterial liquid for 1min, wrapping the inflorescences with a preservative film, placing the inflorescences in a tray in a side-by-side manner, and placing the inflorescences in darkness at room temperatureOne day. Removing the preservative film, putting back into the incubator for growth, collecting seeds after the silique is ripe, and storing at 4 ℃ after airing.

3. Transgenic plant detection and homozygote acquisition

PCR was performed using the 35S promoter using an upstream primer (35S-universal-F: 5'-AGGAAGGTGGCTCCTACAAATGCCATC-3') and a downstream specific primer (pBI 121-3HA-PdMYB2R089-R: 5'-GAACGATCGGGGAAATTCCTATAATCCACAAACTTGGTT-3') for the PdMYB2R089 gene. I.e., using the plant directpcrkit kit from nupran biotechnology, inc, the method is as follows:

sample treatment: taking a small tender leaf (about 1-3mm in diameter), placing in 20mu LPlant Direct Lysis Buffer A, and grinding as much as possible; heating at 95 ℃ for 5-10min, centrifuging briefly after heating, and taking 1 mu L of supernatant as a template of a PCR reaction system.

PCR reaction system: plant leaf/crude extract 1. Mu.L, 2X Plant Direct Master Mix. Mu.L, 35S-unitary-F (10. Mu.M) 1. Mu. L, pBI121-3HA-PdMYB2R089-R (10. Mu.M) 1. Mu. L, ddH ₂ O7. Mu.L, the total system was 20. Mu.L.

The PCR reaction procedure was: 98 ℃ for 5min;95 ℃ for 10s, 58-72 ℃ for 15s, 72 ℃ for 1min/kb, and 35 cycles are total; 72 ℃ for 5min; preserving at 4 ℃.

Gel electrophoresis detection: and adding bromophenol blue into the PCR product, mixing uniformly, spotting on 1% agarose gel, and detecting by electrophoresis. The detection result of 1% gel electrophoresis is shown in figure 1, the length of the band is consistent with the length of the target fragment, and the PdMYB2R089 gene is preliminarily proved to be transferred into arabidopsis.

Screening out Arabidopsis positive seedlings by using the screening pressure of antibiotics Km, transplanting, and then carrying out iterative screening to finally obtain a homozygous transgenic homozygous strain (shown in figure 2) of which the offspring does not have character separation, namely PdMYB2R089-OE, and carrying out the next phenotypic observation.

4. Screening of homozygotes

To obtain transgenic offspring that can be stably inherited, positive homozygous plants are screened by antibiotic Km screening pressure, as follows:

preparing an Arabidopsis thaliana 1/2MS culture medium, placing the Arabidopsis thaliana after sterilization, adding Km (50 mg/L) and Cef (400 mg/L) at about 50 ℃, uniformly mixing, and pouring into a flat plate; putting seeds into 1/4-1/6 of the joint part of the bottom of the tube, cleaning with 1mL of sterile water, and removing floating seeds; adding 1mL of 75% alcohol, mixing for 30s, standing for 15s, and sucking the ethanol by a gun; adding 1mL of 10%84 vortex for 3min, standing for 15s, sucking the supernatant with a gun, washing with sterilized water for 4 times, standing each time, and sucking the supernatant; 1mL of 0.05% agaros (agarose) solution suspending the seeds; the seeds are beaten on a culture medium flat plate (500 mu L of each plate), and beaten by a shearing gun head to disperse the seeds as much as possible; standing for 40 minutes, and waiting for water evaporation; sealing the culture dish, wrapping with newspaper, and culturing in dark at 4deg.C for 3 days (inducing seed germination); culturing in a light incubator for 16h with attention to pollution.

After about 10 days, positive seeds should be significantly larger than non-transformed plants and green (non-transgenic plants are smaller and leaves are relatively whitish in color); after the culture medium plate is germinated and grows to 4 leaves (one week to two weeks), large green plants are selected and transplanted to a small flowerpot; when more leaves are available, taking the leaves for transgene detection; collecting seeds, and repeating until the culture medium is large green plantlet, i.e. the screened seeds are homozygote without character separation.

5. Transgenic arabidopsis drought stress treatment

The transgenic arabidopsis drought treatment is mainly carried out on a solid 1/2MS medium added with PEG6000, and a van der Weele et al treatment mode is adopted, and a liquid medium (containing MES buffer) containing PEG6000 with different concentrations is covered on the solid 1/2MS medium without PEG, so that a solid medium containing 5% and 10% PEG6000 required by the experiment is prepared.

1) To observe the phenotype difference of the wild type and the transgenic arabidopsis, the wild type and the transgenic arabidopsis seedlings sown on 1/2MS for about 10 days are transferred to three drought concentration (0%, 5% and 10%) culture media for 6 days, and statistical analysis is carried out on the whole fresh weight and root fresh weight of the PdMYB2R089-OE plants.

The results are shown in FIG. 3a, and the fresh weight of the transgenic Arabidopsis with the overexpression of PdMYB2R089 is obviously higher than that of the wild type Arabidopsis under normal conditions or drought stress. With increasing concentration of PEG6000, the fresh weight of wild type and transgenic Arabidopsis thaliana showed a decreasing trend, but the decreasing amplitude of transgenic Arabidopsis thaliana was significantly smaller than that of wild type Arabidopsis thaliana.

The results are shown in FIG. 3b, in which root fresh weight of wild type Arabidopsis thaliana is significantly reduced under drought stress, especially 10% PEG6000 stress, and root growth is significantly inhibited. The fresh weight of the transgenic arabidopsis is not obviously reduced compared with that of the wild arabidopsis under drought stress.

2) As shown in FIG. 3c, under normal water conditions, the pore opening degrees of the wild type and the transgenic Arabidopsis thaliana are consistent, and the pore diameter of the transgenic Arabidopsis thaliana is obviously reduced after drought stress treatment, and the pore is restored to be before stress after rehydration treatment. As shown in fig. 4, overexpression of PdMYB2R089 can promote plant stomatal movement, close stomata under drought stress, reduce water loss, and thereby enhance plant early tolerance.

3) As shown in the result of FIG. 3d, the MDA content of the harmful substances in the wild type Arabidopsis thaliana is extremely higher than that of the PdMYB2R089-OE transgenic Arabidopsis thaliana (P < 0.001) after one week of water deficiency, and the damage degree of the transgenic plant is obviously lower than that of the wild type Arabidopsis thaliana under drought stress. The soluble protein has no obvious difference (P > 0.05) between the two (FIG. 3 e), and moreover, the POD enzyme activity of the PdMYB2R089-OE transgenic plant is obviously higher than that of the wild-type Arabidopsis thaliana (P < 0.05) (FIG. 3 f). The PdMYB2R089 can effectively remove oxides such as ROS by activating plant antioxidant stress pathways to generate more PODs when facing drought stress, so that MDA is reduced, and the plant is prevented from being damaged by oxidative stress.

4) The study was carried out by transplanting both wild type and transgenic Arabidopsis into soil and placing them in the same culture environment to observe their flowering and fruiting conditions.

The result is shown in fig. 5, the transgenic arabidopsis starts bolting about 15 days after transplanting, starts flowering about 19 days, and has fruiting about 26 days, and the wild arabidopsis just starts bolting at this time, which shows that the overexpression of PdMYB2R089 promotes the arabidopsis to flower and fruit earlier, and shortens the arabidopsis growth cycle.

Claims

1. The application of drought-resistant nanlin895 poplar PdMYB2R089 gene, wherein the nucleotide sequence of the PdMYB2R089 gene is shown as SEQ ID NO. 1.

2. The application of drought-resistant nanlin895 poplar PdMYB2R089 gene according to claim 1, characterized by comprising the following steps:

(1) Constructing a vector of a PdMYB2R089 gene;

(2) Transforming the constructed vector of the PdMYB2R089 gene into a plant;

3. The application of drought-resistant nanlin895 poplar PdMYB2R089 gene according to claim 2, characterized in that the plant is arabidopsis thaliana.

4. The application of drought-resistant nanlin895 poplar PdMYB2R089 gene according to claim 2, characterized in that said vector is a plant expression vector.

5. The application of drought-resistant nanlin895 poplar PdMYB2R089 gene according to claim 4, characterized in that the plant expression vector is pBI121-3HA-PdMYB2R089.

6. The application of drought-resistant arabidopsis 895 poplar PdMYB2R089 gene in regulating the stomata opening of arabidopsis leaves according to claim 1.

7. The application of drought-resistant nanlin895 poplar PdMYB2R089 gene of claim 1 in improving the in vivo POD enzyme activity of arabidopsis.

8. The application of drought-resistant nanlin895 poplar PdMYB2R089 gene of claim 1 in reducing MDA content in arabidopsis thaliana.

9. The application of drought-resistant arabidopsis 895 poplar PdMYB2R089 gene of claim 1 in promoting early flowering of arabidopsis.

10. The application of drought-resistant arabidopsis 895 poplar PdMYB2R089 gene of claim 1 in shortening growth cycle of arabidopsis.