Rose drought-tolerant gene RrMYB2 and application thereof

Abstract

The invention discloses a rose drought-enduring gene RrMYB2 and application thereof. The nucleotide sequence of the RrMYB2 gene is shown as SEQ ID NO.1, and the protein amino acid sequence of the RrMYB2 gene is shown as SEQ ID NO.2. The invention further discloses a preparation method of the rose drought-enduring gene RrMYB2. According to the application method, the drought resistance of the rosa plants is improved by controlling the overexpression of the gene RrMYB2 in the rosa plants. After the RrMYB2 gene of the purple-branch rose is separated, the RrMYB2 gene is transferred into a model plant arabidopsis thaliana and the purple-branch rose, and the drought resistance of the transgenic arabidopsis thaliana and the purple-branch rose is remarkably enhanced, so that the RrMYB2 gene is a positive regulation factor of the drought resistance and has an important application value in the field of drought-resistant and stress-resistant breeding of rose plants.

Description

Technical Field

[0001] The invention belongs to the technical field of plant gene engineering, and in particular is a rose drought-resistant gene RrMYB2 and an application thereof. Background Art

[0002] Rose (Rosa rugosa Thunb.) is a perennial deciduous shrub of the genus Rosa in the family Rosaceae, with rich varieties and pleasant aroma. Rose is rich in flavonoids, volatile oils, phenolic acids, amino acids and other chemical components and nutrients required by the human body. It is widely used in the fields of skin care products, food and medicine. It is an important natural spice plant in the world and a traditional Chinese medicine and food flower with high economic and health value. At present, most roses are planted in the open air at home and abroad, and the management is relatively extensive. In recent years, the growth and even death of rose plants caused by soil drought have seriously affected the yield and quality of fresh roses, which has become a bottleneck problem in the development of modern agricultural industries including rose planting.

[0003] Transcription factors can regulate plant stress resistance, including resistance to low temperature, drought, salinity, etc., by regulating the expression of key genes in different signaling pathways. MYB transcription factors are one of the largest transcription factor families in plants. Multiple drought-resistant MYB genes have been identified in model plants (Arabidopsis, rice, wheat, etc.). These MYB genes regulate or reduce the damage of stress to plants at multiple levels, and play a vital role in the growth and development of plants under drought conditions. Studies have shown that under adverse stress, MYB transcription factors bind to the core sequence TAACTG in the promoter region of many functional genes, thereby activating the expression of stress-responsive genes. SiMYB56 is involved in regulating the tolerance of rice to drought stress during the vegetative and reproductive stages. It mainly increases the plant's resistance to drought by activating the expression of lignin synthesis genes 4CL5H and F5H1 and ABA synthesis genes P5CS1 and LEA7. Another MYB transcription factor in rice, OsMYB60, can directly bind to the promoter of the wax biosynthesis gene OsCER1, activate its expression, and then promote the synthesis of cuticle wax on the leaf surface to enhance the rice's resistance to drought stress.

[0004] It can be seen that MYB transcription factors play a key role in regulating plant drought tolerance, but there is no specific report on MYB proteins regulating drought resistance in roses. Therefore, exploring the MYB members that regulate drought tolerance in roses will help understand the regulatory mechanism of drought tolerance in roses, which has guiding significance for the resistance breeding of rose plants, provides a basis for breeding new varieties of excellent roses with high drought tolerance and economic value, and promotes the development of the rose industry. Summary of the invention

[0005] In view of the problem that rose plants grow weak or even die due to soil drought, screening and regulating rose drought genes and applying them to drought-resistant variety breeding is an important way to obtain drought-resistant roses. In this regard, the present invention provides a rose drought-resistant gene RrMYB2 and its application.

[0006] The purpose of the present invention is to provide a rose drought-tolerant gene RrMYB2, whose nucleotide sequence is shown in SEQ ID NO. 1 and whose amino acid sequence is shown in SEQ ID NO. 2. RrMYB2 is a MYB transcription factor that responds to drought stress in purple-branched roses and is a transcription factor of R2R3-MYB.

[0007] Another object of the present invention is to provide an application of the rose drought-tolerant gene RrMYB2 in regulating the drought resistance of rose plants. By controlling the overexpression of the gene RrMYB2 in rose plants, the drought resistance of rose plants can be improved. The specific application method is:

[0008] Step 1), constructing an overexpression vector of gene RrMYB2;

[0009] Step 2), introducing the overexpression vector of the gene RrMYB2 into Agrobacterium to obtain the overexpression strain of the gene RrMYB2;

[0010] Step 3), using the strain overexpressing the gene RrMYB2, obtain the rose plant overexpressing the gene RrMYB2.

[0011] Furthermore, in step 1), the plant overexpression vector pFAST-R05 is used to construct an overexpression vector of the gene RrMYB2 to obtain the overexpression vector 35S:RrMYB2.

[0012] Furthermore, in step 2), the Agrobacterium is GV3101 Agrobacterium or K599 Agrobacterium rhizogenes.

[0013] K599 rhizogenes Agrobacterium introduced with the overexpression vector of the gene RrMYB2 can induce rose plants to produce transgenic hairy roots, thereby improving the drought resistance of rose plants.

[0014] Further, the genus includes roses and Chinese roses.

[0015] Technical solution: The present invention

[0016] Beneficial effects: The present invention uses purple-branched rose as material, clones the RrMYB2 gene, detects the expression pattern of the RrMYB2 gene under drought treatment, and improves the drought resistance of the overexpression strain by genetically transforming Arabidopsis and purple-branched rose, proving that it is a positive regulatory factor for drought resistance, and has important application value for cultivating new drought-resistant rose germplasm. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 is the expression level of RrMYB2 in leaves of drought-treated plants within 24 h;

[0018] Figure 2 This is the plasmid map of the plant overexpression vector pFAST-R05;

[0019] Figure 3 This is a diagram showing the drought tolerance phenotype and physiological index detection of Arabidopsis thaliana overexpressing RrMYB2;

[0020] Figure 4 This is a graph showing the drought resistance phenotype and physiological index detection of purple rose with RrMYB2 overexpression. DETAILED DESCRIPTION

[0021] The technical solution of the present invention is described in detail below through embodiments, but the protection scope of the present invention is not limited to the embodiments.

[0022] Example 1: RrMYB2 gene cloning

[0023] In this example, the cDNA of Rosea purpurogena was used as a template to amplify the gene coding region and obtain the nucleotide sequence of the RrMYB2 gene.

[0024] 1. Primer design

[0025] Design upstream and downstream primers containing the complete open reading frame of the RrMYB2 gene:

[0026] SEQ ID NO.3, Primer F: 5'-ATGGATGTTGATCAGTACTTA-3'

[0027] SEQ ID NO.4, Primer R: 5'-GTCGCTATTAAGCTGTTGTTG-3'

[0028] 2. Obtaining the RrMYB2 gene

[0029] 2.1. Total RNA from leaves and roots of wild rose was extracted using FastPure Plant Total RNA Isolation Kit (Vazyme, Nanjing). The specific steps are as follows:

[0030] 1) Preheat Buffer PRL to 65°C in a water bath and add 5% β-mercaptoethanol.

[0031] 2) Take about 0.3 g of rose root in a mortar, grind it with liquid nitrogen, add 500 μl Buffer PRL, and vortex vigorously for 60 seconds.

[0032] 3) Place the lysis mixture in a 65°C water bath for 5 min, centrifuge at 12,000 rpm for 10 min, pipette the supernatant into a new 1.5 ml centrifuge tube, add an equal volume of Ethanol absolute and mix by pipetting.

[0033] 4) Place the FastPure gDNA-Fiter ColumnⅡ collection column into the collection tube, transfer the above mixed solution into it, centrifuge at 12000 rpm for 2 minutes, and discard the filtrate.

[0034] 5) Place the collection column in a new Collection Tubes 2ml, add 500μl Buffer PRLPlus, centrifuge at 12000rpm for 30sec, add 0.5 times the volume of Ethanol absolute to the filtrate, and mix immediately by pipetting.

[0035] 6) Transfer the mixture to FastPure RNA Column IV, centrifuge at 12000 rpm for 2 min, and discard the filtrate.

[0036] 7) Add 500 μl Buffer PRW2 to FastPure RNA Column IV, centrifuge at 12,000 rpm for 30 seconds, and discard the filtrate.

[0037] 8) Repeat step 7).

[0038] 9) Place the FastPure RNA Column IV back into the collection tube and centrifuge at 12000 rpm for 2 min.

[0039] 10) Transfer FastPure RNA Column IV to a new RNase-free Collection Tubes 1.5ml centrifuge tube and drop 30μl of RNase-free ddH 2 O, place at room temperature for 2 min, and centrifuge at 12000 rpm for 1 min.

[0040] 11) The collected RNA was packaged into 1 μg portions and stored in a -80°C refrigerator.

[0041] 2.2. Reverse transcribe RNA into cDNA using HiScript III 1st Strand cDNA Synthesis Kit (Vazyme). The specific steps are as follows:

[0042] 1) RNA template denaturation: Add RNase-Free ddH to 5 μg Total RNA. 20 to 8 μl, incubate at 65°C for 5 min, and place on ice for 2 min.

[0043] 2) Removal of genomic DNA: Add 2 μl 5×gDNA wiper Mix to the product of the previous step, mix well by pipetting, and react at 42°C for 2 min.

[0044] 3) Obtaining the first-strand cDNA: Add 2 μl 10×RT Mix, 2 μl HiScriptⅢEnzymeMix, and 1 μl Oligo(dT) to the product of the previous step 20 VN and 5 μl RNase-Free ddHO 2 O, pipetting and mixing. The reaction was carried out in three steps at 25°C for 5 min, 37°C for 45 min, and 85°C for 5 sec to obtain cDNA.

[0045] 2.3. Isolation of RrMYB2 gene

[0046] 1) PCR amplification of RrMYB2 gene: Add 25 μl PrimeSTAR Max Premix (2X), 2 μl Primer F and Primer R, 1 μl cDNA and 20 μl RNase-Free ddH in a PCR tube. 2 O, pipetting and mixing. Amplification reaction was performed according to the program of 95°C, 3 min; 95°C, 10 sec, 55°C, 5 sec (35 cycles); 72°C, 5 sec; 72°C, 3 min.

[0047] 2) Use FastPure Gel DNA Extraction Mini Kit to obtain purified PCR products.

[0048] 2.4. Clone the RrMYB2 gene into the vector using the pENTR / D-TOPO kit (invitrogen) as follows:

[0049] 1) Connecting vector: 1 μl 5ng freshly recovered RrMYB2 gene product, 0.5 μl D-TOPO vector, 1 μl Salt solution, 1 μl RNase-Free ddH2O, a total of 3 μl system. Incubate at 25℃ for 5 minutes to obtain the recombinant product.

[0050] 2) E. coli transformation: Thaw the -70℃ frozen E. coli competent cells on ice, add 5μl of the ligation product, mix well, incubate on ice for 30min, heat shock at 42℃ for 90sec, incubate on ice for 3min, add 800μl of LB liquid culture medium, resuscitate at 37℃&100rpm for 1h, remove the liquid by centrifugation, mix the remaining bacterial liquid, spread on a plate (LB screening solid culture medium containing Kan), and culture overnight at 37℃.

[0051] 3) Positive clone screening and sequencing analysis: Single colonies were selected from the screening culture plate and inoculated into LB liquid culture medium. The culture was shaken at 37°C and 250 rpm for 6 h, and the culture solution was tested by PCR. The positive culture solution was selected for sequencing, and the RrMYB2 gene was successfully isolated, and the entry vector D-TOPO:RrMYB2 was obtained.

[0052] The nucleotide sequence of the RrMYB2 gene is shown in SEQ ID NO. 1. The amino acid sequence of the RrMYB2 gene is shown in SEQ ID NO. 2.

[0053] Example 2: Simulating drought treatment of purple rose by PEG powder

[0054] 1. Prepare 6 purple roses of the same growth period and prepare PEG6000 powder into a 20% aqueous solution.

[0055] 2. Different strains of purple rose were watered with 20 ml of 20% PEG6000 solution at 0h, 1h, 3h, 6h, 12h, and 24h respectively, and placed in a long-day incubator.

[0056] 3. After the last plant was treated, leaf samples of six purple roses were taken at the same time for subsequent experiments.

[0057] Example 3: Exploring the spatiotemporal expression pattern of the RrMYB2 gene by fluorescence quantitative PCR technology:

[0058] Design of fluorescent quantitative PCR primers based on the exon region of RrMYB2 gene:

[0059] SEQ ID NO.5, upstream primer: 5'-GCTGCAGACTGAGATGGTTG-3'

[0060] SEQ ID NO. 6, downstream primer: 5'-AAGCTGTTCTTGGAGGGTGA-3'.

[0061] Real-time fluorescence quantitative PCR was performed using SYBR Premix Ex Taq (Takara) and fluorescence quantitative PCR instrument CFX96TM (Bio-RAD). Bio-Rad CFX Manager software was used for data processing. Three technical replicates were performed for each biological replicate, and the average of the three parallel data was taken to reduce errors.

[0062] like Figure 1 As shown, in rose plants, drought treatment was carried out according to the method of Example 2. Within 24 hours of drought treatment, the expression level of RrMYB2 in leaves first increased and then decreased with time, reaching a peak at 6 hours. This shows that in purple-branched rose, RrMYB2 response to drought stress is a long-term process.

[0063] Example 4: Construction of RrMYB2 overexpression vector

[0064] Using the plant overexpression vector pFAST-R05 ( Figure 2 ) construct an RrMYB2 overexpression vector, and the construction method is as follows:

[0065] 1) Obtain the overexpression vector using the LR Clonase Enzyme Mix kit (invitrogen): 1 μl entry vector D-TOPO:RrMYB2 plasmid, 1 μl pFAST-R05 vector plasmid, 1 μl LR Enzyme, 2 μl TE Buffer, 1 μl RNase-Free ddH 2 O, a total of 5 μl system. Incubate at 25°C for 1 hour to obtain the recombinant product. The E. coli transformation and positive clone screening methods are the same as in Example 1. The positive bacterial solution is sent to Sangon Biotech Co., Ltd. (Nanjing) for sequencing to obtain the overexpression vector 35S:RrMYB2.

[0066] 3) Agrobacterium transformation: Take GV3101 Agrobacterium competent cells and Agrobacterium K599 competent cells stored at -80°C and thaw them at room temperature, add 0.01-1 μg of plasmid DNA for every 100 μl competent cells, stir the bottom of the tube by hand to mix, let it stand on ice for 5 minutes, liquid nitrogen for 5 minutes, 37°C water bath for 5 minutes, ice bath for 5 minutes, add 700 μl of YEB liquid culture medium without antibiotics and mix, culture at 28°C with shaking for 2-3 hours, centrifuge at 6000 rpm for one minute to collect the bacteria, spread them on YEB plates containing spectinomycin and rifampicin resistance, and culture them upside down at 28°C incubator for 2-3 days to grow positive colonies.

[0067] Example 5: Genetic transformation of Arabidopsis thaliana and observation of drought-tolerant phenotype of RrMYB2-overexpressing Arabidopsis thaliana plants

[0068] 1. Infect Arabidopsis thaliana by inflorescence dip method, and observe RFP fluorescence of the primary seeds obtained based on the RFP tag on the pFAST-R05 expression vector. That is, seeds with RFP fluorescence observed under a stereo microscope are positive seeds. The main steps and applied reagents are as follows:

[0069] I. Abbreviations of reagents and solutions

[0070] The abbreviations of the plant hormones used in the culture medium in this example are as follows: Spec (Spectinomycin); Arabidopsis transformation auxiliary reagent (Silwet L-77).

[0071] II. Agrobacterium-mediated genetic transformation steps

[0072] (1) Cultivation of Agrobacterium

[0073] The positive colonies obtained in Example 4 were picked and inoculated into liquid LB medium (10 g / L peptone + 5 g / L yeast extract + 10 g / L NaCl + 50 mg / L Spec) and cultured overnight at 28°C and 200 rpm shaking until the bacterial solution concentration OD 600 The value is 1.5-2.0.

[0074] (2) Arabidopsis inflorescence dip dyeing method

[0075] The shaken bacterial suspension was centrifuged at 4500 rpm for 10 min, and 5% sucrose solution (with 20 μl / 100 ml of surfactant Silwet L-77 added) was used. The Arabidopsis inflorescence was immersed in the resuspended solution for about 30 sec, shielded from light and kept moist, and placed horizontally for 12-24 h.

[0076] (3) Screening of positive seedlings

[0077] The infected Arabidopsis thaliana was placed in an incubator for normal culture, and the harvested seeds were screened by observing the RFP fluorescence under a stereo microscope; the screened T1 generation plants were sown and transplanted to a light incubator for growth and waiting for harvest. The operation was repeated until the T3 generation homozygous plants were obtained.

[0078] 2. Sow the seeds of Arabidopsis thaliana overexpressing RrMYB2 and wild-type Arabidopsis thaliana on 1 / 2MS medium, and after growing two true leaves, transfer them to the substrate for culture and grow for two weeks. Then, natural drought treatment was carried out. If no water was applied for about 20 days, drought phenotypes would appear. Figure 3As shown, WT represents the wild-type Arabidopsis plant, and RrMYB2-OE1 and RrMYB2-OE2 represent the numbers of two Arabidopsis plants overexpressing RrMYB2. (a) is the phenotype of the plant after 10 days of drought treatment, and (b) is the identification of whether the RrMYB2 gene is overexpressed in the wild-type plant and the transgenic plant. (c) is the survival rate statistics of the wild-type plants and transgenic plants after 20 days of natural drought treatment. (d) is the water loss rate analysis of the wild-type plants and transgenic plants. Through phenotypic observation, it was found that the overexpression strain grew well, while the wild type showed wilting. By measuring the water loss rate and survival rate, it was found that the overexpression strain lost water more slowly than the wild type, and the survival rate was 100%, while the survival rate of the wild type was 0%. The above evidence shows that overexpression of RrMYB2 promotes drought tolerance of Arabidopsis plants.

[0079] Example 6: Cut-dip-budding (CBD) method to induce hairy roots for homologous transformation of Rose hippocastanum

[0080] The non-lignified side branches of Rose rose were cut into 4-5 cm cuttings, the bottom of which was dipped in the positive K599 Agrobacterium colony obtained in Example 4 and colonized in vermiculite. After culturing for 3 weeks at 25°C under long-day conditions (light:dark=16h:8h), a large number of transgenic hairy roots were obtained, and the overexpression efficiency was verified by fluorescence quantification.

[0081] Example 7: Identification of Drought Tolerance of Rosea japonica Thunb. Overexpressing RrMYB2

[0082] According to the method of Example 6, three RrMYB2 overexpressing plants were obtained, which were numbered 35S:RrMYB2-1, 35S:RrMYB2-2, and 35S:RrMYB2-3. After the pFAST-R05 empty vector was transferred into K599 Agrobacterium, the control plant 35S:Empty was obtained according to Example 6. After 15 days of natural drought treatment, the results were as follows: Figure 4 As shown in the figure, compared with the control, the chlorophyll fluorescence of the RrMYB2 overexpression plant had a higher actual photosynthetic rate value. The root physiological indicators under drought stress were measured and it was found that the content of SOD (superoxide dismutase) and CAT (catalase) in the RrMYB2 overexpression root system was significantly higher than that in the control group, indicating that the overexpression strain had a stronger ability to resist osmotic stress; 2 O 2 and O 2 - The content was lower than that in the control group, indicating that the cell membrane of the overexpression strain was less damaged.

[0083] Figure 4The four rows of plants in (a) are represented from top to bottom; the first row: plants without drought treatment; the second row: plants after 15 days of natural drought treatment; the third row: chlorophyll fluorescence images of plants without drought treatment; the fourth row: chlorophyll fluorescence images of plants after 15 days of natural drought treatment. Figure 4 (b) shows the RrMYB2 overexpression efficiency in three different 35S:RrMYB2 lines compared with the control plants. Figure 4 (c) shows the changes in the chlorophyll fluorescence parameter Y(II), i.e., the actual photosynthetic efficiency content of PSII, in the control plants and three 35S:RrMYB2 plants before and after drought treatment. Figure 4 (d)-(g) show the H content in the roots of each plant after 15 days of natural drought treatment. 2 O 2 , O 2 - , SOD, CAT content.

[0084] As described above, although the present invention has been shown and described with reference to specific preferred embodiments, it should not be construed as limiting the present invention itself. Various changes in form and details may be made without departing from the spirit and scope of the present invention.

Claims

1. A rose drought tolerance gene RrMYB2, characterized in that: The nucleotide sequence of the gene RrMYB2 is shown in SEQ ID NO.1, and the amino acid sequence of the protein expressed by the gene RrMYB2 is shown in SEQ ID NO.

2.

2. Use of the rose drought-resistant gene RrMYB2 according to claim 1 in regulating the drought resistance of rose plants.

3. The use according to claim 2, characterized in that: By controlling the overexpression of the gene RrMYB2 in rose plants, the drought resistance of rose plants can be improved.

4. The use according to claim 2, characterized in that: The specific application method is: Step 1), constructing an overexpression vector of gene RrMYB2; Step 2), introducing the overexpression vector of the gene RrMYB2 into Agrobacterium to obtain the overexpression strain of the gene RrMYB2; Step 3), using the strain overexpressing the gene RrMYB2, obtain the rose plant overexpressing the gene RrMYB2.

5. The use according to claim 4, characterized in that: In step 1), the plant overexpression vector pFAST-R05 is used to construct an overexpression vector of the gene RrMYB2 to obtain the overexpression vector 35S:RrMY B2.

6. The use according to claim 4, characterized in that: In step 2), the Agrobacterium is GV3101 Agrobacterium or K599 Agrobacterium rhizogenes.

7. The use according to claim 2, characterized in that: The genus Rosa includes roses and Chinese roses.

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