Drought-resistant gene rrmyb2 of rose and application thereof

Abstract

The application discloses a rose drought-resistant gene RrMYB2 and application thereof, wherein the nucleotide sequence of the RrMYB2 gene is shown as SEQ ID NO. 1, and the protein amino acid sequence is shown as SEQ ID NO. 2. The application method is to improve the drought resistance of rose plants by controlling the overexpression of the gene RrMYB2 in the rose plants. After the RrMYB2 gene of the Rosa rubus cv. is separated, the RrMYB2 gene is introduced into a model plant Arabidopsis thaliana and the Rosa rubus cv., the drought resistance of the transgenic Arabidopsis thaliana and the Rosa rubus cv. is significantly enhanced, and it is indicated that the RrMYB2 gene is a positive regulation factor of drought resistance, and has important application value in the drought-resistant and stress-resistant breeding field of rose plants.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of plant genetic engineering, and particularly relates to a rose drought-tolerant gene RrMYB2 and application thereof. BACKGROUND

[0002] Rose (Rosa rugosa Thunb.) is a perennial deciduous shrub of the genus Rosa in the Rosaceae family, has rich varieties, and has a pleasant fragrance. Rose flowers are rich in flavonoids, volatile oils, phenolic acids, amino acids and other chemical components and nutrients needed by the human body, are widely used in skin care products, food and medicine fields, are important natural perfume plants in the world and traditional medicinal and edible flowers in China, have extremely high economic and health values, and at present, rose planting at home and abroad mostly adopts open cultivation and management is relatively extensive. In recent years, rose plant growth is weakened or even death caused by soil drought, which seriously affects the yield and quality of rose fresh flowers, and has become a bottleneck problem for the development of modern agricultural industry including rose planting.

[0003] Transcription factors can regulate plant stress resistance, including low temperature, drought, salt and alkali, by regulating the expression of key genes in different signal pathways. MYB transcription factors are one of the largest transcription factor families in plants. In model plants (Arabidopsis, rice, wheat, etc.), a plurality of drought-resistant MYB genes have been identified. These MYB genes regulate or reduce the damage of stress to plants through multiple levels, and play a crucial role in the growth and development of plants under drought conditions. Studies have shown that under 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 periods, and it mainly increases the resistance of plants to drought by activating the expression of lignin synthesis genes 4CL5H and F5H1 and ABA synthesis genes P5CS1 and LEA7. Another MYB transcription factor, OsMYB60, in rice can directly bind to the promoter of the wax biosynthesis gene OsCER1, activate its expression, and then promote the synthesis of cutin wax on the leaf surface to enhance the drought stress resistance of rice.

[0004] It can be seen that MYB transcription factors play a key role in regulating plant drought tolerance, but there is currently no specific report on MYB protein regulating drought resistance in roses. Therefore, exploring MYB members that regulate drought tolerance in roses helps to understand the regulation mechanism of drought tolerance in roses, has guiding significance for resistance breeding of Rosa plants, provides a basis for breeding excellent rose varieties with high drought tolerance and economic value, and promotes the development of the rose industry. SUMMARY

[0005] In view of the problem that soil drought causes the growth of rose plants to be weak or even die, screening a drought-regulating gene of rose and applying it to drought-tolerant variety breeding is an important way to obtain drought-tolerant roses.

[0006] The present application provides a drought-tolerant rose gene RrMYB2, the nucleotide sequence of which is shown in SEQ ID NO. 1, and the amino acid sequence of which is shown in SEQ ID NO. 2.

[0007] Another object of the present application is to provide the application of the drought-tolerant rose gene RrMYB2 in regulating drought resistance of Rosa plants.

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

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

[0010] Step 3), using the overexpression strain of the gene RrMYB2 to obtain an overexpression Rosa plant of the gene RrMYB2.

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

[0012] Further, in step 2), the Agrobacterium is GV3101 Agrobacterium or K599 rhizobium Agrobacterium.

[0013] The K599 rhizobium Agrobacterium into which the overexpression vector of the gene RrMYB2 is introduced can induce the Rosa plant to produce transgenic hairy roots and improve the drought resistance of the Rosa plant.

[0014] Further, the Rosa plant includes rose and Chinese rose.

[0015] Technical scheme: The present application provides a drought-tolerant rose gene RrMYB2, the nucleotide sequence of which is shown in SEQ ID NO. 1, and the amino acid sequence of which is shown in SEQ ID NO. 2.

[0016] Beneficial effects: The present application uses purple branch rose as a material, clones the RrMYB2 gene, detects the expression pattern of the RrMYB2 gene under drought treatment, improves the drought resistance of the overexpression strain through genetic transformation of Arabidopsis and purple branch rose, proves that it is a positive regulation factor of drought resistance, and has important application value for cultivating new drought-tolerant rose germplasm. BRIEF DESCRIPTION OF DRAWINGS

[0017] Figure 1 The expression amount of RrMYB2 in leaves within 24 hours of drought treatment of the plants;

[0018] Figure 2 is a plant overexpression vector pFAST-R05 plasmid map;

[0019] Figure 3 is a RrMYB2 overexpression Arabidopsis drought tolerance phenotype and physiological index detection diagram;

[0020] Figure 4 is a RrMYB2 overexpression purple branch rose drought tolerance phenotype and physiological index detection diagram. DETAILED DESCRIPTION

[0021] The technical solutions of the present application will be described in detail below through examples, but the protection scope of the present application is not limited to the examples.

[0022] Example 1: RrMYB2 gene cloning

[0023] In this example, the cDNA of purple branch rose is used as a template to amplify the coding region of the gene, and the nucleotide sequence of RrMYB2 gene is obtained.

[0024] 1. Primer design

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

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

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

[0028] 2. Obtain RrMYB2 gene

[0029] 2.1, extract total RNA from wild rose leaves and roots by FastPure Plant Total RNA Isolation Kit kit (Vazyme, Nanjing), the specific steps are as follows:

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

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

[0032] 3) The lysis mixture was placed in a 65°C water bath for 5 min, centrifuged at 12000 rpm for 10 min, and the supernatant was transferred to a new 1.5 ml centrifuge tube. An equal volume of absolute ethanol was added and mixed well.

[0033] 4) The FastPure gDNA-Fiter Column II collection column was placed in a collection tube, and the above mixture was transferred into it. Centrifugation was performed at 12000 rpm for 2 min, and the filtrate was discarded.

[0034] 5) The collection column was placed in a new Collection Tubes 2 ml, 500 μl Buffer PRLPlus was added, and centrifugation was performed at 12000 rpm for 30 sec. A 0.5-fold volume of absolute ethanol was added to the filtrate, and immediately mixed well.

[0035] 6) The mixture was transferred to the FastPure RNA Column IV shoujizh, centrifuged at 12000 rpm for 2 min, and the filtrate was discarded.

[0036] 7) 500 μl Buffer PRW2 was added to the FastPure RNA Column IV, centrifuged at 12000 rpm for 30 sec, and the filtrate was discarded.

[0037] 8) Repeat step 7).

[0038] 9) The FastPure RNA Column IV adsorption column was placed back into the collection tube and centrifuged at 12000 rpm for 2 min.

[0039] 10) The FastPure RNA Column IV was transferred to a new RNase-free Collection Tubes 1.5 ml centrifuge tube. 30 μl of RNase-free ddH2O was added to the center of the adsorption column membrane, and it was placed at room temperature for 2 min, and then centrifuged at 12000 rpm for 1 min.

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

[0041] 2.2, RNA was reverse transcribed into cDNA by HiScript III 1st Strand cDNA Synthesis Kit(Vazyme) kit, the specific operation steps are as follows:

[0042] 1) RNA template denaturation: 5 μg of Total RNA was added with RNase-Free ddH2O to 8 μl, incubated at 65°C for 5 min, and placed on ice for 2 min.

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

[0044] 3) First-strand cDNA synthesis: Add 2 μl 10x RT Mix, 2 μl HiScript III Enzyme Mix, 1 μl Oligo(dT) 20 VN, and 5 μl RNase-Free ddH2O to the product of the previous step, mix by pipetting. Perform the reaction according to the following three-step program: 25°C for 5 min, 37°C for 45 min, and 85°C for 5 sec to obtain the 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 ddH2O to a PCR tube, mix by pipetting. Perform the amplification reaction according to the following program: 95°C for 3 min, 95°C for 10 sec, 55°C for 5 sec (for 35 cycles), 72°C for 5 sec, and 72°C for 3 min.

[0047] 2) Obtain the purified PCR product using the FastPure Gel DNA Extraction Mini Kit kit.

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

[0049] 1) Ligation of the vector: 1 μl 5 ng of freshly recovered RrMYB2 gene product, 0.5 μl D-TOPO vector, 1 μl Salt solution, and 1 μl RNase-Free ddH2O, for a total of 3 μl system. Incubate at 25°C for 5 min to obtain the recombination product.

[0050] 2) E. coli transformation: Thaw the E. coli competent cells stored at -70°C on ice, add 5 μl of the ligation product, mix, and then incubate on ice for 30 min, heat shock at 42°C for 90 sec, incubate on ice for 3 min, add 800 μl of LB liquid medium, recover at 37°C & 100 rpm for 1 h, centrifuge to remove the liquid, mix the remaining bacterial solution, plate (LB selection solid medium containing Kan), and incubate at 37°C overnight.

[0051] 3) Positive clone screening and sequencing analysis: single colony was picked from the screening plate and inoculated in LB liquid medium, 37°C & 250rmp shaking for 6h, bacterial liquid PCR detection was performed, positive bacterial liquid sequencing was selected, RrMYB2 gene was successfully isolated, and entry vector D-TOPO:RrMYB2 was obtained.

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

[0053] Example 2: Simulating drought treatment of R. rugosa by PEG powder

[0054] 1. Prepare 6 R. rugosa plants of the same growth period, and prepare a 20% aqueous solution of PEG6000 powder.

[0055] 2. Irrigate the different strains of R. rugosa with 20ml of 20% PEG6000 solution at 0h, 1h, 3h, 6h, 12h, and 24h, respectively, and place them in a long-day incubator.

[0056] 3. After the last strain is treated, take leaf samples from the six R. rugosa plants at the same time for subsequent experiments.

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

[0058] Design 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 fluorescent quantitative PCR was performed using SYBR Premix Ex Taq (Takara) and fluorescent quantitative PCR instrument CFX96TM (Bio-RAD), and Bio-Rad CFX Manager software was used for data processing. Each biological repeat was performed three times in technical repeat, and the average value was taken in three parallel data to reduce error.

[0062] As Figure 1As shown, in the rose plant, the expression of RrMYB2 in the leaves was up-regulated and then down-regulated with time within 24 h of drought treatment, and reached a peak at 6 h, according to the method of Example 2. It is shown that in the R. rugosa, RrMYB2 responds to drought stress is a long-term process.

[0063] Example 4: Construction of RrMYB2 overexpression vector

[0064] The plant overexpression vector pFAST-R05 (Invitrogen) was used to construct the RrMYB2 overexpression vector, and the construction method was as follows: Figure 2

[0065] 1) The overexpression vector was obtained by using the LR Clonase Enzyme Mix kit (Invitrogen): 1 μl of the entry vector D-TOPO:RrMYB2 plasmid, 1 μl of the pFAST-R05 vector plasmid, 1 μl of the LR Enzyme, 2 μl of the TE Buffer, and 1 μl of the RNase-Free ddH2O, a total of 5 μl of the system. Incubate at 25°C for 1 h to obtain the recombination product. The E. coli transformation and positive clone screening method are the same as in Example 1. The positive bacteria liquid was sent to Shengong Biotechnology Co., Ltd. (Nanjing) for sequencing, and the overexpression vector 35S:RrMYB2 was obtained.

[0066] 3) Agrobacterium transformation: Take the -80°C stored GV3101 Agrobacterium competent cells and Agrobacterium K599 competent cells, melt at room temperature, add 0.01-1 μg of plasmid DNA to each 100 μl of competent cells, mix well by tapping the tube bottom with hands, and then sequentially stand on ice for 5 minutes, stand in liquid nitrogen for 5 minutes, 37°C water bath for 5 minutes, ice bath for 5 minutes, add 700 μl of YEB liquid medium without antibiotics, mix well, and incubate at 28°C for 2-3 hours. Centrifuge at 6000 rpm for one minute to collect the bacteria, and then spread on YEB plates containing spectinomycin and rifampicin resistance. Invert and incubate in a 28°C incubator for 2-3 days, and grow positive colonies.

[0067] Example 5: Genetic transformation of Arabidopsis thaliana and observation of drought tolerance phenotype of RrMYB2 overexpression Arabidopsis thaliana plants

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

[0069] I. Abbreviations of reagents and solutions

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

[0071] II. Agrobacterium-mediated genetic transformation procedure

[0072] (1) Agrobacterium culture

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

[0074] (2) Arabidopsis inflorescence dipping method

[0075] The shaken bacterial solution was centrifuged at 4500 rpm for 10 min, and the Arabidopsis inflorescences were soaked in the resuspension solution for about 30 seconds, and then placed horizontally in the dark and moistened for 12-24 h.

[0076] (3) Screening of positive seedlings

[0077] The infected Arabidopsis was placed in a normal incubator for normal culture, and the harvested seeds were observed under a microscope for RFP fluorescence for screening; the screened T1 generation plants were sown and transplanted into a light incubator for growth and waiting for seed harvesting. The operation was repeated until T3 generation homozygous plants were obtained.

[0078] 2, The RrMYB2 overexpression Arabidopsis and wild type Arabidopsis seeds were sown on 1 / 2MS medium, and then moved to the substrate for culture after two true leaves grew out and grew for two weeks. Then natural drought treatment was carried out, and about 20 days without watering would appear drought phenotype. The results are shown in Figure 3 (a) is the phenotype of the plants after 10 days of drought treatment, (b) is the identification of whether RrMYB2 gene is overexpressed in wild type plants and transgenic plants, (c) is the survival rate statistics of wild type plants and transgenic plants after 20 days of natural drought treatment, (d) is the water loss rate analysis of wild type plants and transgenic plants. It is found through phenotype observation that the overexpression strain grows well, and the wild type shows wilting. Through determination of water loss rate and survival rate, it is found that the water loss rate of the overexpression strain is slower than that of the wild type, and the survival rate is 100%, while the survival rate of the wild type is 0%. The above evidence shows that overexpression of RrMYB2 promotes the drought resistance of Arabidopsis plants.

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

[0080] Using the non-lignified lateral branches of R. fruticosum, cut into 4-5 cm cuttings, dip the bottom into the positive K599 colonies obtained in Example 4, and plant in vermiculite under long-day conditions (light:dark = 16h:8h) at 25°C for 3 weeks to obtain a large number of transgenic hairy roots. The overexpression efficiency was verified by fluorescence quantification.

[0081] Example 7: Drought tolerance identification of RrMYB2 overexpression R. fruticosum

[0082] According to the method of Example 6, three RrMYB2 overexpression plants were obtained, numbered 35S:RrMYB2-1, 35S:RrMYB2-2, 35S:RrMYB2-3; after the pFAST-R05 empty vector was transformed into K599 Agrobacterium, the control plant 35S:Empty was obtained according to Example 6. After 15 days of natural drought treatment, the results are shown in Figure 4 Compared with the control, the chlorophyll fluorescence of the RrMYB2 overexpression plants had higher actual photosynthetic rate values. The determination of root physiological indicators under drought stress found that the SOD (superoxide dismutase) and CAT (catalase) contents in the RrMYB2 overexpression roots were significantly higher than those in the control group, indicating that the overexpression lines had stronger osmotic stress resistance; the H2O2 and O2 - contents in the RrMYB2 overexpression roots were lower than those in the control group, indicating that the damage to the cell membrane of the overexpression lines was lower.

[0083] Figure 4 In (a), the four rows of plants from top to bottom represent; the first row: plants without drought treatment; the second row: plants after 15 days of natural drought treatment; the third row: chlorophyll fluorescence image of plants without drought treatment; the fourth row: chlorophyll fluorescence image of plants after 15 days of natural drought treatment. Figure 4 In (b), the RrMYB2 overexpression efficiency in the three different 35S:RrMYB2 lines compared with the control plant is shown. Figure 4 In (c), the change in chlorophyll fluorescence parameter Y(II), i.e. PSII actual photosynthetic efficiency content, in the control plant and the three 35S:RrMYB2 plants before and after drought treatment is shown. Figure 4 In (d)-(g), the H2O2, O2 - , SOD, CAT contents in the roots of each plant after 15 days of natural drought treatment are shown.

[0084] While the application has been described and illustrated with reference to specific preferred embodiments, it is not intended that it be limited to these particulars. Various changes in form and detail can be made thereto without departing from the spirit and scope of the application.

Claims

1. A rose drought-tolerant gene RrMYB2, characterized in that the nucleotide sequence of the gene RrMYB2 is shown as SEQ ID NO. 1, and the amino acid sequence of the expressed protein of the gene RrMYB2 is shown as SEQ ID NO.

2.

2. The rose drought tolerance gene of claim 1 RrMYB2 use in modulating drought tolerance in roses, characterized in that, Controlling genes in rose plants RrMYB2 Overexpression, increasing drought resistance in roses.

3. Use according to claim 2, characterized in that, The application specifically method is: Step 1), construction of the overexpression vector of the gene RrMYB2 ; Step 2), introducing the overexpression vector of the gene RrMYB2 into Agrobacterium to obtain an overexpression strain of the gene RrMYB2 . Step 3) Obtain overexpression rose plants of the gene RrMYB2 using the overexpression strain of the gene RrMYB2 .

4. Use according to claim 3, characterized in that, In step 1), the overexpression vector of the gene was constructed using the plant overexpression vector pFAST-R05 RrMYB2 The overexpression vector 35S: RrMYB2 .

5. Use according to claim 3, characterized in that, In step 2), the agrobacterium is GV3101 agrobacterium or K599 rhizogenic agrobacterium.

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