Rose salt-tolerant gene RrC2H2 and application thereof

Abstract

The invention discloses a rose salt-tolerant gene RrC2H2 and application thereof, and belongs to the technical field of plant genetic engineering, the nucleotide sequence of the RrC2H2 gene is as shown in SEQ ID NO.1, and the protein amino acid sequence of the RrC2H2 gene is as shown in SEQ ID NO.2. The RrC2H2 gene is cloned by taking the wild rose as a material, the expression mode of the RrC2H2 gene under salt treatment is detected, the salt tolerance of overexpressed arabidopsis thaliana is improved through genetic transformation, the RrC2H2 gene of the wild rose is separated and transferred into a model plant arabidopsis thaliana, the salt tolerance of transgenic arabidopsis thaliana is remarkably enhanced, it is proved that the RrC2H2 gene is a salt tolerance positive regulation factor, and the RrC2H2 gene of the wild rose is converted into the model plant arabidopsis thaliana. The method has an important application value in the field of salt-resistant and stress-resistant breeding of rosa plants.

Description

Technical Field

[0001] The invention belongs to the technical field of plant genetic engineering, and in particular is a rose salt-tolerant gene RrC2H2 and an application thereof. Background Art

[0002] Rose (Arugosa Thunb.) is a deciduous shrub of the genus Rosa in the Rosaceae family. It is one of the oldest natural spice plants in the world. Its flowers are rich in aromatic substances such as terpenes and are widely used in spice industry materials or edible scented teas. The market demand at home and abroad is increasing. Wild roses are native to China and are a national second-level endangered plant. They are naturally distributed in the coastal areas of Northeast China, the Russian Far East, the Korean Peninsula and Japan. The special living environment makes wild roses have strong salt tolerance and are precious materials for studying the physiological basis of rose salt tolerance. At present, the cultivated area of ​​roses in my country is expanding year by year. Due to the adaptive evolution caused by long-term directional selection of ornamental traits, cultivated rose varieties can only tolerate mild salt stress, which limits their promotion and application and industrial development.

[0003] Zinc finger proteins are a class of proteins that bind Zn 2+ A transcription factor that folds itself into a short and stable finger-like structure. It plays an important role in gene expression regulation, cell differentiation, embryonic development and enhancing plant stress resistance. 2+ According to the different spatial structures, Krishna et al. classified zinc finger proteins into eight types: C2H2, C2HC, C2HC5, C3HC4, C3H, C4, C4HC3, C6 and C8. C2H2 zinc finger proteins belong to TFⅢ type and are currently the most widely distributed and studied type of zinc finger proteins in eukaryotic genomes.

[0004] Studies have found that C2H2 zinc finger proteins have been widely reported in plants such as Arabidopsis, petunia, wheat, cotton, soybean and rice. In Arabidopsis, 176 C2H2 zinc finger proteins have been reported, of which 189, 109, 118 and 47 C2H2 zinc finger proteins have been identified in rice, poplar, tobacco and wheat, respectively. Functional analysis has shown that C2H2 zinc finger proteins play an important role in plant leaf morphogenesis, flower development and resistance to biotic and abiotic stresses. C2H2 zinc finger proteins have multiple functions in plant resistance to salt stress. Studies have reported that ABA plays a vital role in plant osmotic stress response. Under salt stress, the ABA signal transduction pathway is activated, inducing the expression of stress-related genes, among which C2H2 zinc finger proteins play an important role. At the same time, salt stress can lead to the accumulation of reactive oxygen species and cause oxidative damage to cells. C2H2 zinc finger proteins can regulate the expression of genes related to reactive oxygen scavenging and reduce the accumulation of H2O2 under salt stress. In addition, maintaining ion balance is also a way for C2H2 zinc finger proteins to improve plant salt tolerance.

[0005] There is no research on the regulation of salt tolerance by the rose C2H2 gene family and its members. Exploring the C2H2 members that regulate rose salt tolerance will help us understand the regulatory mechanism of rose salt tolerance, which has guiding significance for the resistance breeding of Rosaceae plants, provides a basis for breeding new rose varieties with high salt tolerance and economic value, and promotes the development of the rose industry. Summary of the invention

[0006] In order to solve the problems in the prior art, the present invention proposes a rose salt-tolerance gene RrC2H2 and an application thereof.

[0007] The present invention provides a rose salt-tolerant gene RrC2H2, whose nucleotide sequence is shown in SEQ ID NO. 1. The expression protein of the rose salt-tolerant gene RrC2H2, whose amino acid sequence is shown in SEQ ID NO. 2. The overexpression vector containing the rose salt-tolerant gene RrC2H2 is pNC-Cam1304: RrC2H2.

[0008] The overexpression vector is assembled with a Nimble Cloning Frame, which can quickly assemble gene fragments in one step to obtain an expression frame composed of 35S promoter-RrC2H2-NOS terminator, and can ensure that the RrC2H2 gene is stably and continuously expressed in large quantities in host cells.

[0009] The present invention provides rose salt-tolerance gene RrC2H2, expression protein of rose salt-tolerance gene RrC2H2, and application of overexpression vector containing rose salt-tolerance gene RrC2H2 in breeding new varieties of rose, Chinese rose and other plants of genus Rosa for improving salt tolerance.

[0010] Beneficial effects: The present invention uses wild rose as material, clones the RrC2H2 gene, detects the expression pattern of the RrC2H2 gene under salt treatment, improves the salt tolerance of overexpressed Arabidopsis through genetic transformation, separates the RrC2H2 gene of wild rose, and transfers it into the model plant Arabidopsis, and the salt tolerance of the transgenic Arabidopsis is significantly enhanced, proving that it is a positive regulatory factor for salt tolerance, and has important application value in the field of salt-tolerant and stress-resistant breeding of Rosa plants. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] Figure 1 This is the spatiotemporal expression pattern of the RrC2H2 gene;

[0012] Figure 2 This is the plasmid map of the plant overexpression vector pNC-Cam1304;

[0013] Figure 3 The salt tolerance phenotype of RrC2H2-overexpressing Arabidopsis thaliana;

[0014] Figure 4 This is a diagram showing the physiological index detection of Arabidopsis thaliana with overexpression of RrC2H2. DETAILED DESCRIPTION

[0015] 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.

[0016] Example 1: RrC2H2 gene cloning

[0017] The wild rose root cDNA was used as a template to amplify the gene coding region and obtain the gene nucleotide sequence.

[0018] 1.1. Primer design: Design upstream and downstream primers containing the complete open reading frame of the RrC2H2 gene:

[0019] SEQ ID NO.3, Primer F: 5'-ATGATAACAGTGCCAAACTT-3',

[0020] SEQ ID NO.4, Primer R: 5'-TCAAAGCTTTAAAGTAAGAT-3'

[0021] 1.2 Gene Isolation

[0022] Step 1: Extract total RNA from leaves and roots of wild rose using FastPure Plant Total RNA Isolation Kit (Vazyme, Nanjing); the specific steps are as follows:

[0023] 1) Preheat Buffer PRL to 65°C in a water bath and add 5% β-mercaptoethanol.

[0024] 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.

[0025] 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.

[0026] 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.

[0027] 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.

[0028] 6) Transfer the mixture to FastPure RNA Column IV, centrifuge at 12000 rpm for 2 min, and discard the filtrate.

[0029] 7) Add 500 μL Buffer PRW2 to FastPure RNA Column IV, centrifuge at 12,000 rpm for 30 seconds, and discard the filtrate.

[0030] 8) Repeat step 7).

[0031] 9) Place the FastPure RNA Column IV back into the collection tube and centrifuge at 12000 rpm for 2 min.

[0032] 10) Transfer FastPure RNA Column IV to new RNase-free Collection Tubes, add 30 μL of RNase-free ddH2O, let stand at room temperature for 2 minutes, and centrifuge at 12000 rpm for 1 minute.

[0033] 11) The collected RNA was packaged into 1 μg portions and stored in a -80°C refrigerator.

[0034] Step 2: Reverse transcribe RNA into cDNA using HiScript III 1st Strand cDNA Synthesis Kit (Vazyme, Nanjing). The specific steps are as follows:

[0035] 1) RNA template denaturation: Add RNase-Free ddH2O to 8μl of 5μg Total RNA, incubate at 65℃ for 5min, and place on ice for 2min.

[0036] 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.

[0037] 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 ddH2O were pipetted and mixed. 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.

[0038] Step 3, isolating the RrC2H2 gene;

[0039] 1) PCR amplification of RrC2H2 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 and mix well by pipetting. Perform amplification reaction according to the following procedure: 95°C, 3 min; 95°C, 10 sec, 55°C, 5 sec (35 cycles); 72°C, 5 sec; 72°C, 3 min.

[0040] 2) Use FastPure Gel DNA Extraction Mini Kit to obtain purified PCR products.

[0041] Step 4: Clone the RrC2H2 gene into the vector using pEASY-Blunt Cloning Kit (Quanshijin, Beijing), as follows:

[0042] 1) Connect the vector: Add 1 μL pEASY-Blunt Zero Cloning Vector to 50 ng PCR product, add RNase-Free ddH2O to 5 μL, and incubate at 25°C for 10 min to obtain the connection product.

[0043] 2) E. coli transformation: Thaw the -70℃ frozen E. coli competent cells on ice, add 5 μL of the ligation product, mix well, incubate on ice for 30 min, heat shock at 42℃ for 90 sec, incubate on ice for 3 min, add 800 μL of LB liquid culture medium, resuscitate at 37℃&100 rpm for 1 h, centrifuge to remove the liquid, mix the remaining bacterial liquid, plate (LB screening solid culture medium containing Kan), and culture overnight at 37℃.

[0044] 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 bacterial solution was tested by PCR. The positive bacterial solution was selected for sequencing, and the rose salt-tolerance gene RrC2H2 was successfully isolated. Its nucleotide sequence is shown in SEQ ID NO.1, and the expression protein of the rose salt-tolerance gene Rr C2H2, its amino acid sequence is shown in SEQ ID NO.2.

[0045] Example 2: Exploring the spatiotemporal expression pattern of the RrC2H2 gene by fluorescence quantitative PCR technology

[0046] Design of upstream and downstream primers for fluorescence quantitative PCR based on the exon region of RrC2H2 gene:

[0047] SEQ ID NO. 5, upstream primer: 5′-GCCAATGCCACCAATGCCAA-3′;

[0048] SEQ ID NO. 6, downstream primer: 5'-ACCTCGGATTCCCTCACCCA-3'.

[0049] Real-time fluorescence quantitative PCR was performed using SYBR Premix Ex Taq (Takara) and fluorescent quantitative PCR instrument CFX96TM (Bio-RA D), and data were processed using Bio-Rad CFX Manager software. Three technical replicates were performed in each biological replicate, and the average of the three sets of parallel data was taken to reduce errors. Figure 1 As shown in the figure, in the rose root system, the expression of RrC2H2 in the root system first increased and then decreased with time within 24 hours of salt treatment, reaching a peak at 6 hours, and the expression level increased by about 1.4 times; after 3d and 7d of salt treatment, the expression level of RrC2H2 in the root system increased by about 2 times. This shows that in the root system of wild rose, the response of RrC2H2 to salt stress is a long-term process.

[0050] Example 3: Construction of RrC2H2 overexpression vector

[0051] Using the plant overexpression vector pNC-Cam1304 ( Figure 2) construct an RrC2H2 overexpression vector, and the construction method is as follows:

[0052] 1) Obtain the RrC2H2 gene fragment fused with the NC linker and design the RrC2H2 primer with the NC linker:

[0053] SEQ ID NO.7, upstream primer with NC linker RrC2H2: 5′-AGTGGTCTCTGTCCAGTCCTATGATAACAGTGCCAA-3′;

[0054] SEQ ID NO. 8, downstream primer with NC linker RrC2H2: 5'-GGTCTCAGCAGACCACAAGTAAGCTTTAAAGTAAGA-3'.

[0055] PCR amplification was performed using the target fragment of the RrC2H2 gene obtained in Example 1 as a template. The PCR amplification and product recovery process were the same as in Example 1.

[0056] 2) Obtain the recombinant vector using the Nimble Cloning kit (NC Biotech):

[0057] 50 ng of the recovered fragment and 1 μL of pNC-Cam1304 vector were mixed with RNase-Free ddH2O to 5 μL and incubated at 25°C for 10 min to obtain the recombinant product. The E. coli transformation and positive clone screening methods were the same as in Example 1. The positive bacterial solution was sent to Sangon Biotech Co., Ltd. (Nanjing) for sequencing to obtain the recombinant overexpression vector pNC-Cam1304:RrC2H2.

[0058] 3) Agrobacterium transformation: Take the competent Agrobacterium EHA105 stored at -80℃ and thaw it at room temperature, add 0.01-1μg of the recombinant overexpression vector pNC-Cam1304:RrC2H2 for every 100μL competent medium, stir the bottom of the tube by hand to mix, let it stand on ice for 5 minutes, liquid nitrogen for 5 minutes, 37℃ water bath for 5 minutes, ice bath for 5 minutes, add 700mL of YEB liquid culture medium without antibiotics, shake and culture at 28℃ for 2-3 hours, centrifuge at 6000rpm for one minute to collect the bacteria, spread on YEB plates containing kanamycin resistance, and invert to culture in a 28℃ incubator for 2-3 days to grow positive colonies.

[0059] Example 4: Arabidopsis genetic transformation

[0060] Arabidopsis thaliana is infected by the inflorescence dip method, and transgenic plants are obtained by screening transformed seedlings with hygromycin resistance.

[0061] The abbreviations of the plant hormones used in the culture medium are as follows: Kan (Kanamycin); Hyg (Hygromycin); Arabidopsis transformation assistant reagent (Silwet L-77).

[0062] The steps of Agrobacterium-mediated genetic transformation are as follows:

[0063] (1) Cultivation of Agrobacterium: Positive colonies were picked and inoculated into liquid LB medium (10 g / L peptone + 5 g / L yeast extract + 10 g / L NaCl + 50 mg / L Kan), and cultured overnight at 28°C and 200 rpm in a shaking incubator until the bacterial solution concentration OD 600 The value is 1.5-2.0.

[0064] (2) Arabidopsis inflorescence dip staining method: 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-7) was added. 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.

[0065] (3) Screening of positive seedlings: The infected Arabidopsis thaliana was placed in an incubator for normal culture. The harvested seeds were sterilized on a clean bench with 70% alcohol for 15-30 seconds and 2% sodium hypochlorite for 15 minutes. After being washed with sterile water 3-4 times, they were spread on a 1 / 2MS medium containing resistance (1 / 2MS + 30g / L sucrose + 7g / L agar + 20mg / L Hyg) for screening. The screened T1 generation plants were tested for PCR positive and transplanted to a light incubator for harvest. The operation was repeated until T3 generation homozygous plants were obtained.

[0066] Example 5: Observation of salt tolerance phenotype of RrC2H2 overexpressing Arabidopsis plants

[0067] RrC2H2-overexpressing Arabidopsis and wild-type Arabidopsis were seeded on 1 / 2MS medium containing 0 mM, 25 mM, and 75 mM NaCl concentrations and grown for two weeks.

[0068] Phenotypic observations Figure 3 As shown in the figure, WT represents wild-type Arabidopsis, and RrC2H2 represents RrC2H2 overexpressing Arabidopsis. Through phenotypic observation, it was found that on the medium of 1 / 2MS sodium chloride with a sodium chloride concentration of 0mM, the growth state of each plant was good, the growth trend was consistent, and the leaves showed healthy emerald green ( Figure 3 In the medium with a sodium chloride concentration of 25 mM, the roots of Arabidopsis thaliana overexpressing RrC2H2 were longer and had more root branches than those of wild-type Arabidopsis thaliana ( Figure 3In the medium containing 75 mM sodium chloride, the growth of wild-type Arabidopsis and RrC2H2-overexpressing Arabidopsis was inhibited: the leaf tips wilted and turned yellow, and the plants developed slowly ( Figure 3 The above evidences indicate that overexpression of RrC2H2 promotes salt tolerance in Arabidopsis plants.

[0069] The contents of peroxidase POD, glutathione GSH, malondialdehyde MDA and proline Pro in RrC2H2 transgenic plants treated with 75 mM sodium chloride were determined. Figure 4 shown. Figure 4 In the figure, WT represents wild-type Arabidopsis, and OE refers to RrC2H2 transgenic plants treated with 75 mM sodium chloride. The results showed that the POD content of RrC2H2 transgenic plants was significantly different from that of the control plants, the MDA content was significantly different, and the GSH and Pro index were not significantly different. Since the POD content is related to the cytoplasmic reactive oxygen scavenging mechanism, it is speculated that the reactive oxygen content of RrC2H2 transgenic plants is higher. The MDA content of the RrC2H2 transgenic strain is 4.7 times that of the control group CK, indicating that cell damage is the main difference in salt tolerance between the transgenic strain and the wild-type strain.

[0070] 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 salt tolerance gene RrC2H2, characterized in that: Its nucleotide sequence is shown in SEQ ID NO.

1.

2. An expression protein of the salt-tolerant gene RrC2H2 according to claim 1, characterized in that: Its amino acid sequence is shown in SEQ ID No.

2.

3. An overexpression vector containing the salt-tolerant gene RrC2H2 according to claim 1, characterized in that: The overexpression vector is pNC-Cam1304:RrC2H2.

4. Use of the salt-tolerant gene RrC2H2 according to claim 1 in the breeding of new varieties of rose plants.

5. Use of the expression protein of the salt-tolerant gene RrC2H2 according to claim 2 in the breeding of new varieties of rose plants.

6. Use of the overexpression vector of the salt-tolerant gene RrC2H2 according to claim 3 in the breeding of new varieties of rose plants.

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