A rose salt-tolerant gene RrC2H2 and application thereof

Abstract

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

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of plant genetic engineering, and particularly relates to a rose salt-tolerant gene RrC2H2 and application thereof. BACKGROUND

[0002] Rosa rugosa Thunb. is a deciduous shrub of Rosaceae, and is one of the oldest natural perfume plants in the world. The flower of R. rugosa is rich in terpenes and other aromatic substances, and is widely used in perfume industry materials or edible scented tea. The demand for R. rugosa in the domestic and international markets is increasing. Wild R. rugosa is a national second-class endangered protected plant, and is naturally distributed in the northeast coastal areas of China, the Far East of Russia, the Korean Peninsula and Japan. The special living environment leads wild R. rugosa to have strong salt tolerance, and is a precious material for studying the physiological basis of salt tolerance of R. rugosa. At present, the cultivation area of R. rugosa is expanding year by year. Due to the adaptive evolution caused by long-term directional selection of ornamental traits, the cultivated R. rugosa varieties can only tolerate mild salt stress, which limits the popularization and application and industrial development of the cultivated R. rugosa varieties.

[0003] Zinc finger protein is a kind of transcription factor which is self-folded and forms a short and stable finger-like structure by binding Zn 2+ . Zinc finger protein plays an important role in gene expression regulation, cell differentiation, embryonic development and enhancement of plant stress resistance. According to the different spatial structures formed by histidine and cysteine residues around Zn 2+ , Krishna et al. divide zinc finger proteins into eight types, i.e. C2H2, C2HC, C2HC5, C3HC4, C3H, C4, C4HC3, C6 and C8. C2H2 type zinc finger protein belongs to TF Ⅲ type, and is a kind of zinc finger protein which is most widely distributed and studied in the eukaryotic genome at present.

[0004] The research found that C2H2 zinc finger proteins have been widely reported in Arabidopsis, petunia, wheat, cotton, soybean and rice. In Arabidopsis, 176 C2H2 zinc finger proteins have been reported, and 189, 109, 118 and 47 C2H2 zinc finger proteins have been identified in rice, poplar, tobacco and wheat. Functional analysis shows that C2H2 zinc finger proteins play an important role in plant leaf morphogenesis, flower development and resistance to biological and non-biological stress. The multifunctionality of C2H2 zinc finger proteins in plant resistance to salt stress has been reported. ABA plays a crucial role in plant osmotic stress response, and ABA signal transduction pathway is activated under salt stress, inducing the expression of stress-related genes, in which C2H2 zinc finger proteins play an important role. At the same time, salt stress can lead to the accumulation of reactive oxygen species, causing oxidative damage to cells, and C2H2 zinc finger proteins can regulate the expression of reactive oxygen species clearance related genes, reducing the accumulation of H2O2 under salt stress. In addition, maintaining ion balance is also a method for C2H2 zinc finger proteins to improve plant salt tolerance.

[0005] The regulation of rose C2H2 gene family and its members on salt tolerance has not been studied, and exploring the C2H2 members of rose salt regulation is helpful to understand the regulation mechanism of rose salt tolerance, has guiding significance for Rosaceae plant resistance breeding, provides a basis for cultivating high salt tolerance and economic value of excellent rose new varieties, and promotes the development of rose industry. SUMMARY

[0006] In order to solve the problems in the prior art, the present application provides a rose salt tolerance gene RrC2H2 and its application.

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

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

[0009] The present application provides the application of rose salt tolerance gene RrC2H2, expression protein of rose salt tolerance gene RrC2H2 and overexpression vector containing rose salt tolerance gene RrC2H2 in rose, Chinese rose and other Rosaceae plant cultivation new variety breeding to improve salt tolerance.

[0010] Beneficial effects: The present application takes wild rose as the material, clones RrC2H2 gene, and detects the expression mode of RrC2H2 gene under salt treatment, improves the salt tolerance of overexpressed Arabidopsis thaliana through genetic transformation, separates the RrC2H2 gene of wild rose, and transplants it into model plant Arabidopsis thaliana, and the salt tolerance of transgenic Arabidopsis thaliana is significantly enhanced, which proves that it is a positive regulation factor of salt tolerance, and has important application value in the field of breeding rose plant salt-tolerant and stress-resistant breeding. BRIEF DESCRIPTION OF DRAWINGS

[0011] Figure 1 is a space-time expression pattern diagram of RrC2H2 gene;

[0012] Figure 2 is a plant overexpression vector pNC-Cam1304 plasmid map;

[0013] Figure 3 is a salt tolerance phenotype of RrC2H2 overexpressed Arabidopsis thaliana;

[0014] Figure 4 is a physiological index detection diagram of RrC2H2 overexpressed Arabidopsis thaliana. DETAILED DESCRIPTION

[0015] The technical scheme 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.

[0016] Example 1: RrC2H2 gene cloning

[0017] The gene coding region is amplified by taking the root system cDNA of wild rose as the template, and the nucleotide sequence of the gene is obtained.

[0018] 1.1, primer design, the upstream and downstream primers containing the complete open reading frame of RrC2H2 gene are designed:

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

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

[0021] 1.2, gene separation

[0022] Step 1, extract total RNA of wild rose leaves and root system by FastPure Plant Total RNA Isolation Kit kit (Vazyme, Nanjing); the specific steps are as follows:

[0023] 1) Buffer PRL was preheated to 65°C in a water bath, and 5% β-mercaptoethanol was added.

[0024] 2) About 0.3 g of rose root system was taken in a mortar, grinded with liquid nitrogen, and 500 μL Buffer PRL was added. The mixture was vortexed for 60 sec.

[0025] 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 Ethanol absolute was added and mixed by vortexing.

[0026] 4) The FastPure gDNA-Fiter Column II was placed in a collection tube, and the above mixture was transferred into it. The mixture was centrifuged at 12000 rpm for 2 min, and the filtrate was discarded.

[0027] 5) The collection column was placed in a new Collection Tubes 2 ml, 500 μL Buffer PRLPius was added, and the mixture was centrifuged at 12000 rpm for 30 sec. An equal volume of Ethanol absolute was added to the filtrate, and the mixture was immediately mixed by vortexing.

[0028] 6) The mixture was transferred to the FastPure RNA Column IV, centrifuged at 12000 rpm for 2 min, and the filtrate was discarded.

[0029] 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.

[0030] 8) Step 7) was repeated.

[0031] 9) The FastPure RNA Column IV was placed back in the collection tube, and centrifuged at 12000 rpm for 2 min.

[0032] 10) The FastPure RNA Column IV was transferred to a new RNase-free Collection Tubes, 30 μL of RNase-free ddH2O was added dropwise, and the mixture was left at room temperature for 2 min, and centrifuged at 12000 rpm for 1 min.

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

[0034] Step 2, reverse transcription of RNA to cDNA by HiScript III 1st Strand cDNA Synthesis Kit (Vazyme, Nanjing) kit, the specific operation steps are as follows:

[0035] 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.

[0036] 2) Removal of genomic DNA: 2 μL of 5×gDNA wiper Mix was added to the product of the previous step, mixed by blowing, and reacted at 42°C for 2 min.

[0037] 3) First strand cDNA acquisition: 2 μL of 10×RT Mix, 2 μL of HiScript III Enzyme Mix, 1 μL of Oligo(dT) 20 , and 5 μL of RNase-Free ddH2O were added to the product of the previous step, mixed by blowing, and reacted according to the three-step procedure of 25°C for 5 min, 37°C for 45 min, and 85°C for 5 sec to obtain cDNA.

[0038] Step 3, isolation of RrC2H2 gene;

[0039] 1) PCR amplification of RrC2H2 gene: 25 μL of PrimeSTAR Max Premix (2X), 2 μL of Primer F and Primer R, 1 μL of cDNA, and 20 μL of RNase-Free ddH2O were added to the PCR tube, mixed by blowing, and reacted according to the procedure of 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.

[0040] 2) Purified PCR product was obtained using FastPure Gel DNA Extraction Mini Kit.

[0041] Step 4, cloning of RrC2H2 gene into vector by pEASY-Blunt Cloning Kit (TransGen Biotech, Beijing), steps as follows:

[0042] 1) Ligation of vector: 50 ng of PCR product was added with 1 μL of pEASY-Blunt Zero Cloning Vector, RNase-Free ddH2O was added to 5 μL, and the ligation product was obtained by incubation at 25°C for 10 min.

[0043] 2) E. coli transformation: E. coli competent cells stored at -70°C were thawed on ice, 5 μL of ligation product was added, mixed and then incubated in ice bath for 30 min, 42°C heat shock for 90 sec, ice bath for 3 min, 800 μL of LB liquid medium was added, 37°C & 100 rpm recovery for 1 h, centrifugal suction of liquid, mixing of remaining bacterial liquid, plating (LB screening solid medium containing Kan), 37°C overnight culture.

[0044] 3) Positive clone screening and sequencing analysis: single colony was selected from the screening culture plate and inoculated in LB liquid medium, 37°C & 250 rpm shaking for 6 h, bacterial liquid PCR detection was performed, positive bacterial liquid sequencing was selected, and the salt-tolerant gene RrC2H2 of rose was successfully isolated, the nucleotide sequence of which is shown as SEQ ID NO. 1, and the expression protein of the salt-tolerant gene RrC2H2 of rose, the amino acid sequence of which is shown as SEQ ID NO. 2.

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

[0046] The fluorescence quantitative PCR upstream and downstream primers were designed 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 fluorescence quantitative PCR instrument CFX96TM (Bio-RAD), and Bio-Rad CFX Manager software was used for data processing. Each biological repeat was repeated three times, and the average value of the three parallel data was taken to reduce the error. The results are shown in Figure 1 As shown in the results, in the rose root system, the expression amount of RrC2H2 in the root system increased with time within 24 h of salt treatment, and then decreased after up-regulation, reaching a peak at 6 h, with an up-regulation of about 1.4 times; after 3 d and 7 d of salt treatment, the expression amount of RrC2H2 in the root system increased by about 2 times. It is shown that in the wild rose root system, RrC2H2 responds to salt stress for a long time.

[0050] Example 3: Construction of RrC2H2 overexpression vector

[0051] The plant overexpression vector pNC-Cam1304 (Invitrogen) was used Figure 2) Construct RrC2H2 overexpression vector, the construction method is as follows:

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

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

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

[0055] The RrC2H2 gene fragment obtained in Example 1 is used as a template for PCR amplification, and the PCR amplification and product recovery process are the same as in Example 1.

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

[0057] 50 ng of the recovered fragment is mixed with 1 μL of pNC-Cam1304 vector, RNase-Free ddH2O is added to 5 μL, and the mixture is incubated at 25°C for 10 min to obtain a recombination product. The E. coli transformation and positive clone screening method are the same as in Example 1. The positive bacterial solution is sent to GenScript Biotech (Nanjing) for sequencing, and the recombinant overexpression vector pNC-Cam1304: RrC2H2 is obtained.

[0058] 3) Agrobacterium transformation: Take the Agrobacterium EHA105 competent cells stored at -80°C and melt at room temperature. Add 0.01-1 μg of recombinant overexpression vector pNC-Cam1304: RrC2H2 to each 100 μL of competent cells, mix well with hand, and then sequentially incubate on ice for 5 min, in liquid nitrogen for 5 min, in a 37°C water bath for 5 min, and in an ice bath for 5 min. Add 700 μL of antibiotic-free YEB liquid medium, and incubate at 28°C for 2-3 hours. Centrifuge at 6000 rpm for 1 min to collect the bacteria, and then spread on a YEB plate containing kanamycin. Invert the plate in a 28°C incubator and culture for 2-3 days to grow positive colonies.

[0059] Example 4: Genetic transformation of Arabidopsis thaliana

[0060] Infect Arabidopsis thaliana by floral dip method, and obtain transgenic plants by screening transformed seedlings with hygromycin resistance.

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

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

[0063] (1) Agrobacterium culture: positive colonies are inoculated into liquid LB medium (10 g / L peptone + 5 g / L yeast extract + 10 g / L NaCl + 50 mg / L Kan) and cultured at 28°C, 200 rpm overnight until the bacterial solution concentration OD 600 is 1.5-2.0.

[0064] (2) Arabidopsis inflorescence dipping method: the shaken bacterial solution is centrifuged at 4500 rpm for 10 min, and the Arabidopsis inflorescence is soaked in the resuspension solution for about 30 seconds, and is placed horizontally in the dark and moistened for 12-24 hours.

[0065] (3) Screening of positive seedlings: the infected Arabidopsis is placed in a normal incubator for culture, and the harvested seeds are disinfected by 70% alcohol for 15-30 seconds and 2% sodium hypochlorite for 15 minutes, washed with sterile water for 3-4 times, and then plated on 1 / 2MS medium containing resistance (1 / 2MS + 30 g / L sucrose + 7 g / L agar + 20 mg / L Hyg) for screening; the screened T1 generation plants are subjected to PCR positive detection and transplanted to a light incubator for seed collection. Repeat the operation until the T3 generation homozygous plants are obtained.

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

[0067] RrC2H2 overexpression Arabidopsis and wild type Arabidopsis are sowed on 1 / 2MS medium containing 0 mM, 25 mM and 75 mM concentration of NaCl, and grown for two weeks.

[0068] The results of phenotype observation are shown in Figure 3 The figure WT represents wild type Arabidopsis, and RrC2H2 represents RrC2H2 overexpression Arabidopsis. It is found through phenotype observation that the growth state of each plant is good and the growth trend is consistent on the 1 / 2MS sodium chloride medium with a sodium chloride concentration of 0 mM, and the leaves present a healthy emerald green color Figure 3 On the medium with a sodium chloride concentration of 25 mM, the roots of RrC2H2 overexpression Arabidopsis are longer than those of wild type Arabidopsis, and the root branches are more Figure 3B) in the presence of 75 mM NaCl. In the medium containing 75 mM NaCl, the growth of wild-type Arabidopsis and RrC2H2 overexpressing Arabidopsis was inhibited: the leaf tips withered and turned yellow, and the plants grew slowly Figure 3 C) in the presence of 75 mM NaCl. The above evidence shows that overexpression of RrC2H2 promotes the salt tolerance of Arabidopsis plants.

[0069] The contents of peroxidase POD, glutathione GSH, malondialdehyde MDA and proline Pro of RrC2H2 transgenic plants treated with 75 mM NaCl were determined, and the results are shown in Table 4. Figure 4 Figure 4 In Table 4, WT represents wild-type Arabidopsis, and OE refers to RrC2H2 transgenic plants treated with 75 mM NaCl. The results show that the POD content of RrC2H2 transgenic plants is significantly different from that of the control plants, the MDA content is significantly different, and the GSH and Pro contents are 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 RrC2H2 transgenic strains is 4.7 times that of the control group CK, indicating that cell damage is the main difference in salt tolerance between transgenic strains and wild-type strains.

[0070] As described above, although the present application has been shown and described with reference to specific preferred embodiments, it is to be understood that various modifications and changes can be made to it without departing from the spirit and scope of the application.​

Claims

1. A salt-tolerant gene for roses RrC2H2 Its characteristics are, Its nucleotide sequence is shown in SEQ ID NO.

1.

2. A salt-tolerant gene as described in claim 1 RrC2H2 The expressed protein is characterized by, Its amino acid sequence is shown in SEQ ID No.

2.

3. A rose salt tolerance gene as described in claim 1 RrC2H2 The application of the overexpression vector in improving the salt tolerance of roses is characterized by, The overexpression vector is pNC-Cam1304:RrC2H2.

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