Rose salt-tolerance gene RrGTγ-4 and its application

Abstract

The invention discloses a wild rose salt-tolerant gene RrGT c‑4 and its applications, RrGTγ‑4 The nucleotide sequence of the gene is shown in SEQ ID NO.1, and the amino acid sequence of the protein is shown in SEQ ID NO.2. RrGTγ‑4 After the gene was isolated, it was transferred into the model plant Arabidopsis thaliana. The salt tolerance of the transgenic Arabidopsis thaliana was significantly enhanced, indicating that RrGTγ‑4 The gene is a positive regulatory factor of salt tolerance and has important application value in the field of breeding roses for salt tolerance and stress resistance.

Description

Technical Field

[0001] The present invention belongs to the technical field of plant genetic engineering, and specifically relates to a rose salt-tolerant gene RrGTγ-4 and its applications. Background Art

[0002] Rosaceae Rosa genus deciduous shrub wild rose ( Rosa rugosa Thunb. is a Class II endangered plant species naturally distributed in the coastal areas of Northeast China, the Russian Far East, the Korean Peninsula, and Japan. It is well adapted to high-salinity habitats and exhibits strong salt tolerance, making it a valuable parent material for the development of salt-tolerant varieties of the genus Rosa. Cultivated roses, known for their excellent ornamental and quality qualities and rich in aromatic substances such as rose essential oil, are widely used in the fragrance industry. However, long-term artificial selection has resulted in cultivated varieties with poor salt tolerance. However, my country's main rose-producing areas are rich in saline soil resources, which remain underutilized, hindering the promotion and development of the rose industry.

[0003] The Trihelix transcription factor family was discovered because its conserved domains can specifically bind to GT elements required for light response, thereby exerting positive or negative regulatory functions. This class of proteins responds to biotic and abiotic stresses and participates in developmental processes such as flowers, stomata, and seeds [1, 2]. Recent studies have divided triple-helical transcription factors into five subfamilies based on their distinct domain structures: SIP1, GT-1, GT-2, GTγ, and SH4. The DNA binding domain of GT factors in the Trihelix transcription factor family contains three conserved tandem helical structures. The GT-2 subfamily contains two Trihelix conserved domains (DNA binding domains), while the SH4 subfamily lacks a fourth α-helical domain and carries an extended third triple helix. Other subfamilies contain a conserved domain, a fourth α-helical domain, and a distinct α-helical domain.

[0004] Previous studies have shown that Trihelix transcription factors play a crucial role in plant growth and development. In recent years, research on their responses to abiotic stress has increased significantly. To date, with the exception of SH4, members of each subfamily have been reported to be involved in abiotic stress responses. Research on the GT-1 and SIP1 subfamilies has primarily focused on changes in ABA sensitivity, while the GTγ subfamily focuses on salt tolerance. Research on the GT-2 subfamily is more comprehensive, encompassing drought, cold, and salt stresses. Furthermore, whole-genome analyses of Trihelix have been conducted in model species such as Arabidopsis thaliana, rice, and soybean [4-6]. Related studies have also been published in species such as Brassica rapa, buckwheat, and bamboo [7-13]. Whole-genome analyses of wheat [14, 15] have also been conducted under both normal environments and abiotic stresses.

[0005] The regulation of salt tolerance by the rose Trihelix gene family and its members has not yet been studied. Exploring the Trihelix members that regulate rose salt tolerance has guiding significance for resistance breeding of Rosa plants, providing a basis for breeding new rose varieties with high salt tolerance and economic value, and promoting the development of the rose industry. Summary of the Invention

[0006] The purpose of the present invention is to provide a rose salt-tolerant gene to address the deficiencies in the prior art. RrGTγ-4 And its application, the first purpose is to provide rose salt tolerance gene RrGTγ-4 Another purpose is to find rose salt tolerance genes RrGTγ-4 application.

[0007] The object of the present invention is achieved in this way: a rose salt-tolerant gene RrGTγ-4 , whose nucleotide sequence is shown in SEQ ID NO.1. RrGTγ-4 The expressed protein has an amino acid sequence as shown in SEQ ID NO.2.

[0008] Containing the rose salt-tolerant gene RrGTγ-4 That is, it contains the rose salt-tolerant gene RrGTγ-4 Plant overexpression vector pNC-Cam1304-MCS35S: RrGTγ-4 The vector is equipped with a Nimble Cloning Frame, which can quickly assemble gene fragments in one step to obtain the 35S promoter- RrGTγ-4 -NOS terminator overexpression cassette, which can ensure RrGTγ-4 The gene is stably and continuously expressed in large quantities in the host cells.

[0009] The rose salt-tolerant gene RrGTγ-4 Application in improving salt tolerance of roses.

[0010] Through the present invention, the present invention discloses a wild rose salt-tolerant gene RrGTγ-4 and its applications, RrGTγ-4 The nucleotide sequence of the gene is shown in SEQ ID NO.1, and the amino acid sequence of the protein is shown in SEQ ID NO.2. RrGTγ-4 After the gene was isolated, it was transferred into the model plant Arabidopsis thaliana. The salt tolerance of the transgenic Arabidopsis thaliana was significantly enhanced, indicating that RrGTγ-4 The gene is a positive regulatory factor of salt tolerance and has important application value in the field of breeding roses for salt tolerance and stress resistance.

[0011] Beneficial effect: The present invention uses wild rose as material to clone RrGTγ-4 Gene, tested RrGTγ-4 The gene expression pattern under salt treatment improved the salt tolerance of overexpressing Arabidopsis through genetic transformation, proving that it is a positive regulatory factor for salt tolerance and has important application value for breeding new salt-tolerant rose germplasm. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 yes RrGTγ-4 Gene spatiotemporal expression pattern diagram;

[0013] Figure 2 This is the plasmid map of the plant overexpression vector pNC-Cam1304-MCS35S;

[0014] Figure 3 yes RrGTγ-4 Overexpression of salt tolerance phenotype in Arabidopsis;

[0015] Figure 4 yes RrGTγ-4 Statistics of Arabidopsis phenotypes overexpressing the gene. DETAILED DESCRIPTION

[0016] The present invention will be further described below with reference to specific embodiments. Example 1

[0017] 1. RrGTγ-4 Gene cloning

[0018] The cDNA of wild rose leaves was used as a template to amplify the gene coding region fragment, connect it to the cloning vector and sequence it to obtain the gene nucleotide sequence.

[0019] I. Primer Design

[0020] Based on the Rose transcript, design RrGTγ-4 Upstream and downstream primers containing the complete open reading frame of the gene

[0021] Primer F: 5' - TGCAGAGATTAGACTATGACA -3'

[0022] Primer R: 5' - ATACATATTATCCGGTGAGGT -3'

[0023] II. Gene Isolation

[0024] Total RNA was extracted from leaves and roots of wild rose using the FastPure Plant Total RNA Isolation Kit (Vazyme). The specific steps are as follows:

[0025] 1) Spray RNase and nucleic acid remover in the clean bench to be used to eliminate the interference of RNase in subsequent experiments.

[0026] 2) Preheat Buffer PRL to 65°C in a water bath and add 5% β-mercaptoethanol.

[0027] 3) Take about 0.3 g of sample in a mortar, add liquid nitrogen to quickly freeze and grind, add 500 μl of Buffer PRL preheated at 65°C, and immediately vortex vigorously for 60 seconds to fully lyse.

[0028] 4) Incubate the lysis mixture in a 65°C water bath for 5 min, inverting 1-2 times to aid lysis, and then centrifuge at 12,000 rpm for 10 min.

[0029] 5) Pipette the supernatant into a new 1.5 ml RNase-free centrifuge tube, add 0.5 times the volume of supernatant Ethanol absolute, and immediately mix by pipetting.

[0030] 6) Place FastPure gDNA-Fiter Column II into a collection tube and transfer the above mixture into it. Centrifuge at 12,000 rpm for 2 minutes and discard the filtrate.

[0031] 7) Place FastPure gDNA-Fiter Column II into new 2 ml Collection Tubes, add 500 μl Buffer PRLPlus, and centrifuge at 12,000 rpm for 30 seconds.

[0032] 8) Add 0.5 times the volume of supernatant to the filtrate and mix immediately by pipetting.

[0033] 9) Transfer the mixture to FastPure RNA Column IV, centrifuge at 12,000 rpm for 2 minutes, and discard the filtrate.

[0034] 10) Add 500 μl of Buffer PRW2 to FastPure RNA Column IV, centrifuge at 12,000 rpm for 30 seconds, and discard the filtrate.

[0035] 11) Repeat step 10)

[0036] 12) Place the FastPure RNA Column IV back into the collection tube and centrifuge at 12,000 rpm for 2 minutes.

[0037] 13) Transfer FastPure RNA Column IV to a new 1.5ml RNase-free Collection Tube. Add 30 μl of RNase-free ddH2O to the center of the column membrane. Incubate at room temperature for 2 minutes and centrifuge at 12,000 rpm for 1 minute.

[0038] 14) The collected RNA was aliquoted into 1 μg portions and stored at -80°C.

[0039] RNA was reverse transcribed into cDNA using the HiScript III 1st Strand cDNA Synthesis Kit (Vazyme). The specific steps are as follows:

[0040] 1) Denaturation of RNA template: Add RNase-free ddH2O to 8 μl of 5 μg total RNA, incubate at 65°C for 5 min, and place on ice for 2 min.

[0041] 2) Removal of genomic DNA: Add 2 μl of 5×gDNA wiper mix to the product from the previous step, mix thoroughly by pipetting, and incubate at 42°C for 2 min.

[0042] 3) First-strand cDNA acquisition: Add 2 μl 10×RT Mix, 2 μl HiScript III Enzyme Mix, and 1 μl Oligo (dT) to the product from the previous step. 20 VN and 5 μl RNase-free ddH2O were mixed by pipetting. The reaction was performed in three steps: 25°C for 5 min, 37°C for 45 min, and 85°C for 5 sec to obtain cDNA.

[0043] Separation RrGTγ-4 Gene

[0044] 1) PCR amplification RrGTγ-4 Gene: Add 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 to a PCR tube and mix thoroughly by pipetting. Amplify the reaction using the following protocol: 95°C for 3 min; 95°C for 10 sec; 55°C for 5 sec (35 cycles); 72°C for 5 sec; and 72°C for 3 min.

[0045] 2) Purify the PCR product using the FastPure Gel DNA Extraction Mini Kit.

[0046] The pEASY-Blunt Cloning Kit (Full Gold) RrGTγ-4 The gene is cloned into the vector as follows:

[0047] 1) Connect the vector: add 1 μl to 50 ng PCR product pEASY -Blunt Zero Cloning Vector, add RNase-Free ddH2O to 5 μl, and incubate at 25°C for 10 min to obtain the ligation product.

[0048] 2) E. coli Transformation: Thaw freshly prepared or -70°C frozen Trans1-T1 competent cells on ice; add 5 μl of the ligation product to 100 μl of competent cells, mix gently, and incubate on ice for approximately 30 minutes; heat shock in a 42°C water bath for 90 seconds, then quickly place on ice for 3-5 minutes; add 800 μl of LB liquid medium and shake at 37°C at 100 rpm for 1 hour; centrifuge at 4000 rpm for 3 minutes, aspirate the upper 800 μl of medium, and mix the remaining culture; spread the culture onto LB selection solid medium containing Kan and incubate inverted at 37°C overnight.

[0049] 3) Positive clone screening and sequencing analysis: Single colonies were selected from the screening culture plate and inoculated into LB liquid medium, and the culture was shaken at 37°C and 250 rpm for 6 hours. After PCR detection of the culture solution using the 6-hour culture solution as a template, the positive culture solution was sent to Shanghai Sangon Biotechnology Co., Ltd. for sequencing verification to confirm the correct sequence. RrGTγ-4 Gene. Example 2

[0050] Fluorescence quantitative PCR technology RrGTγ-4 Gene spatiotemporal expression patterns:

[0051] based on RrGTγ-4 Fluorescence quantitative PCR upstream primer 5'-TGGTCAGCTATGCCCACCATGAAGCTG -3' and downstream primer 5'-AGCGCTGTCGTCAGCACCAAACC -3' were designed for the exon region of the gene. PhD) designed the internal reference upstream primer 5'-GCCGACGCGCTGGTTTGATT-3' and downstream primer 5'-CCAGTCCTTCTCCGCCATCCC-3'. Real-time fluorescence quantitative PCR was performed using SYBR Premix Ex Taq (Takara) and a fluorescence quantitative PCR instrument CFX96TM (Bio-RAD). Data were processed using Bio-Rad CFX Manager software. 2 -△△ The Ct method was used for calculation. Three technical replicates were performed for each biological replicate, and the average of the three sets of parallel data was taken to reduce the error.

[0052] like Figure 1 As shown, in the rose root system, salt treatment for 0.5 h RrGTγ-4 No response to stress, 2 h after salt treatment RrGTγ-4 The expression level reached an ultra-high level (about 669 times) which was significantly higher than that of the control group (about 19 times). After 4h and 8h of salt treatment, the expression level gradually decreased to 9.8 times, close to the control group. This shows that in the roots of wild roses, RrGTγ-4 could respond strongly to salt stress in the short term (p < 0.01).

[0053] Example 3

[0054] RrGTγ-4 Overexpression vector construction and Arabidopsis genetic transformation

[0055] Using the plant overexpression vector pNC-Cam1304-MCS35S ( Figure 2 ) Build RrGTγ-4 The overexpression vector was constructed as follows:

[0056] 1) Obtain NC splice fusion RrGTγ-4 Gene fragment: designed with NC linker RrGTγ-4 Primers 5'-AGTGGTCTCTGTCCAGTCCTTGCAGAGATTAGACTATGACA-3' and 5'-GGTCTCAGCAGACCACAAGTATACATATTATCCGGTGAGGT-3' were used to prepare the primers obtained in Example 1. RrGTγ-4 The target gene fragment was used as a template for PCR amplification. The PCR amplification and product recovery process were the same as in Example 1.

[0057] 2) Using the Nimble Cloning Kit (NC Biotech), the recombinant vector was obtained: 50 ng of the recovered fragment was mixed with 1 μl of the pNC-Cam1304-MCS35S vector, and RNase-free ddH2O was added to 5 μl. The mixture was incubated at 25°C for 10 min to obtain the recombinant product. E. coli transformation and positive clone screening were performed as in Example 1. The positive bacterial solution was sent to Sangon Biotech (Nanjing) for sequencing to obtain the recombinant overexpression vector pNC-Cam1304-MCS35S. RrGTγ-4 .

[0058] 3) Agrobacterium transformation: Thaw competent Agrobacterium tumefaciens EHA105 culture medium stored at -80°C at room temperature. Add 0.01-1 μg of plasmid DNA per 100 μl competent medium and mix thoroughly by hand by flicking the bottom of the tube. Incubate on ice for 5 minutes, in liquid nitrogen for 5 minutes, in a 37°C water bath for 5 minutes, and in an ice bath for 5 minutes. Add 700 ml of antibiotic-free YEB liquid medium and mix thoroughly. Incubate at 28°C with shaking for 2-3 hours. Harvest the culture by centrifugation at 6000 rpm for one minute. Spread the culture onto a YEB plate containing kanamycin resistance and incubate upside down at 28°C for 2-3 days to allow positive colonies to grow. Example 3

[0059] Arabidopsis thaliana was infected by the inflorescence dip method, and transgenic plants were obtained by screening for hygromycin-resistant transformed seedlings. The main steps and reagents used are as follows:

[0060] I. Reagent and Solution Abbreviations

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

[0062] II. Agrobacterium-mediated genetic transformation steps

[0063] (1) Cultivation of Agrobacterium

[0064] 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 with a shaker at 200 rpm until the bacterial concentration reached OD 600 The value is 1.5-2.0.

[0065] (2) Arabidopsis inflorescence dip method

[0066] Centrifuge the shaken bacterial suspension at 4500 rpm for 10 min, and use 5% sucrose solution (with 20 μl / 100 ml of surfactant Silwet L-7). Soak the Arabidopsis inflorescence in the resuspended solution for about 30 sec, shield from light, keep moist, and place horizontally for 12-24 h.

[0067] (3) Screening of positive seedlings

[0068] Infected Arabidopsis plants were cultured normally in an incubator. Harvested seeds were sterilized in a clean bench by immersing them in 70% alcohol for 15-30 seconds and 2% sodium hypochlorite for 15 minutes. After washing 3-4 times with sterile water, they were plated onto resistant 1 / 2 MS medium (1 / 2 MS + 30 g / L sucrose + 7 g / L agar + 20 mg / L Hydration) for selection. T1 plants that were positive in the selected generation were tested by PCR and transferred to a lighted incubator for harvest. This procedure was repeated until homozygous T3 plants were obtained. Example 4

[0069] RrGTγ-4 Observation of salt-tolerance phenotype in overexpressing Arabidopsis plants

[0070] Will RrGTγ-4 Overexpressing Arabidopsis and wild-type Arabidopsis were seeded on 1 / 2 MS medium containing 150 mM NaCl and grown for two weeks. Figure 3 ) found that the root length of the overexpressing plants was significantly longer than that of the wild type, and the statistical index was significantly different among different transgenic lines (p < 0.1, Figure 4 There was no significant difference in leaf morphology, but the number of leaves in the overexpression plants was significantly higher than that in the wild-type plants (p<0.01, Figure 4 The above evidence shows that overexpression RrGTγ- 4 Promoted salt tolerance in Arabidopsis plants.

[0071] SEQ ID NO.1

[0072]

[0073] SEQ ID NO.2

[0074] MEPNGIGGGLFPEMRSGMLGLEMPLHQTQNPPNPQNPHHHMHQPQMVSYAHQEADHNHHPQAHQSAKHGYPYATKPKQITLSDEDEPGFGADDSAGENKRKISPWQRMKWTDTMVRLLIMAVFYIGDEGGSEGPDPTGKKKSGGGLLQKKGKWKSVSRAMMEKGFYVSPQQCEDKFNDLNKRYKRVNDILGKGTACKVVENQGLLEKMELSSKMKDEVKKLLNSKHLFFREMCAYHNSCGHGTVGAAGASGAHNSSPEDPSEVQLQQQQQQPQQERCFHVSENAHVVANSGRTETEGSKMFKAESGGEDEDEDYEDDGSEDDDDEEEEGVVEGGSRGQIGHGHEDEDENEHERAKRPRKGSCFSGSSHKMLMQQLGSEVSGVIQDVSKSPWEKKQWMKARLIQLEEQQVNYQYQSFELEKQRLKWVKYSSKKERDMETAKLENERRRLENERMLLLVRQKELELLDLHHHQQQQHQQQNSSNKRSDPSSLTG

Claims

1. Rose salt tolerance gene RrGTγ-4 Application in enhancing salt tolerance of Arabidopsis thaliana, characterized in that, The gene sequence of SEQ ID NO.1 was introduced into the plant overexpression vector pNC-Cam1304-MCS35S, and the RrGTγ-4 protein was stably expressed in Arabidopsis cells mediated by Agrobacterium infection to improve salt tolerance.

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