Rose salt sensitive site gene RrSPL9 and application thereof

Abstract

The invention discloses a rose salt sensitive site gene RrSPL9 and application thereof, and belongs to the technical field of plant genetic engineering, the nucleotide sequence of the gene RrSPL9 is as shown in SEQ ID NO.1, and the protein amino acid sequence of the gene RrSPL9 is as shown in SEQ ID NO.2. The RrSPL9 reduces the salt tolerance of transgenic roses and arabidopsis thaliana, the miRNA resistance target RrSPL9mut transgenic phenotype is more prominent, which indicates that the RrSPL9 gene is a key salt-sensitive site, the RrSPL9 gene is expected to greatly enhance the salt tolerance of roses after being knocked out or silenced, and the RrSPL9 gene has important application value in the field of salt-tolerant stress-resistant breeding.

Description

Technical Field

[0001] The invention belongs to the technical field of plant genetic engineering, and in particular is a rose salt-tolerant gene RrSPL9 and an application thereof. Background Art

[0002] Wild rose (Rosarugosa Thunb.) has a strong salt tolerance and is a precious parent material for breeding salt-tolerant varieties of the genus Rosa. Cultivated roses have excellent ornamental and quality traits but weak salt tolerance. However, my country has abundant saline soil resources that cannot be fully utilized, which limits the rose planting industry.

[0003] SPL transcription factors are plant-specific transcription factors that contain a highly conserved DNA binding domain, namely the SBP domain, which is a zinc finger structure. AmSBP1 and AmSBP2 were first found in snapdragon to bind to the promoter element of SQUAMOSA to regulate flower development. All SPL transcription factors have a nuclear localization signal sequence at the C-terminus of the SBP domain, which partially overlaps with the second zinc ion binding site, allowing the SPL protein to enter the nucleus for expression. Studies have shown that the plant miR156-SPL module is involved in the control of plant phase transitions, organ development, and morphological construction. In Arabidopsis, miR156 is significantly induced by salt stress. Overexpression of miR156 improves the tolerance of plants to salt and drought while delaying flowering time. Overexpression of miR156 resistance SPL targets results in the opposite phenotype, which is sensitive to salt and drought and blooms early. Rice overexpressing miR156 or resistance SPL targets has a phenotype similar to Arabidopsis. Further studies have found that SPL9 affects anthocyanin metabolism by regulating dihydroflavonol reductase DFR. Anthocyanin accumulation improves plant resistance to stress. The miR156-SPL9-DFR pathway dynamically links adversity and developmental regulation. A similar situation occurs under heat stress. Heat stress induces miR156. SPL downregulation improves plant adaptability but slows growth. Under low temperature conditions, plants overexpressing miR156 inhibit SPL3 translation more efficiently, resulting in the inability of the FT promoter to initiate transcription, which significantly delays flowering. miR156-SPL9-FT allows plants to balance environmental temperature and developmental processes. It can be seen that miR156-SPL links abiotic stress with plant growth and development, allowing plants to flexibly control stress responses and growth and development.

[0004] At present, the regulation of salt tolerance by the rose SPL gene family and its members has not been reported. Cloning and developing SPL members that regulate rose salt tolerance will have guiding significance for the resistance breeding of Rosa plants, and can provide a basis for breeding new rose varieties with high salt tolerance and economic value, thus promoting the development of the rose industry. Summary of the invention

[0005] In order to solve the problem that the regulation of rose SPL gene family and its members on salt tolerance in the prior art is still unclear, the present invention proposes a rose salt tolerance gene RrSPL9 and application thereof.

[0006] In a first aspect, the present invention provides a rose salt sensitive site gene RrSPL9, whose nucleotide sequence is shown in SEQ ID No.1.

[0007] In a second aspect, the present invention provides an expression protein of the gene RrSPL9, whose amino acid sequence is shown in SEQ ID No.2.

[0008] In a second aspect, the present invention provides an application of the gene RrSPL9, and breeds salt-tolerant rose varieties by knocking out the salt-sensitive site gene RrSPL9 in roses.

[0009] The salt-sensitive locus gene RrSPL9 in rose was knocked out by constructing a double-targeting vector. The double-targeting vector construction method is as follows:

[0010] The two gRNA sequences and gRNA backbone sequence shown in SEQ ID NO.6 and SEQ ID NO.7 were directly synthesized into a double targeting sequence, gRNA1-backbone sequence-gRNA2, and recombined through the NC cloning frame of the pNC-HSE401 plasmid to obtain the pNC-HSE401:RrSPL9 double targeting vector.

[0011] In a third aspect, the present invention discloses a method for obtaining the RrSPL9 gene:

[0012] Total RNA was extracted from the roots of wild roses, and mRNA was separated. The mRNA was reverse transcribed into cDNA using reverse transcriptase, and PCR amplification primers as shown in SEQ ID No. 3 and SEQ ID No. 4 were used to obtain the RrSPL9 gene.

[0013] In a fourth aspect, the present invention discloses a recombinant overexpression vector containing gene RrSPL9, and the construction method of the recombinant overexpression vector is:

[0014] The RrSPL9 gene fragment was amplified by PCR to add the NC cloning frame to obtain the overexpression frame of 35S promoter-RrSPL9-NOS terminator, which was then recombined with the plant overexpression plasmid Cam1304-MCS35S to obtain the recombinant overexpression vector Cam1304-MCS35S:RrSPL9.

[0015] In a fifth aspect, the present invention discloses a recombinant overexpression vector constructed based on the gene RrSPL9, and the construction steps are as follows:

[0016] 1) Obtaining the miRNA-resistant target RrSPL9 mut , the steps are as follows:

[0017] Design RrSPL9 mutation primers as shown in SEQ ID NO.5;

[0018] Using the RrSPL9 mutant primer as shown in SEQ ID NO.5 and the reverse primer as shown in SEQ ID No.4, a round of PCR amplification reaction was performed on RrSPL9;

[0019] The first round PCR product was used as a large primer, and the forward primer shown in SEQ ID No. 3 was used as a paired primer to perform a second round of PCR amplification reaction to obtain the miR156 resistance target RrSPL9 mut , whose nucleotide sequence is shown in SEQ ID NO.6;

[0020] 2) Resistance to miRNA target RrSPL9 mut Add the NC cloning frame through PCR amplification reaction to obtain a product with an NC linker;

[0021] 3) The product with the NC linker was recombined with the plant overexpression plasmid Cam1304-MCS35S to obtain the recombinant plant overexpression vector Cam1304-MCS35S: RrSPL9 mut .

[0022] In a sixth aspect, the present invention discloses the use of the expression protein and recombinant overexpression vector of the above-mentioned gene RrSPL9 in plant salt tolerance or rose resistance breeding.

[0023] Beneficial effects: Compared with the prior art, the present invention uses wild rose as material, clones the RrSPL9 gene, and after transferring it into Arabidopsis and rose, it significantly inhibits the salt tolerance of transgenic plants, indicating that it is a salt-sensitive site. A gene knockout vector is further constructed, and the salt tolerance of rose is improved by knocking out the RrSPL9 salt-sensitive site, which has important application value in salt tolerance. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 is the plasmid map of the plant overexpression vector Cam1304-MCS35S;

[0025] Figure 2 is the subcellular localization map of RrSPL9;

[0026] Figure 3 is the salt-sensitive phenotype of RrSPL9 transgenic Arabidopsis seedlings;

[0027] Figure 4is the salt-sensitive phenotype of RrSPL9 transgenic Arabidopsis plants;

[0028] Figure 5 It is the salt-sensitive phenotype of RrSPL9-overexpressing ‘purple branch’ rose;

[0029] Figure 6 This is the plasmid map of the gene editing vector pNC-HSE401. DETAILED DESCRIPTION

[0030] 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.

[0031] Example 1

[0032] Clone the wild rose RrSPL9 gene, design and synthesize cloning primers based on the coding region:

[0033] Primer F(SEQ ID No.3):5'-ATGGGCTCGACCTCTATGAC-3'

[0034] Primer R (SEQ ID No. 4): 5'-TTAAAGAGACCAATGCATGTG-3';

[0035] Total RNA was extracted from the roots of wild roses, and reverse transcriptase was used to reverse transcribe the mRNA into cDNA. The RrSPL9 gene was isolated by amplifying the gene coding region based on the above cloning primers. The specific isolation steps are as follows:

[0036] 1) PCR amplification: Add 25 μL PrimeSTAR Max Premix (2X), 2 μL Primer F and Primer R, 1 μL wild rose cDNA and 20 μL RNase-Free ddH2O to a PCR tube and mix well. 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.

[0037] 2) Product purification: Use FastPure Gel DNA Extraction Mini Kit to obtain purified PCR products.

[0038] 3) Connecting vector: Add 1 μL pEASY-Blunt Zero Cloning Vector to 50 ng of purified product, add RNase-Free ddH2O to 5 μL, and incubate at 25°C for 10 min to obtain the connecting product.

[0039] 4) E. coli transformation: thaw 100 μL Trans1-T1 competent cells on ice, add 5 μL ligation product, mix gently, ice bath for 20 min, 42°C water bath for 90 sec, ice bath for 3 min, add 800 μL LB liquid culture medium, shake at 37°C & 100 rpm for 1 h, centrifuge at 4000 rpm for 3 min, aspirate the supernatant, mix the remaining bacterial liquid, smear on LB screening solid culture medium containing kanamycin, and culture upside down at 37°C overnight.

[0040] 5) Positive identification and sequencing: A single colony was selected and inoculated into LB liquid culture medium, and the culture was shaken at 37°C and 250 rpm for 6 hours. The culture solution after 6 hours of culture was used as a template for PCR detection, and the culture solution with positive bands was selected and sent to Shanghai Sangon Biotechnology Co., Ltd. for sequencing. The correct sequence was verified and the RrSPL9 gene was successfully isolated.

[0041] The nucleotide sequence of RrSPL9 is shown in SEQ ID No.1, and the amino acid sequence of the expressed protein of RrSPL9 is shown in SEQ ID No.2.

[0042] SEQ ID NO.1:

[0043]

[0044] SEQ ID NO.2

[0045] MGSTSMTTTDSGGSSSSPPNNSSTEESLNGLKFGRKIYFEDAALGPNPPKSGSGSAGSSSSGATPPKKQRSGGGGGLVHQGQPPRCQVEGCNVDLSGVKAYYSRHKVCAMHSKTPTVIVAGLEQRFCQQCSRFHQLPEFDQGKRSCRRRLAGHNERRRKPQPGSMMSTRFGRLSSSIYENTSRVGSFLMDF TAYPRAGRDEWPTARTSERPPGNQTVTATGKFLQHPWQSNPENTRSDLYLQGSAGGTSYPGPGIPPGECVTGVTDSSCALSLLSNQPWDSRNRGVGTNAFINTQGAPVAQSANVGTINPFPTTSWGFKGNEAGSSSHQMLPDLGLGQISQPHNSHYSSELELSQQGRRQQNMELGNSRGYDSSSQHMHWSL

[0046] Example 2

[0047] The ORF of RrSPL9 contained a conserved miR156 response element. Multi-point mutation was performed using megaprimer PCR to obtain the miRNA-resistant target RrSPL9. mut , to prevent the expression of RrSPL9 from being cleaved by miRNA, the specific steps are as follows:

[0048] Mutation primer design: Based on the codon degeneracy, synonymous mutation primers were designed to introduce as many mutation sites as possible, and at least 10 nt of complementary bases were retained on the flanks. The RrSPL9 mutation primer (SEQ ID NO.5) is:

[0049] 5'-GTCACTGACTCGAGCTGCGCATTGAG CTTATTAAGCAACCAACCA TGGGACTCT-3'.

[0050] 2) First round of PCR: Amplify the first round of PCR product fragments using RrSPL9 mutant primer (SEQ ID NO.5) and the RrSPL9 coding region reverse primer Primer R (SEQ ID NO.4) of Example 1, using a high-fidelity PCR reaction system, including 10×LA PCR Buffer 5.0 μL; 2.5 mM dNTP Mixture 8.0 μL; 25 mM Mg 2+5.0 μL; LA Taq DNA Polymerase (5U / μL) 0.5 μL; RrSPL9 mutant primer and Primer R primer (10 μM) 2 μL each; template 1 μL, amplification procedure is the same as Example 1.

[0051] 3) Second round of PCR: The first round of PCR product was used as a large primer, and the RrSPL9 ORF forward primer Primer F in Example 1 was used as a paired primer to perform a second round of amplification (the same steps as above), introduce multiple site mutations, purify the product, transform Escherichia coli, and then clone and sequence (the same steps as in Example 1) to obtain the miR156 resistance target RrSPL9 mut .

[0052] miR156 resistance target RrSPL9 mut The nucleotide sequence is shown in SEQ ID NO.6, the underline indicates the mutated miRNA action site, and the lowercase indicates the mutated base.

[0053] SEQ ID NO.6:

[0054] ATGGGCTCGACCTCTATGACCACTACTGACTCAGGAGGCAGCTCCTCCTCCCCACCCAACAACTCCTCCACCGAGGAGTCCCTCAACGGCCTCAAATTCGGCCGCAAAATCTACTTTGAGGACGCTGCTCTCGGACCCAACCCACCCAAATCCGGGTCAGGGTCTGCCGGCTCTTCCTCCTCCGGGGCCACGCCGCCCAAGAAGCAGAGGAGTGGTGGCGGTGGTGGGTTGGTCCATCAGGGCCAGCCTCCGAGGTGCCAGGTGGAGGGCTGCAACGTAGATCTGAGTGGTGTTAAAGCTTACTATTCCAGGCACAAAGTGTGTGCCATGCACTCCAAGACCCCTACTGTTATTGTTGCCGGCCTTGAACAGCGGTTTTGCCAGCAGTGTAGCAGATTTCATCAACTTCCTGAATTTGACCAAGGGAAACGTAGTTGCCGTAGACGTCTGGCTGGTCATAATGAGCGTAGGAGAAAGCCACAACCTGGATCCATGATGTCTACACGTTTTGGCCGTCTCTCTTCGTCTATTTATGAAAACACCAGCCGAGTTGGAAGCTTTCTGATGGACTTCACTGCATACCCAAGGGCTGGGAGAGATGAATGGCCAACTGCAAGAACTTCTGAGCGGCCACCTGGAAATCAAACTGTCACTGCCACCGGAAAGTTTCTTCAACATCCATGGCAGAGCAACCCTGAGAATACTCGATCTGATCTTTACCTGCAAGGGTCAGCCGGTGGTACCAGTTATCCTGGTCCTGGAATTCCTCCGGGAGAATGCGTCACAGGAGTCACTGACTCGAGC TGcGCatTgagctTatTaagcAATCAACCATGGGACTCTAGAAACCGCGGTGTTGGGACAAATGCCTTCATAAACACCCAAGGGGCACCCGTGGCCCAATCAGCTAATGTTGGCACCATCAATCCATTTTCCAACCACATCATGGGGTTTTCAAGGGAAATGAGGCTGGTAGCAGTTCGCATCAG ATGCTTCCTGATCTGGGTCTTGGTCAAATTTCGCAGCCTCATAACAGTCATTACTCTAGTGAGCTCGAGTTGTCTCAACAGGGTAGGAGGCAGCAAAATATGGAACTTGGAAACTCCAGGGGCTACGACTCCTCCAGTCAACACATGCATTGGTCTCTTTAA

[0055] Example 3

[0056] Using the plant overexpression plasmid Cam1304-MCS35S ( Figure 1 ) and the subcellular localization plasmid p35s-GF P to construct the required transgenic vector and subcellular localization vector, and the construction steps are as follows:

[0057] 1) Introduction of linker: RrSPL9 and RrSPL9 obtained in Examples 1 and 2 mut The fragment was purified by PCR to add an NC connector, and the product with the connector was recovered in the same manner as in Example 1.

[0058] 2) Recombinant vector: 50 ng of the recovered product, 1 μL of Cam1304-MCS35S vector, and 1 μL of Nimble Cloning recombinase were pipetted and mixed, and RNase-Free ddH2O was added to make up to 5 μL, and incubated at 25°C for 10 min to obtain the recombinant product. The E. coli transformation and cloning sequencing methods were the same as in Example 1 to obtain the recombinant overexpression vectors Cam1304-MCS35S:RrSPL9 and Cam1304-MCS35S:RrSPL9 mut The same method was used to construct the recombinant subcellular localization vector p35s-GFP:RrSPL9.

[0059] 3) Agrobacterium transformation: 100 μL of frozen EHA105 Agrobacterium competent cells were thawed at room temperature, 100 ng of recombinant vector was added, and the cells were gently mixed. The cells were sequentially placed in an ice bath 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. 700 μL of YEB liquid culture medium was added, and the cells were cultured at 28°C with shaking for 2-3 hours. The bacterial solution was collected by centrifugation at 6000 rpm for one minute, and spread on a YEB plate containing kanamycin. The cells were cultured at 28°C for 2 days, and positive colonies were identified by PCR. The identification steps were the same as those in Example 1.

[0060] The vector is assembled with an NC cloning frame, which can quickly assemble genes in a one-step method to obtain an overexpression frame of 35S promoter-RrSPL9-NOS terminator, thereby ensuring that the RrSPL9 gene is stably and continuously expressed in large quantities in host cells.

[0061] Example 4

[0062] The steps for observing the subcellular localization of RrSPL9 are as follows:

[0063] 1) Obtaining infection solution: Place the positive colony in 4 mL LB resistance medium (containing kanamycin and rifampicin), culture overnight at 200 rpm and 28°C, transfer to a new 50 mL LB resistance medium, shake at 200 rpm / min and 28°C until the OD 600 The cells were collected by centrifugation at 5000 rpm for 10 min and resuspended in an appropriate amount of resuspension solution (10 mM magnesium chloride, 10 mM sodium fatty acid methyl ester sulfonate, 200 μL acetosyringone, pH = 5.7) until the OD 600 is 1.0, and the infection solution is obtained.

[0064] 2) After standing for 2 hours in the dark at 28°C, the infection solution was injected into the back of 4-week-old Nicotiana benthamiana leaves using a syringe without a needle. After 48 hours of long-day culture at 22°C, the fluorescence of the infected leaves was observed using a laser confocal microscope and photographed ( Figure 2 ), and found that its fluorescence signal was located in the cell nucleus, indicating that RrSPL9 was localized in the cell nucleus, which was consistent with the characteristics of transcription factors.

[0065] Example 5

[0066] Transgenic plants were obtained by infecting Arabidopsis thaliana with the inflorescence dip method, and their salt stress phenotypes were observed. The main steps are as follows:

[0067] 1) Obtaining infection solution: Pick positive colonies (positive colonies after Agrobacterium transformation in Example 3) and inoculate them into liquid LB medium (10 g / L peptone + 5 g / L yeast extract + 10 g / L NaCl + 50 mg / L kanamycin), and culture them overnight at 28°C and 200 rpm in a shaking incubator until the bacterial solution concentration OD 600When the value is 1.5-2.0, the bacteria are collected by centrifugation at 4500 rpm for 10 min and resuspended in 5% sucrose solution (containing 20 μL / 100 ml of surfactant Silwet L-7) to obtain the infection solution.

[0068] 2) Inflorescence dip: Soak the Arabidopsis inflorescence in the infection solution for about 30 seconds, shade it from light and keep it moist, and place it horizontally for 12-24 hours. Transfer it to an incubator for normal culture, and harvest the seeds after about 1 month.

[0069] 3) Transgenic Arabidopsis screening: On a clean bench, the seeds were disinfected with 70% alcohol for 15 seconds, 2% sodium hypochlorite for 15 minutes, washed 3-4 times with sterile water, and then spread on 1 / 2MS solid medium containing hygromycin (containing 30 g / L sucrose) for screening. The plants that germinated and grew normally were T1 generation positive plants. After PCR detection, each positive line was numbered and grown and harvested. The screening and seed collection were repeated for 2 rounds to obtain T3 generation lines, which were homozygous transgenic Arabidopsis.

[0070] 4) Seedling stage phenotype: The obtained RrSPL9 and RrSPL9 mut Transgenic Arabidopsis and wild-type Arabidopsis were sown in ordinary 1 / 2MS medium and 1 / 2MS medium containing 120mM and 150mM NaCl, respectively. Phenotypic observation was performed after 14 days of growth ( Figure 3 In A, WT represents wild-type Arabidopsis, and OE refers to transgenic Arabidopsis) we found that RrSPL9 and RrSPL9 mut The overexpression plants had no significant difference in growth status compared with the wild type on normal culture medium, but showed slight yellowing and obvious wilting and yellowing on 120mM salt culture medium. The root length of the transgenic plants was 1.4 and 1.03 times shorter than that of the wild type ( Figure 3 The chlorophyll fluorescence of transgenic plants RrSPL9 and RrSPL9 showed lower actual photosynthetic rate values, which were 1.02 and 1.03 times lower than those of the control group (wild-type Arabidopsis). mut Wilting and necrosis occurred, and the root length was 2.8 and 1.5 times shorter than that of the wild type.

[0071] 5) Plant phenotype: Arabidopsis plants grown for 30 days were watered with 200 mM NaCl (200 ml every 2 days) and the phenotype was observed after 14 days ( Figure 4 ), and found that under water irrigation conditions, the transgenic plants and wild-type plants grew well with no significant difference. Under saline irrigation conditions, the salt damage phenotype of wilting and necrosis of the overexpressing plants was significantly stronger than that of the wild-type ( Figure 4 Middle A), photosynthetic determination of aboveground parts ( Figure 4 RrSPL9 and RrSPL9 were also found in B and Cmut The actual photosynthetic rates of the overexpression plants were reduced by 1.34 and 1.98 times compared with those of the wild-type plants.

[0072] In summary, overexpression of RrSPL9 reduced the salt tolerance of Arabidopsis thaliana, and RrSPL9 is a negative regulator of salt tolerance.

[0073] Example 6

[0074] Homologous transformation was performed by the Cut-dip-budding (CBD) method and the phenotype was observed. The steps are as follows:

[0075] 1) Colony acquisition: RrSPL9 mut The overexpression vector was transformed into K599 Agrobacterium competent cells to obtain transformed positive colonies, and the specific steps were the same as those in Example 3.

[0076] 2) CBD method for rose transformation: The non-lignified side branches of the 'Purple Branch' rose were cut into 4-5 cm cuttings, and the bottom was dipped in a positive K599 Agrobacterium colony, which was then colonized in vermiculite and cultured at 25°C with 14 hours of light for 4 weeks to obtain a large number of adventitious roots, mainly transgenic hairy roots (identified by GFP tag observation).

[0077] 3) Phenotypic observation: The transgenic roses and control plants were irrigated with 120 mM NaCl (300 ml per day) for 7 days and compared with the control group. Figure 5 As shown, the growth status of the two is the same under the condition of no salt water irrigation, but under the condition of salt water irrigation, RrSPL9 mut Overexpression roses showed obvious wilting and yellowing, and their chlorophyll photosynthetic rate value was significantly lower than that of the control group.

[0078] The above results indicate that RrSPL9 is a salt-sensitive site. mut It is not subject to post-transcriptional inhibition by miRNA, and its overexpression has a particularly pronounced salt-sensitive phenotype.

[0079] Example 7

[0080] The steps for constructing the gene editing vector and testing its efficiency are as follows:

[0081] 1) Construction of double-targeting vector:

[0082] According to the gRNA principle, the salt-sensitive site RrSPL9 was searched and the best two gRNA sequences (gRNA A1 and gRNA2), direct and backbone sequences ( Figure 5 , gRNA scaffold) to synthesize a double targeting sequence, the sequence order is: gRNA1-gRNAscaffold-gRNA2. Figure 5 ) were recombined with the NC cloning frame to obtain the pNC-HSE401:RrSPL9 double targeting vector.

[0083] gRNA1 (SEQ ID NO.7): 5-GGCCGTTGAGGGACTCCTCGGTGG-3

[0084] gRNA2 (SEQ ID NO.8): 5-GGAGTGGTGGCGGTGGTGGG-3

[0085] The skeleton sequence is a commonly used gRNA scaffold for plants (SEQ ID NO.9): 5-GTTTTAGAGC TAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTG GCACCGAGTCGGTGC-3

[0086] 2) Knockout efficiency test: Rose protoplasts were separated by enzymatic hydrolysis and resuspended to 10 5 / mL cell density, 5μg of the targeting vector was transformed into 100mL of protoplasts by the PEG polyethylene glycol method, and after 36h, the protoplast DNA was extracted by a plant DNA extraction kit. Using DNA as a template, the RrSPL9 site was amplified, and cloned and sequenced to detect whether it was edited, and the steps were the same as in Example 1. The results showed that the editing efficiency of the double targeting vector exceeded 50%, and 20 of the 30 random clones were frameshift mutations caused by deletions, achieving RrSPL9 site knockout, indicating that pNC-HSE401: RrSPL9 has a high knockout efficiency and is suitable for subsequent transgenic knockout plants.

[0087] The present invention can quickly assemble two gRNAs in one step to obtain a RrSPL9 multi-target targeting vector, thereby ensuring efficient knockout of the RrSPL9 gene in the rose genome.

[0088] The double-targeting vector was transformed into rose cuttings by the CBD method (the specific steps are the same as in Example 6) to create rose hairy roots with the salt-sensitive site RrSPL9 gene knocked out. Rose plants carrying RrSPL9 knocked-out hairy roots are expected to significantly improve salt tolerance and be applied to planting in saline-alkali tidal flats, which has application value in plant salt tolerance breeding.

[0089] The above description is only illustrative rather than restrictive of the present invention. Those skilled in the art will understand that many modifications, changes or equivalents may be made without departing from the spirit and scope defined by the appended claims, but all will fall within the scope of protection of the present invention.

Claims

1. A rose salt sensitive locus gene RrSPL9, characterized in that: Its nucleotide sequence is shown in SEQ ID No.

1.

2. The expression protein of the gene RrSPL9 according to claim 1, characterized in that: Its amino acid sequence is shown in SEQ ID No.

2.

3. The use of the gene RrSPL9 according to claim 1, characterized in that: Breed salt-tolerant rose varieties by knocking out the salt-sensitive locus gene RrSPL9 in roses.

4. The use according to claim 3, characterized in that: The salt-sensitive locus gene RrSPL9 in rose was knocked out by constructing a double-targeting vector. The double-targeting vector construction method is as follows: The two gRNA sequences and gRNA backbone sequence shown in SEQ ID NO.6 and SEQ ID NO.7 were directly synthesized into a double targeting sequence, gRNA1-backbone sequence-gRNA2, and recombined through the NC cloning frame of the pNC-HSE401 plasmid to obtain the pNC-HSE401:RrSPL9 double targeting vector.

5. The gene RrSPL9 according to claim 1, characterized in that The method for obtaining the RrSPL9 gene is: Total RNA was extracted from the roots of wild roses, and mRNA was separated. The mRNA was reverse transcribed into cDNA using reverse transcriptase, and PCR amplification primers as shown in SEQ ID No. 3 and SEQ ID No. 4 were used to obtain the RrSPL9 gene.

6. A recombinant overexpression vector containing the gene RrSPL9 according to claim 1, characterized in that: The steps for constructing the recombinant overexpression vector are as follows: The RrSPL9 gene fragment was amplified by PCR to add the NC cloning frame to obtain the overexpression frame of 35S promoter-RrSPL9-NOS terminator, which was then recombined with the plant overexpression plasmid Cam1304-MCS35S to obtain the recombinant overexpression vector Cam1304-MCS35S:RrSPL9.

7. A recombinant overexpression vector constructed based on the gene RrSPL9 according to claim 1, characterized in that: The steps for constructing the recombinant overexpression vector are as follows: 1) Obtaining the miRNA-resistant target RrSPL9 mut , the steps are as follows: Design RrSPL9 mutation primers as shown in SEQ ID NO.5; Using the RrSPL9 mutant primer as shown in SEQ ID NO.5 and the reverse primer as shown in SEQ ID No.4, a round of PCR amplification reaction was performed on RrSPL9; The first round PCR product was used as a large primer, and the forward primer shown in SEQ ID No. 3 was used as a paired primer to perform a second round of PCR amplification reaction to obtain the miR156 resistance target RrSPL9 mut , whose nucleotide sequence is shown in SEQ ID NO.6; 2) Resistance to miRNA target RrSPL9 mut Add the NC cloning frame through PCR amplification reaction to obtain a product with an NC linker; 3) The product with NC linker was recombined with plant overexpression plasmid Cam1304-MCS35S to obtain recombinant overexpression vector Cam1304-MCS35S: RrSPL9 mut .

8. Use of the expression protein according to claim 2, the recombinant overexpression vector according to claim 6, or the recombinant overexpression vector according to claim 7 in plant salt tolerance or rose resistance breeding.

Images