RrJMJ12 gene for regulating and controlling drought resistance of rose and application of RrJMJ12 gene

Abstract

The invention provides an RrJMJ12 gene for regulating and controlling the drought resistance of roses and application of the RrJMJ12 gene, and belongs to the technical field of plant genetic engineering, the nucleotide sequence of the RrJMJ12 gene is shown as SEQ ID NO.1; the amino acid sequence of the RrJMJ12 gene is as shown in SEQ ID NO. 2. The RrJMJ12 gene provided by the invention enriches the gene resources of rose drought-tolerant breeding, has important application value in the field of rose plant drought-tolerant stress-resistant breeding, improves the drought resistance of rose plants by interfering with the RrJMJ12 gene, and is suitable for being applied to rose plant breeding of root-related phenotypes.

Description

Technical Field

[0001] The invention belongs to the technical field of plant genetic engineering, and in particular relates to an RrJMJ12 gene for regulating drought resistance of roses and an application thereof. Background Art

[0002] Rose (Rosa rugosa Thunb.) is a perennial deciduous shrub of the genus Rosa in the Rosaceae family, with rich varieties and pleasant aroma. Rose is rich in flavonoids, volatile oils, phenolic acids, amino acids and other chemical components and nutrients required by the human body. It is widely used in skin care products, food and medicine. It is an important natural spice plant in the world and a traditional Chinese medicine and food flower with high economic and health value. At present, most roses are planted in the open air at home and abroad, and the management is relatively extensive. In recent years, with global warming, the intensification of the greenhouse effect and the shortage of water resources, about 40% of the land in the world belongs to arid or semi-arid areas. Soil drought will directly cause the growth of rose plants to weaken or even die, seriously affecting the yield and quality of fresh roses, and has become a bottleneck problem in the development of modern agricultural industries including rose planting. Therefore, screening genes that regulate drought tolerance in roses and cultivating new varieties of roses that are more drought-tolerant is the fundamental way to deal with drought environments at present.

[0003] The JMJ family is a class of histone demethylases containing the JmjC domain, which are mainly involved in the growth and development processes such as the regulation of flowering time and circadian rhythm in plants, as well as the response of plants to stresses such as drought, temperature, and pathogens. The JMJ family members are divided into five subfamilies based on the differences in protein structure: KDM5 / JARID1, KDM4 / JARID3, KDM3 / J ARID2, JMJD6, and JmjCdomain-only. At present, 21 and 20 histone demethylases containing the Jmj domain have been identified in Arabidopsis and rice, respectively. AtJMJ17 is an H3K4 demethylase in the KDM5B subfamily of the JMJ family. Under normal circumstances, AtJMJ17 binds to the dehydration stress and abscisic acid response gene OST1, removes the H3K4me3 mark at the gene site, and can inhibit its expression. The H3K9 demethylase AtJMJ27, a member of the KDM3 / JHDM2 subfamily, needs to interact with the 26S proteasome subunit RPN1a to negatively regulate the accumulation of AtJMJ27. Under drought conditions, the abundance of RPN1a decreases, promoting the accumulation of JMJ27 and activating the expression of GOLS2 and RD20 in response to drought stress. Studies have also reported that overexpression of the H3K4 demethylase OsJMJ703 in rice makes transgenic rice sensitive to drought stress, while knocking out OsJMJ703 enhances rice tolerance to drought stress.

[0004] There is no research on the regulation of drought tolerance by the rose JMJ family and its members. Exploring the JMJ members that regulate drought tolerance in roses will help us understand the regulatory mechanism of rose drought tolerance, which has guiding significance for the resistance breeding of Rosa plants, provides a basis for breeding new rose varieties that are drought-resistant and have economic value, and promotes the development of the rose industry. Summary of the invention

[0005] At present, most roses are cultivated outdoors. To address the problem of weak growth or even death of rose plants caused by soil drought, screening and regulating rose drought genes and applying them to drought-resistant variety breeding is an important way to obtain drought-resistant roses. In this regard, the present invention provides an RrJMJ12 gene for regulating rose drought resistance and its application.

[0006] The purpose of the present invention is to provide a RrJMJ12 gene for regulating the drought resistance of roses, 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. RrJMJ12 is a histone demethylase gene that responds to drought stress in purple-branched roses.

[0007] Another object of the present invention is to provide an application of the RrJMJ12 gene in regulating the drought resistance of Rosa plants, wherein the application method is to improve the drought resistance of Rosa plants by interfering with the RrJMJ12 gene. The Rosa plants include roses, Chinese roses, etc. The specific steps of improving the drought resistance of Rosa plants by interfering with the RrJMJ12 gene are as follows:

[0008] 1) Construct an interference fragment for interfering with the RrJMJ12 gene. The interference fragment is 394 bp in total and its sequence is shown in SEQ ID NO.3.

[0009] 2) Using the interference fragment and the plant interference vector pFAST-R03, constructing the interference vector RrJMJ12-RNAi of the RrJMJ12 gene;

[0010] 3) introducing the interference vector RrJMJ12-RNAi into K599 Agrobacterium rhizogenes;

[0011] 4) Introducing K599 rhizogenes Agrobacterium into rose plants to obtain interference plants, wherein the K599 rhizogenes Agrobacterium can induce rose plants to produce transgenic hairy roots.

[0012] The method for improving the drought resistance of rose plants by interfering with the RrJMJ12 gene can be used for breeding of rose plants.

[0013] Beneficial effects: The present invention uses purple rose as material, clones the RrJMJ12 gene, detects the expression pattern of the RrJMJ12 gene under drought treatment, and can improve the drought resistance of roses by interfering with the RrJMJ12 gene in roses. The RrJMJ12 gene provided by the present invention enriches the gene resources for rose drought resistance breeding, has important application value in the field of drought resistance and stress resistance breeding of rose plants, and is suitable for application in the breeding of rose plants with root-related phenotypes. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 This is the spatiotemporal expression pattern of the RrJMJ12 gene;

[0015] Figure 2 This is the plasmid map of the plant interference vector pFAST-R03;

[0016] Figure 3 This is the drought tolerance phenotype and physiological index detection diagram of the RrJMJ12 gene interference strain; DETAILED DESCRIPTION

[0017] 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.

[0018] Example 1: RrJMJ12 gene cloning

[0019] In this example, the cDNA of Rosea purpurogena was used as a template to amplify the gene coding region and obtain the nucleotide sequence of the RrJMJ12 gene.

[0020] 1. Primer design

[0021] Design upstream and downstream primers containing the complete open reading frame of the RrJMJ12 gene:

[0022] SEQ ID NO.4, Primer F: 5'-ATGGCAGCTTCGGAGCAGCCCCA-3'

[0023] SEQ ID NO.5, Primer R: 5'-TCGCGATCGCCCCTTACCTTT-3'

[0024] 2. Obtaining the RrJMJ12 gene

[0025] 2.1. Total RNA was extracted from the leaves of Rosella purpurogenum using the FastPure Plant Total RNA Isolation Kit (Vazyme, Nanjing). The specific steps are as follows:

[0026] 1) Preheat Buffer PRL to 65°C in a water bath and add 5% β-mercaptoethanol.

[0027] 2) Take about 0.3 g of rose leaves in a mortar, grind with liquid nitrogen, add 500 μl of Buffer PRL, and vortex vigorously for 60 seconds.

[0028] 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.

[0029] 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.

[0030] 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.

[0031] 6) Transfer the mixture to FastPure RNA Column IV, centrifuge at 12000 rpm for 2 min, and discard the filtrate.

[0032] 7) Add 500 μl Buffer PRW2 to FastPure RNA Column IV, centrifuge at 12,000 rpm for 30 seconds, and discard the filtrate.

[0033] 8) Repeat step 7.

[0034] 9) Place the FastPure RNA Column IV back into the collection tube and centrifuge at 12000 rpm for 2 min.

[0035] 10) Transfer FastPure RNA Column IV to a new RNase-free Collection Tubes 1.5ml centrifuge tube, add 30μl of RNase-free ddH2O to the center of the adsorption column membrane, let it stand at room temperature for 2 minutes, and centrifuge at 12000rpm for 1 minute.

[0036] 11) The collected RNA was packaged into 1 μg portions and stored in a -80°C refrigerator.

[0037] 2.2. Reverse transcribe RNA into cDNA using HiScript III 1st Strand cDNA Synthesis Kit (Vazyme). The specific steps are as follows:

[0038] 1) Denaturation of RNA template: Add RNase-Free ddH2O to 8μl of 5μg Total RNA, incubate at 65℃ for 5min, and place on ice for 2min.

[0039] 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.

[0040] 3) Obtaining the first-strand cDNA: Add 2 μl 10×RT Mix, 2 μl HiScriptⅢE nzymeMix, and 1 μl Oligo(dT) to the product of the previous step 20 VN and 5 μl RNase-Free ddH2O were mixed by pipetting, and 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.

[0041] 2.3. Isolation of RrJMJ12 gene

[0042] 1) Using the upstream and downstream primers designed above, PCR amplify the RrJMJ12 gene: add 25μl PrimeSTARMax Premix (2X), 2μl Primer F and Primer R, 1μl cDNA and 20μl RNase-Free ddH2O to a PCR tube and mix well by pipetting. Perform the amplification reaction according to the program of 95℃, 3min; 95℃, 10sec, 55℃, 5sec (35 cycles); 72℃, 5sec; 72℃, 3min.

[0043] 2) Use FastPure Gel DNA Extraction Mini Kit to obtain purified PCR products.

[0044] 2.4. The RrJMJ12 gene was cloned into the vector using pEASY-Blunt Cloning Kit (Quanshijin, Beijing) as follows:

[0045] 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.

[0046] 2) E. coli transformation: Thaw the -70℃ frozen E. coli competent cells on ice, add 5μl of the ligation product, mix well, incubate on ice for 30min, heat shock at 42℃ for 90sec, incubate on ice for 3min, add 800μl of LB liquid culture medium, resuscitate at 37℃&100rpm for 1h, remove the liquid by centrifugation, mix the remaining bacterial liquid, spread on a plate (LB screening solid culture medium containing Kan), and culture overnight at 37℃.

[0047] 3) Positive clone screening and sequencing analysis: A single colony was selected from the screening culture plate and inoculated into LB liquid culture medium, and the culture was shaken at 37°C and 250 rpm for 6 hours. The bacterial solution was tested by PCR, and the positive bacterial solution was sequenced. The RrJMJ12 gene was successfully isolated and obtained, and its nucleotide sequence is shown in SEQ ID NO.1. The amino acid sequence of the RrJMJ12 gene is shown in SEQ ID NO.2.

[0048] Example 2: Exploring the spatiotemporal expression pattern of the RrJMJ12 gene by fluorescence quantitative PCR technology

[0049] Based on the exon region of the RrJMJ12 gene, the upstream primer (SEQ ID NO.6) 5'-TCGGACTGAGCACATTAGGG-3' and the downstream primer (SEQ ID NO.7) 5'-CTGACACAA ACCGGAAGGTC-3' of fluorescent quantitative PCR were designed. Real-time fluorescent quantitative PCR was performed using SYBR Premix Ex Taq (Takara) and fluorescent quantitative PCR instrument CFX 96TM (Bio-RAD), and Bio-Rad CFX Manager software was used to reduce errors.

[0050] like Figure 1 As shown, different strains of purple-branched rose were irrigated with 20 ml of 20% PEG6000 solution at 0h, 1h, 3h, 6h, 12h, and 24h, respectively. In the rose strain, the expression level of RrJMJ12 in the plant was significantly downregulated within 24h of PEG (polyethylene glycol) treatment, indicating that RrJMJ12 negatively responded to drought stress.

[0051] Example 3: Construction of RrJMJ12 interference vector

[0052] Using the plant interference vector pFAST-R03 (such as Figure 2 The RrJMJ12 interference vector was constructed as shown in the figure. The construction method is as follows:

[0053] 1) Obtain the interference fragment, use the target fragment of the RrJMJ12 gene obtained in Example 1 as a template, design the interference fragment forward primer and reverse primer with a CACC linker: forward primer (SEQ ID NO.8) 5'-CACCGTGAGCCTATCCAGGTTGCAA-3', reverse primer (SEQ ID NO.9) 5'-AGTCTGACAAGTTTGTTTCG-3', perform PCR amplification, and the PCR amplification and product recovery process are the same as in Example 1. The interference fragment is 394 bp in total, and its sequence is shown in SEQ ID NO.3.

[0054] 2) Use pENTR / D-TOPO kit (invitrogen) to obtain entry vector: 1 μl 5ng freshly recovered interference fragment product, 0.5 μl D-TOPO vector, 1 μl Salt solution, 1 μl RNase-Free ddH2O, a total of 3 μl system. Incubate at 25°C for 5 min to obtain the recombinant product. The E. coli transformation and positive clone screening methods are the same as in Example 1. The positive bacterial solution is sent to Sangon Biotech Co., Ltd. (Nanjing) for sequencing to obtain the entry vector D-TOPO:RrJMJ12.

[0055] 3) The interference vector was obtained using the LR Clonase Enzyme Mix kit (invitrogen): 1 μl entry vector D-TOPO:RrJMJ12 plasmid, 1 μl pFAST-R03 vector plasmid, 1 μl LR Enzyme, 2 μl TE Buffer, 1 μl RNase-Free ddH2O, a total of 5 μl system. The recombinant product was obtained by incubation at 25°C for 1 hour. 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 interference vector RrJMJ12-RNAi.

[0056] 4) Agrobacterium transformation: Thaw the competent Agrobacterium K599 culture medium stored at -80°C at room temperature, add 0.01-1 μg of plasmid DNA to every 100 μl competent medium, stir the bottom of the tube by hand to mix, let stand 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 YEB liquid medium without antibiotics, culture at 28°C with shaking for 2-3 hours, collect the bacteria by centrifugation at 6000 rpm for one minute, spread on YEB plates containing spectinomycin and rifampicin resistance, and culture inverted at 28°C incubator for 2-3 days to grow positive colonies.

[0057] Example 4: Cut-dip-budding (CBD) method to induce hairy roots for homologous transformation of purple rose and

[0058] The non-lignified side branches of Rose rose were cut into 4-5 cm cuttings, the bottom was dipped in positive K599 Agrobacterium colonies, and colonized in vermiculite. After culturing for 3 weeks at 25°C under long-day conditions (light: dark = 16h:8h), a large number of transgenic hairy roots were obtained, and the interference efficiency was verified by fluorescence quantification.

[0059] Example 5: RrJMJ12 interferes with the identification of drought tolerance of purple rose

[0060] According to the method of Example 4, three RrJMJ12 interference plants were obtained, and were numbered RrJMJ12-RNAi-1, RrJMJ12-RNAi-2, and RrJMJ12-RNAi-3 respectively; after the pFAST-R03 empty vector was transferred into K599 Agrobacterium, the control plant Empty-RNAi was obtained according to the method of Example 4.

[0061] After 15 days of natural drought treatment, the results are as follows Figure 3 As shown in the figure, compared with the control group, the chlorophyll fluorescence of the RrJMJ12-interferenced plants had a higher actual photosynthetic rate value. The physiological indicators of the roots under drought stress were measured and it was found that the contents of SOD (superoxide dismutase) and CAT (catalase) in the RrJMJ12-interferenced roots were significantly higher than those in the control group, indicating that the interference strains had a stronger ability to resist osmotic stress; the H2O2 and O2 - The content was lower than that in the control group, indicating that the cell membrane of the interference strain was less damaged.

[0062] Figure 3 The four rows of plants in (a) are represented from top to bottom; the first row: plants without drought treatment; the second row: plants after 15 days of natural drought treatment; the third row: chlorophyll fluorescence images of plants without drought treatment; the fourth row: chlorophyll fluorescence images of plants after 15 days of natural drought treatment. Figure 3 (b) shows the interference efficiency of RrJMJ12 in three different RrJMJ12-RNAi lines compared with the control plants. 3) Figure 3 (c) shows the chlorophyll fluorescence parameter Y(II), i.e. the actual photosynthetic efficiency content change of PSII in the control plants and three RrJMJ12-RNAi plants before and after drought treatment. 4) Figure 3 (d)-(g) show the H2O2 and O2 in the roots of each plant after 15 days of natural drought treatment. - Content of SOD and CAT.

[0063] 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 may be made to the form and details without departing from the spirit and scope of the present invention.

Claims

1. A RrJMJ12 gene for regulating drought resistance of rose, characterized in that: The nucleotide sequence of the RrJMJ12 gene is shown in SEQ ID NO.1, and the amino acid sequence of the protein expressed by the RrJMJ12 gene is shown in SEQ ID NO.

2.

2. Use of the RrJMJ12 gene according to claim 1 in regulating drought resistance of Rosa plants.

3. The use according to claim 2, characterized in that: Improving drought resistance of Rosa plants by interfering with the RrJMJ12 gene.

4. The use according to claim 3, characterized in that: The specific steps of improving the drought resistance of rose plants by interfering with the RrJMJ12 gene are as follows: 1) constructing an interference fragment for interfering with the RrJMJ12 gene, the interference fragment is 394 bp in total, and its sequence is shown in SEQ ID NO.3; 2) Using the interference fragment and the plant interference vector pFAST-R03, constructing the interference vector RrJMJ12-RNAi of the RrJMJ12 gene; 3) Introducing the interference vector RrJMJ12-RNAi into K599 Agrobacterium rhizogenes; 4) Introducing K599 rhizogenes Agrobacterium into Rosa plants to obtain interference plants.

5. The use according to claim 2, characterized in that: The genus Rosa includes roses and Chinese roses.

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