Application of FvJAZ8 gene in regulating the occurrence of strawberry runners

Abstract

The present invention belongs to the technical field of molecular biology and genetic engineering, and specifically relates to the application of FvJAZ8 gene in regulating the occurrence of strawberry runners. The coding region sequence of the FvJAZ8 gene is shown in SEQ ID NO.1. The present invention also provides a recombinant plant expression vector for silencing the FvJAZ8 gene. The recombinant plant expression vector is introduced into the diploid forest strawberry Hawaii 4, which can significantly reduce the number of its runners, indicating that FvJAZ8 can effectively regulate the occurrence of strawberry runners, and the results can provide a basis for studying the occurrence law and molecular regulation mechanism of strawberry runners.

Description

Technical Field

[0001] The invention belongs to the technical fields of molecular biology and genetic engineering, and particularly relates to application of FvJAZ8 gene in regulating the occurrence of strawberry runners. Background Art

[0002] Strawberry is a perennial herb. Its fruit is sweet, bright in color, unique in flavor and rich in nutritional value, and is well-loved by consumers. The runner is the main reproductive organ of strawberry. In production, the main method of vegetative propagation of runners is to produce seedlings. Runner propagation can better maintain the characteristics of the variety. Therefore, the reproductive ability of runners is crucial to the production of strawberries.

[0003] Gibberellic acid (GA) is a commonly used plant growth regulator. Studies have shown that under long-day conditions, exogenous application of gibberellin (GA) can promote the growth of strawberry runners and the elongation of petioles, while inhibiting flowering. Tenreira et al. found that the forest strawberry Yellow Wonder cannot form runners due to the functional loss of the GA synthase gene FveGA20ox4. GA promotes plant growth by inhibiting the activity of DELLA proteins. DELLA proteins are the core factors in the GA signaling pathway that inhibit GA-mediated plant growth and development. Through forward genetics studies, researchers conducted gene localization studies on strawberry mutants and wild types that can continuously produce runners, and through gene function verification, they found that a DELLA protein encoding gene RGA1 has an inhibitory effect on the formation of strawberry runners (Li et al., 2021). Mouhu et al. discovered the gene FveSOC1 that promotes GA synthesis and indirectly increases the number of strawberry runners. In addition, some researchers have found that the number of branches and runners of the forest strawberry lam mutant is significantly reduced. Studies have shown that the LAM gene can promote the formation of strawberry runners (Feng et al., 2021).

[0004] The Jasmonate ZIM-domain (JAZ) transcription factor has a highly conserved TIFY(F / Y)XG motif and belongs to the TIFY gene family. JAZ is a key negative regulator in the jasmonic acid signaling pathway and also affects other signaling pathways and growth and metabolic processes in plants. However, there are few reports on JAZ regulating the occurrence of strawberry runners. Summary of the invention

[0005] The applicant's research team found in the previous study that the forest strawberry 'Ruegen', which does not produce runners, can induce the occurrence of runners by exogenous spraying of gibberellins. Later, the research team found that the number of runners of the micropropagated seedlings of cultivated strawberry 'Yanli' was significantly higher than that of ordinary seedlings (seedlings bred by runner propagation). At the same time, transcriptome data analysis showed that the expression level of FvJAZ8 in the stem tip of the micropropagated seedlings of 'Yanli' was 16.5 times that of ordinary seedlings. Therefore, we speculate that the FvJAZ8 gene can participate in the occurrence of strawberry runners. In order to verify this conjecture, the function of the gene was studied to clarify its role in the occurrence of strawberry runners, laying a theoretical foundation for the study of strawberry runner formation.

[0006] The technical solution of the present invention is as follows:

[0007] The first object of the present invention is to provide an application of the FvJAZ8 gene in regulating the occurrence of strawberry runners, wherein the coding region sequence of the FvJAZ8 gene is shown in SEQ ID NO.1.

[0008] Furthermore, the amino acid sequence of the protein encoded by the FvJAZ8 gene is shown in SEQ ID NO.2.

[0009] Furthermore, the amplification primer sequence of the FvJAZ8 gene is shown in SEQ ID NO.3-4.

[0010] The second object of the present invention is to provide a recombinant plant expression vector for silencing the FvJAZ8 gene.

[0011] The third object of the present invention is to provide a recombinant bacterium containing the recombinant plant expression vector.

[0012] The fourth object of the present invention is to provide the use of the recombinant plant expression vector or the recombinant bacteria in reducing the number of strawberry runners.

[0013] Furthermore, the strawberry is a diploid forest strawberry.

[0014] The fourth object of the present invention is to provide a method for reducing the number of strawberry runners, comprising: introducing the recombinant plant expression vector into diploid forest strawberry.

[0015] Furthermore, the introduction is carried out by Agrobacterium-mediated method.

[0016] The beneficial effects of the present invention are:

[0017] The present invention first discovered the application of the FvJAZ8 gene in regulating the occurrence of strawberry runners. The strawberry FvJAZ8 gene was silenced in the forest strawberry 'Hawaii 4' with runners, and its expression in the strawberry plants was reduced. By comparing the strawberry plants with silenced genes and the control plants, it was found that the number of runners in the FvJAZ8 gene silenced plants was significantly reduced. This shows that FvJAZ8 can effectively regulate the occurrence of strawberry runners, and can provide a basis for studying the occurrence law and molecular regulation mechanism of strawberry runners. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 This is the amplification result of the FvJAZ8 gene coding region sequence.

[0019] Figure 2 This is the electrophoresis diagram for PCR identification of transgenic plants.

[0020] Figure 3 is the relative expression level of FvJAZ8 gene in transgenic plants.

[0021] Figure 4 Phenotypic analysis of RNAi-interference FvJAZ8 transgenic strawberry plants and wild-type control plants. DETAILED DESCRIPTION

[0022] The present invention is described in detail below in conjunction with the accompanying drawings and specific examples, but should not be construed as limiting the present invention. Unless otherwise specified, the technical means used in the following examples are conventional means well known to those skilled in the art, and the materials, reagents, etc. used in the following examples, unless otherwise specified, can be obtained from commercial sources.

[0023] Example 1: Cloning of the strawberry FvJAZ8 gene

[0024] The diploid forest strawberry 'Ruegen' was used as the test material.

[0025] RNA extraction: The total RNA of the test materials was extracted using the modified CTAB method. The entire operation process followed the CTAB RNA extraction process, and then the total RNA was used as a template for reverse transcription to obtain cDNA.

[0026] Gene cloning: Using the cDNA as a template, PCR amplification was performed using primers FvJAZ8-F and FvJAZ8-R to obtain a 393 bp target fragment, such as Figure 1 shown.

[0027] FvJAZ8-F (SEQ ID NO.3): 5′-ATGAGGAGGAACTGCAATTTG-3′;

[0028] FvJAZ8-R (SEQ ID NO. 4): 5′-CTAGTGATTGTAGGGATGAGATG-3′.

[0029] The PCR product was recovered by gel recovery, and the recovered target fragment was connected to the pMD18-T vector (purchased from TaKaRa) to obtain the pMD-T-FvJAZ8 plasmid, which was then transformed into Escherichia coli competent cells TOP10 (purchased from Beijing Tiangen Biotechnology Co., Ltd.), positive single colonies were screened, plasmids were extracted, and the sequencing sequence was shown in SEQ ID NO.1.

[0030] After comparison, the coding region sequences of FvJAZ8 in forest strawberry 'Ruegen' and 'Hawaii 4' were consistent, and the amino acid sequence of the encoded protein is shown in SEQ ID NO.2.

[0031] SEQ ID NO.1: ATGAGGAGGAACTGCAATTTGGAGTCCCGCCTTAATA CTTCCAGTGCTAATTCCGGCTTCCCTCTTCGCAACCACCGCGACCGCCCCGCCGTTCTGGAATCGAGTTCTGGAACACCGCAGCAGCAGCCATTGACGATCTTCTACGACGGAGTGATGTGTGTTTCTGATGTTACAGAGCTTCAGGCTAGAGCGATTCTTTTTCTTGCAAACAAAGA AATGGAGGAAAGGTTGAAGACCAGTCCAAGTCCGAGTCCTTGTGCGTCGACTTCTGAACCATCTTCACCGAGCCTGCACTCCCCAATATGCAGCCCTTCCATGAAGAAGTCACTACAGACGTTTTTGCAGAAGAGGAAGTATCGGATTCAAGCAACATCTCATCCCTACAATCACTAG.

[0032] SEQ ID NO. 2: MRRNCNLELRLNTSSANSGFPLRNHRDRPAVLESSSGTP QQQPLTIFYDGVMCVSDVTELQARAILFLANKEMEERLKTSPSPSPCASTSEP SSPSLHSPICSPSMKKSLQTFLQKRKYRIQATSHPYNH.

[0033] Example 2: Construction of recombinant plant expression vector RNAi-FvJAZ8

[0034] 1. Use Primer Primer 5.0 software to analyze and design primers for amplifying the forward and reverse fragments of FvJAZ8:

[0035] FvJAZ8-RNAi-F1 (SEQ ID NO.5): 5′-TCTAGATTCCCTCTTCGCAACCA C-3′;

[0036] FvJAZ8-RNAi-R1 (SEQ ID NO.6): 5′-CCCGGGTTGAATCCGATACTTC CTCT-3′;

[0037] FvJAZ8-RNAi-F2 (SEQ ID NO.7): 5′-GGTACCTTGAATCCGATACTTCC TCT-3′;

[0038] FvJAZ8-RNAi-R2 (SEQ ID NO. 8): 5′-GAGCTCTTCCCTCTTCGCAACC AC-3′.

[0039] 2. Obtaining the forward fragment:

[0040] Using pMD-T-FvJAZ8 as template, PCR reaction was performed; the PCR reaction system was:

[0041] Take 1 μL of pMD-T-FvJAZ8 plasmid and add 0.2 μL of Ex Taq enzyme, 2 μL of 10×Ex Buffer, and 2.5 mmol·L of dNTPs. -1 ) 1.6 μL, forward and reverse primers (FvJAZ8-RNAi-F1 / FvJAZ8-RNAi-R1) 0.5 μL each, and finally make up to 20 μL with water;

[0042] PCR reaction procedure:

[0043] 95 for 5 min; 95°C for 30 s, 58°C for 30 s, 72°C for 90 s, 35 cycles; 72°C for 7 min, stored at 4°C; the product was subjected to agarose gel electrophoresis;

[0044] The nucleic acid was recovered using a Nucleic Acid Purification Kit (TakaRa).

[0045] After recovery, the recovered PCR product and the pRI101-RNAi vector were digested with restriction endonucleases XbaⅠ and SmaⅠ, respectively, and then purified, recovered and ligated again.

[0046] The PCR reaction system was as follows: 7 μL of recovered PCR product, 1 μL of T4 DNA ligase, 1 μL of 10× T4 DNA ligase buffer, and 1 μL of T4 vector;

[0047] PCR reaction program: 16°C ligation for 16 h;

[0048] E. coli was transformed in the presence of Amp (60 μg mL -1 ) on LB medium plates and cultured at 37°C for 12-16h. Pick a single white clone and spread it on a new plate containing Amp (60 μg mL -1 ) on LB medium plates (secondary transformation), cultured at 37°C for 12-16h, and then colony PCR amplification was performed. After the verified correct strain was cultured, it was sent to Suzhou Genewise for sequencing. The sequencing was correct and named RNAi-FvJAZ8-positive.

[0049] 3. Obtaining the reverse fragment:

[0050] PCR reaction was performed using pMD-T-FvJAZ8 as template and FvJAZ8-RNAi-F2 and FvJAZ8-RNAi-R2 as primers. After the PCR product was recovered, the recovered product and RNAi-FvJAZ8-positive vector were digested with KpnⅠ and SacⅠ restriction endonucleases, respectively, and purified and recovered again after digestion. After the vector connection was completed, Escherichia coli transformation and colony PCR verification were performed, and then the vector was handed over to Sangon Biotech Co., Ltd. for sequencing. DNAMAN software was used for sequence analysis. When the analysis results were correct, the recombinant plant expression vector (RNAi-FvJAZ8) of silencing FvJAZ8 gene was obtained. RNAi-FvJAZ8 was introduced into Agrobacterium EHA105 competent cells, and the obtained Agrobacterium containing the recombinant plasmid was used to transform forest strawberry.

[0051] Example 3: Transgenic Function Verification - Screening and Phenotypic Analysis of Transgenic Forest Strawberry Plants

[0052] 1. Preparation of infection solution

[0053] Pick the Agrobacterium containing the RNAi-FvJAZ8 recombinant vector and incubate it in a 28°C constant temperature shaking incubator overnight with YEP liquid culture medium containing 25 mg / L rifampicin and 100 mg / L kanamycin for 10-14 hours until it turns orange-yellow. Transfer 1 mL of the bacterial solution to 50 mL of YEP liquid culture medium and shake it in a 28°C shaking incubator until the OD 600 The value is about 0.5. Then collect the cells and resuspend them in MS suspension so that the OD is still between 0.4-0.6 for later use.

[0054] 2. Infection of Explants

[0055] Cut the well-growing diploid 'Hawaii 4' tissue culture strawberry seedlings, quickly cut off the leaves around, leaving small squares of 2-4mm, and quickly put the trimmed explants into the co-cultivation liquid culture medium. After all the leaves are trimmed, transfer the explants to the bacterial suspension to start infection. After 8 minutes, use sterilized filter paper to absorb the bacterial liquid on the surface of the explants, and then quickly spread the explants on the co-cultivation solid culture medium and co-cultivate them in the dark for 3 days.

[0056] Delayed culture: After 3 days of solid co-culture, the explants were transferred to delay medium (200 mg / L Cef, 200 mg / L Tim) and cultured in the dark for 4 days.

[0057] Selective culture: After 4 days of culture on the culture medium, the explants were transferred to the selective culture medium (containing 10 mg / L Kan, 200 mg / L Cef, and 200 mg / L Tim) and kept in the dark until the explants grew plump yellow-green callus. Then they were transferred to the light to promote the growth of adventitious buds.

[0058] Differentiation culture: After the resistant buds appear, they are transferred to differentiation medium (containing 30 mg / L Kan, 200 mg / L Cef, 200 mg / L Tim) for differentiation culture.

[0059] 3. Identification of transgenic plants:

[0060] (1) When the resistant seedlings grew to 7-8 leaves, DNA was extracted from the well-growing positive transgenic lines JAZ8-RNAi#12 and JAZ8-RNAi#14. Non-transgenic 'Hawaii 4' strawberry plants were used as negative controls. 35S / RV was used as primers, and PCR amplification and agarose gel electrophoresis were performed. The results were as follows: Figure 2 As shown, the transgenic plants showed a 1450 bp fragment, while the control plants did not, indicating that the strawberry FvJAZ8 silencing vector was successfully introduced into strawberry.

[0061] (2) When the resistant seedlings grew to 7-8 leaves, RNA was extracted from the young leaves of the transgenic strawberry plants and reversed into cDNA. RT-PCR was performed using FvJAZ8-q F / FvJAZ8-q R as primers to detect the expression level of the FvJAZ8 gene.

[0062] FvJAZ8-q F(SEQ ID NO.9):5'-TCTGGAACACCGCAGCAGCAGCC-3';

[0063] FvJAZ8-q R (SEQ ID NO. 10): 5'-AAGGACTCGGACTTGGACTG-3'.

[0064] The results are as follows Figure 3 As shown, from Figure 3 It can be seen that the expression level of FvJAZ8 in the transgenic plants was significantly lower than that in the control plants, indicating that the FvJAZ8 gene silenced plants were successfully obtained.

[0065] (3) Observation of transgenic strawberry phenotype

[0066] Non-transgenic strawberry tissue culture plants were set as the control group, and plants transferred with the FvJAZ8 silencing vector were set as the experimental group. The two groups of tissue culture plants were transplanted into the artificial light culture room at the same time. Under the same cultivation conditions, the number of runners of the two groups of strawberries was observed and measured separately.

[0067] The results showed that the number of runners in FvJAZ8-silenced plants was significantly reduced compared with the control plants (Hawaii 4 wild type with runners) (Table 1, Figure 4 ). This indicates that the expression of the FvJAZ8 gene affects the occurrence of strawberry runners. This result shows that FvJAZ8 is an important gene involved in the process of strawberry runners.

[0068] Table 1 Comparison of the number of runners between transgenic plants and control plants

[0069]

[0070] Note: Different lowercase letters indicate significant differences at the P<0.05 level.

[0071] References:

[0072] Li W, Wang B, Sun H, Zhang Z. Transcriptome profiling of runner formation induced by exogenous gibberellin in Fragaria vesca. ScientiaHorticulturae. 2021.281:109966.

[0073] Feng J,Cheng L,Zhu Z,Yu F,Dai C,Liu Z,Guo W,Wu

[0074] Although the preferred embodiments of the present invention have been described, those skilled in the art may make other changes and modifications to these embodiments once they have learned the basic creative concept. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments and all changes and modifications that fall within the scope of the present invention.

[0075] Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include these modifications and variations.

Claims

1. Application of the FvJAZ8 gene in regulating the occurrence of strawberry runners, characterized in that: The coding region sequence of the FvJAZ8 gene is shown in SEQ ID NO.1, and the regulation is specifically: reducing the number of strawberry runners by silencing the FvJAZ8 gene.

2. The use according to claim 1, characterized in that: The amino acid sequence of the protein encoded by the FvJAZ8 gene is shown in SEQ ID NO.

2.

3. The use according to claim 1, characterized in that: The amplification primer sequence of the FvJAZ8 gene is shown in SEQ ID NO.3-4.

4. The use according to claim 1, characterized in that: The strawberry is a diploid forest strawberry.

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