Application of nbfkbp13 gene in resistance to tomato yellow leaf curl virus

Abstract

The application discloses application of an NbFKBP13 gene in resisting tomato yellow leaf curl virus and belongs to the technical field of plant disease prevention and treatment. Through genetic engineering technology, the application obtains a stably heritable overexpression NbFKBP13 transgenic N.benthamiana. Phenotype observation finds that the NbFKBP13 gene has no significant influence on the growth and development of the N.benthamiana. Artificial inoculation of TYLCV finds that the NbFKBP13 gene can significantly enhance the resistance of the N.benthamiana to the TYLCV after overexpression. The above research provides an important gene bank and new germplasm resource for virus disease resistance breeding.

Description

Technical Field

[0001] This invention relates to the field of plant disease control technology, and more specifically to the application of the NbFKBP13 gene in resistance to tomato yellow leaf curl virus. Background Technology

[0002] Plant viral diseases are among the most threatening diseases in agricultural production, often referred to as "plant cancer." Tomato yellow leaf curl is a widespread tomato disease worldwide, caused by tomato yellow leaf curl virus (TYLCV), which belongs to the genus Begomovirus in the family Geminiviridae. In recent years, TYLCV has spread to tomato-growing areas in various provinces and cities of my country, seriously jeopardizing the development of my country's tomato industry.

[0003] FKBPs are a class of proteins possessing a domain (FKBP domain) that binds to the immunosuppressants FK506 or rapamycin. They are widely distributed in eukaryotes and prokaryotes, and most exhibit prolyl cis-trans isomerase (PPI) activity. FKBPs are prevalent in plant cells and all contain the FKBP domain. As a prolyl cis-trans isomerase, FKBPs utilize their PPIase activity for rapid protein folding in cells, effectively alleviating endoplasmic reticulum stress. Current research indicates that FKBPs play an important role in plant responses to various stresses. However, the function of FKBPs in plant antiviral immunity remains unclear.

[0004] In summary, how to provide an anti-TYLCV FKBP protein and its application is a problem that urgently needs to be solved by those skilled in the art. Summary of the Invention

[0005] In view of this, the present invention provides the application of the NbFKBP13 gene in resistance to tomato yellow leaf curl virus.

[0006] This invention utilizes genetic engineering technology to obtain a stably heritable transgenic Nicotiana benthamiana overexpressing the NbFKBP13 gene. Phenotypic observation revealed that the NbFKBP13 gene transfection had no significant effect on the growth and development of Nicotiana benthamiana. TYLCV inoculation experiments were conducted on wild-type and NbFKBP13-overexpressing transgenic Nicotiana benthamiana, respectively. The results showed that overexpression of the NbFKBP13 gene significantly enhanced the resistance of Nicotiana benthamiana to TYLCV. This invention provides a new approach for breeding TYLCV-resistant plants and selecting new virus-resistant varieties.

[0007] To achieve the above objectives, the present invention adopts the following technical solution:

[0008] The application of the NbFKBP13 gene in resistance to tomato yellow leaf curl virus, the nucleotide sequence of the NbFKBP13 gene is shown in SEQ ID No. 1.

[0009] Furthermore, the amino acid sequence encoding the NbFKBP13 gene is shown in SEQ ID No. 2;

[0010] MNSVPFSIGTYIPIKHNTTLKSPLLCNKNPQTTADTISNVKSVIKLKEKSELPCLFRRREALSTLGLSFAATFLEALLQPQSNIEAVAEEEATATGCEFTVTPSGLCYCDKV VGYGPEAVKGQLIKAHYVGKLENGQVFDSSYNRGKPLTFRVGVGEVIKGWDQGILGGDGFPPMLTGGKRKLKIPPELGYGMRGAGCRGGSCIIPPNSVLLFDVEFVGKA, SEQ ID No.2.

[0011] Furthermore, the application of overexpression of the NbFKBP13 gene in resistance to tomato yellow leaf curl virus.

[0012] The application of biological materials that enhance the expression of the NbFKBP13 gene in resistance to tomato yellow leaf curl virus, wherein the biological material is any one of the following:

[0013] A: An expression cassette capable of overexpressing the NbFKBP13 gene with a nucleotide sequence as shown in SEQ ID No. 1;

[0014] B: A recombinant vector containing the expression cassette described in A;

[0015] C: Recombinant microorganisms containing the expression cassette described in A or the recombinant vector described in B.

[0016] A method to improve the resistance of Nicotiana benthamiana to tomato yellow leaf curl virus is to enhance the resistance to tomato yellow leaf curl virus by overexpressing the NbFKBP13 gene shown in SEQ ID No. 1 of Nicotiana benthamiana.

[0017] The application of the NbFKBP13 gene in breeding of Tobacco Benedictine foetida for resistance to Tomato Yellow Leaf Curl Virus, the nucleotide sequence of the NbFKBP13 gene is shown in SEQ ID No. 1.

[0018] As can be seen from the above technical solution, compared with the prior art, the beneficial effects achieved by the present invention are as follows:

[0019] This invention constructs an overexpression vector of the NbFKBP13 gene and uses Agrobacterium-mediated transformation to obtain transgenic Nicotiana benthamiana that can be stably inherited. Transgenic Nicotiana benthamiana has no significant effect on the growth and development of Nicotiana benthamiana, but it can significantly enhance the resistance of Nicotiana benthamiana to TYLCV. Attached Figure Description

[0020] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on the provided drawings without creative effort.

[0021] Figure 1 Phenotypic observation of NbFKBP13 overexpression transgenic and wild-type Nicotiana benthamiana in Example 3 of this invention;

[0022] Figure 2 This is the result of the Western blot experiment verifying the NbFKBP13 overexpression transgenic Nicotiana benthamiana in Example 4 of the present invention;

[0023] Figure 3 This is a schematic diagram of the pathogenesis symptoms of NbFKBP13 transgenic and wild-type Nicotiana benthamiana after TYLCV infection in Example 6 of the present invention.

[0024] Figure 4 This invention provides an example of RT-qPCR detection of TYLCV virus accumulation in NbFKBP13-overexpressing transgenic and wild-type Nicotiana benthamiana plants.

[0025] Figure 5 In Example 6 of this invention, Western blot was used to detect the accumulation of TYLCV virus in NbFKBP13-overexpressing transgenic and wild-type Nicotiana benthamiana plants. Detailed Implementation

[0026] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0027] The reagents required for this invention are conventional experimental reagents, purchased from commercially available channels; the experimental methods not mentioned are conventional experimental methods, and will not be described in detail here.

[0028] Example 1

[0029] Construction of a binary expression vector for Nicotiana benthamiana NbFKBP13

[0030] (1) Extraction of total RNA from tobacco leaves and synthesis of cDNA sequence

[0031] Total RNA was extracted from tobacco leaves using TRIzol RNA extraction reagent, according to PrimeScript from TaKaRa. TM The RTreagent Kit with gDNA Eraser (Perfect Real Time) was used to synthesize first-strand cDNA via RNA reverse transcription, following the instructions in the kit's manual.

[0032] (2) Cloning of the NbFKBP13 gene in Nicotiana benthamiana

[0033] Based on the NbFKBP13 gene sequence identified in the Nicotiana benthamiana genome published in the Nicotiana benthamiana genome database, specific primers for amplifying the full sequence of the NbFKBP13 gene were designed using DNAMAN software. Using Nicotiana benthamiana cDNA as a template, PCR amplification was performed using a high-fidelity enzyme.

[0034] NbFKBP13 gene nucleotide sequence:

[0035] , SEQ ID No.1.

[0036] The specific amplification system was as follows: 10 μL of PrimeSTAR Max Premix (2×), 0.5 μL of 10 μM upstream primer (5'-TCTAGAATGAATTCAGTTCCCTT-3', SEQ ID No. 3), 0.5 μL of 10 μM downstream primer (5'-GTCGACAGCCTTGCCAACAAACT-3', SEQ ID No. 4), 100 ng of DNA, and ddH2O added to a final volume of 20 μL.

[0037] The PCR amplification program was as follows: 98℃ pre-denaturation for 10s, cycling parameters were 98℃ denaturation for 10s, 57℃ annealing for 10s, 72℃ extension for 1 min 30s, for 34 cycles, with a final extension at 72℃ for 10 min.

[0038] After the reaction was completed, the PCR product was mixed with the loading buffer and detected by 1.0% agarose gel electrophoresis. The target nucleic acid band in the agarose gel was then cut under UV light and the DNA was recovered using the gel recovery kit from Tiangen Company.

[0039] (3) Construction of a binary expression vector for NbFKBP13 in Nicotiana benthamiana

[0040] The recovered DNA fragments and pCV vector were digested with Xba1 and Sal1, and the NbFKBP13 gene was ligated into the pCV vector using Takara's T4 DNA ligase.

[0041] The ligation system consisted of: 1 μL of 10×Ligation Buffer, 0.2–0.3 pmol of DNA fragment, 0.03 pmol of pCV vector, 1 μL of T4 DNA ligase, and finally, ddH2O to a final volume of 10 μL.

[0042] The ligation reaction was carried out overnight at 4°C. The ligation product was transformed into E. coli DH5α competent cells. Positive clones were selected and sent to the company for sequencing. After sequence alignment, the recombinant vector pCV-NbFKBP13-GFP was obtained.

[0043] Example 2

[0044] Agrobacterium transformation

[0045] Add 3.0 μL of the recombinant vector pCV-NbFKBP13-GFP to 50 μL of Agrobacterium GV3101 competent cells. After incubating on ice for 5 min, transfer the competent cells to liquid nitrogen and let them stand for 5 min. Then transfer them to a 37℃ water bath and let them stand for 5 min. Remove them and place them on ice for 5 min. Add 700 μL of antibiotic-free LB medium and incubate at 28℃ and 200 rpm for 2 h. Centrifuge at 8000 rpm for 3 min, take 100 μL of the supernatant, gently pipette the precipitate, mix well, and spread it on LB solid medium containing Rif+Kana antibiotic. Incubate at 28℃ for 48 h until colonies grow. Pick a single colony and place it in liquid medium containing Rif+Kana antibiotic. Incubate overnight at 28℃ and 200 rpm. PCR detection showed all colonies were positive, yielding the Agrobacterium culture.

[0046] Example 3

[0047] Benedict's tobacco genetic transformation

[0048] Select healthy Benedictine tobacco leaves, sterilize them, and cut them into pieces approximately 0.5cm in size. 2Small pieces of the bacteria were placed in pre-culture medium (MS + 1 mg / L 6-BA + 0.1 mg / L NAA) and cultured for 2 days to promote cell division. Then, the leaves of *Tobacco Benzoinus* were immersed in the *Agrobacterium* cultured in Example 2 for 10 minutes, and excess culture was aspirated. The infected leaves were placed on co-culture medium (MS + 200 μMAS) and cultured in the dark at 25°C for 2 days. They were then transferred to selection medium (MS + 1 mg / L 6-BA + 0.1 mg / L NAA + 50 mg / L kanamycin), and the medium was changed every 2 weeks until resistant buds differentiated. The resistant buds were cut off and transferred to rooting medium (1 / 2 MS + 0.1 mg / L NAA). After culturing for 2–3 weeks, complete plants were obtained. Figure 1 As shown, there was no significant difference in growth phenotype between the NbFKBP13 overexpressing transgenic and wild-type Nicotiana benthamiana. After hardening off, the seedlings were transplanted to a greenhouse.

[0049] Example 4

[0050] Positive identification of transgenic plants

[0051] Total protein was extracted from the positive transgenic plants in Example 3, and 10% SDS-PAGE separating and stacking gels were prepared for protein electrophoresis. After the protein electrophoresis was completed, the protein was transferred to a PVDF membrane and blocked with 5% skim milk at room temperature for 1 hour. Then, it was incubated overnight at 4°C with GFP monoclonal antibody, washed with TBST, and incubated with HRP-labeled secondary antibody at room temperature for 1 hour. After that, TBST was added and the membrane was washed thoroughly 5 times. Super ECLplus chromogenic solution was evenly dropped onto the nitrocellulose membrane, and the membrane was allowed to stand in the dark for 2 minutes. Chemiluminescence imaging was performed using a BIO-RAD gel imaging system, and the images were recorded.

[0052] The results are as follows Figure 2 As shown, the target band can be detected in transgenic Nicotiana benthamiana plants overexpressing NbFKBP13, indicating that the NbFKBP13 protein is stably expressed.

[0053] Example 5

[0054] Artificial inoculation with TYLCV

[0055] TYLCV infectious clonal Agrobacterium, stored at -80℃, was streaked onto LB agar plates containing 100 μg / mL kanamycin sulfate and 20 μg / mL rifampin, and incubated at 28℃ for 48 hours. After single colony formation, typical colonies were picked and inoculated into LB liquid medium containing the same antibiotics, and cultured at 28℃ with shaking at 200 rpm for 12 hours. Subsequently, the bacterial cells were collected by centrifugation at 5000 rpm for 10 minutes, the supernatant was discarded, and the cells were thoroughly resuspended in inoculum resuspension (10 mM MES, 200 μM acetylsalicylic acid, 10 mM MgCl2). The OD of the bacterial suspension was measured and adjusted using a spectrophotometer.600 After activating the solution to 1.0 and allowing it to stand at room temperature for 2-3 hours, inoculate each plant with 1 ml of the solution using a sterile syringe.

[0056] Example 6

[0057] Detection of virus accumulation in Nicotiana benthamiana plants

[0058] Fourteen days after inoculation, observe the disease symptoms of the plants and take photos for recording. The results are as follows: Figure 3 As shown, compared with WT plants inoculated with TYLCV, NbFKBP13-OE plants inoculated with TYLCV exhibited milder leaf curling symptoms.

[0059] Total DNA and total protein were extracted from wild-type and NbFKBP13 transgenic Nicotiana benthamiana plants inoculated with TYLCV. Virus accumulation in wild-type and NbFKBP13 transgenic Nicotiana benthamiana plants was detected by qPCR using TYLCV-specific quantitative detection primers (F: CCCTCAAAGCTCTATGG CAATCGG, SEQ ID No. 5; R: AAGTGAGCGTCTGTGGAACCCTC, SEQ ID No. 6). The TYLCV CP protein content was detected by Western blotting using a TYLCV CP protein-specific antibody.

[0060] The results are as follows Figure 4 , Figure 5 As shown, compared with WT, the accumulation levels of TYLCV CP protein and DNA in NbFKBP13-OE plants were significantly reduced, indicating that NbFKBP13 positively regulates the resistance of Nicotiana benthamiana to TYLCV.

[0061] The various embodiments in this specification are described in a progressive manner, with each embodiment focusing on the differences from other embodiments. The same or similar parts between the various embodiments can be referred to each other.

[0062] The above description of the disclosed embodiments enables those skilled in the art to make or use the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. NbFKBP13 The application of genes in enhancing the resistance of Nicotiana benthamiana to tomato yellow leaf curl virus is characterized by, The NbFKBP13 The nucleotide sequence of the gene is shown in SEQ ID No.

1.

2. The application as described in claim 1, characterized in that, overexpression NbFKBP13 Application of genes in enhancing the resistance of Tomato Yellow Leaf Curl Virus in Nicotiana benthamiana.

3. Improvement of the claim 1 NbFKBP13 The application of gene-expressing biomaterials in enhancing the resistance of Nicotiana benthamiana to tomato yellow leaf curl virus is characterized by, The biomaterial is any one of the following: A: Enables nucleotide sequences as shown in SEQ ID No. 1 NbFKBP13 Expression cassettes for gene overexpression; B: A recombinant vector containing the expression cassette described in A; C: Recombinant microorganisms containing the expression cassette described in A or the recombinant vector described in B.

4. A method for improving the resistance of Nicotiana benthamiana to tomato yellow leaf curl virus, characterized in that, By overexpressing the substance represented by SEQ ID No. 1 in *Ben's tobacco* NbFKBP13 Genes are used to enhance resistance to tomato yellow leaf curl virus.

5. NbFKBP13 The application of genes in breeding *Nicotiana benthamiana* for resistance to tomato yellow leaf curl virus is characterized by... The NbFKBP13 The nucleotide sequence of the gene is shown in SEQ ID No. 1.

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