Application of NbFKBP13 gene in resisting tomato yellow leaf curl virus

Abstract

The invention discloses an application of an NbFKBP13 gene in resisting tomato yellow leaf curl virus. Belongs to the technical field of plant disease control. The overexpression NbFKBP13 transgenic nicotiana benthamiana which can be stably inherited is obtained through a gene engineering technology. Phenotypic observation shows that the NbFKBP13 transgene has no significant influence on the growth and development of nicotiana benthamiana; it is found through artificial inoculation of TYLCV that after overexpression of the NbFKBP13 gene, the resistance of nicotiana benthamiana to TYLCV can be significantly enhanced. The research provides an important gene pool and a new germplasm resource for virus disease resistance breeding.

Description

Technical Field

[0001] The present invention relates to the technical field of plant disease control, and more particularly to the application of the NbFKBP13 gene in resisting tomato yellow leaf curl virus. Background Art

[0002] Plant virus diseases are one of the most threatening diseases in agricultural production and are known as the "cancer of plants". Tomato yellow leaf curl disease is a common tomato disease occurring worldwide. This disease is caused by tomato yellow leaf curl virus (TYLCV), and TYLCV belongs to the genus Begomovirus of the family Geminiviridae. In recent years, TYLCV has spread to tomato-growing areas in various provinces and cities in China, seriously endangering the development of the tomato industry in China.

[0003] FKBP is a class of proteins with a domain (FKBP domain) that can bind to the immunosuppressant FK506 or Rapamycin, and is widely present in eukaryotes and prokaryotes. Most of them have peptidyl-prolyl cis-trans isomerase (PPI) activity. FKBP is widely present in plant cells and all contain the FKBP domain. As a class of peptidyl-prolyl cis-trans isomerases, FKBP uses its PPIase activity in cells for rapid protein folding, which can effectively relieve endoplasmic reticulum stress. Current research findings show that FKBP plays an important role in plant responses to various stress reactions. However, the function of FKBP in plant antiviral immunity is still unclear.

[0004] In summary, how to provide an FKBP protein resistant to TYLCV and its application is an urgent problem 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 resisting tomato yellow leaf curl virus.

[0006] Through genetic engineering techniques, the present invention obtains transgenic Nicotiana benthamiana with stable and heritable overexpression of NbFKBP13. Phenotypic observation shows that the transgenic NbFKBP13 has no significant effect on the growth and development of Nicotiana benthamiana. TYLCV inoculation experiments were respectively carried out on wild-type and NbFKBP13 overexpressing transgenic Nicotiana benthamiana, and the results showed that overexpressing the NbFKBP13 gene can significantly enhance the resistance of Nicotiana benthamiana to TYLCV. The present invention provides new ideas for TYLCV-resistant plant breeding and the selection of new virus-resistant varieties.

[0007] To achieve the above object, the present invention adopts the following technical solutions:

[0008] Use of the NbFKBP13 gene in resisting tomato yellow leaf curl virus, wherein the nucleotide sequence of the NbFKBP13 gene is as shown in SEQ ID No.1.

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

[0010] MNSVPFSIGTYIPIKHNTTLKSPLLCNKNPQTTADTISNVKSVIKLKEKSELPCLFRRREALSTLGLSFAATFLEALLQPQSNIEAVAEEEATATGCEFTVTPSGLCYCDKVVGYGPEAVKGQLIKAHYVGKLENGQVFDSSYNRGKPLTFRVGVGEVIKGWDQGILGGDGFPPMLTGGKRKLKIPPELGYGMRGAGCRGGSCIIPPNSVLLFDVEFVGKA, SEQ ID No.2.

[0011] Furthermore, use of overexpression of the NbFKBP13 gene in resisting tomato yellow leaf curl virus.

[0012] Use of a biological material for increasing the expression of the above-mentioned NbFKBP13 gene in resisting 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 the nucleotide sequence as shown in SEQ ID No.1;

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

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

[0016] A method for increasing the resistance of Nicotiana benthamiana to tomato yellow leaf curl virus, which improves the ability to resist tomato yellow leaf curl virus by overexpressing the expression level of the NbFKBP13 gene shown in SEQ ID No.1 in Nicotiana benthamiana.

[0017] Use of the NbFKBP13 gene in the breeding of Nicotiana benthamiana resistant to tomato yellow leaf curl virus, wherein the nucleotide sequence of the NbFKBP13 gene is as shown in SEQ ID No.1.

[0018] It can be seen from the above technical solutions that, compared with the prior art, the beneficial effects achieved by the present invention are:

[0019] By constructing an overexpression vector of the NbFKBP13 gene and using the Agrobacterium-mediated method, stably heritable transgenic Nicotiana benthamiana was obtained; overexpression of the NbFKBP13 gene had no significant effect on the growth and development of Nicotiana benthamiana, but significantly enhanced the resistance of Nicotiana benthamiana to TYLCV. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for the description of the embodiments or the prior art. Obviously, the drawings in the following description are only the embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can be obtained according to the provided drawings.

[0021] Figure 1 It is a phenotypic observation diagram of overexpressed NbFKBP13 transgenic and wild-type Nicotiana benthamiana in Example 3 of the present invention;

[0022] Figure 2 It is the verification result of the Western blot experiment of overexpressed NbFKBP13 transgenic Nicotiana benthamiana in Example 4 of the present invention;

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

[0024] Figure 4 It is the detection of the TYLCV virus accumulation in overexpressed NbFKBP13 transgenic and wild-type Nicotiana benthamiana plants by RT-qPCR in Example 6 of the present invention;

[0025] Figure 5 It is the detection of the TYLCV virus accumulation in overexpressed NbFKBP13 transgenic and wild-type Nicotiana benthamiana plants by Western blot in Example 6 of the present invention. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0026] The following will clearly and completely describe the technical solutions in the embodiments of the present invention with reference to the drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts belong to the scope of protection of the present invention.

[0027] The reagents required for the present invention are conventional experimental reagents, purchased from commercial channels; the experimental methods not mentioned are conventional experimental methods, which will not be elaborated here one by one.

[0028] Example 1

[0029] Construction of Binary Expression Vector of NbFKBP13 in Nicotiana benthamiana

[0030] (1) Extraction of Total RNA from Nicotiana benthamiana Leaves and Synthesis of cDNA Sequences

[0031] Extract the total RNA of Nicotiana benthamiana leaves with the RNA extraction reagent TRIzol. According to the PrimeScript TM RTreagent Kit with gDNA Eraser(Perfect Real Time) reverse transcription kit of TaKaRa Company, reverse transcribe the RNA to synthesize the first-strand cDNA, and the method refers to the kit instruction manual.

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

[0033] According to the NbFKBP13 gene sequence identified in the Nicotiana benthamiana genome published in the Nicotiana benthamiana genome database, use DNAMAN software to design specific primers for amplifying the full sequence of the NbFKBP13 gene, and use high-fidelity enzyme for PCR amplification with Nicotiana benthamiana cDNA as the template.

[0034] Nucleotide sequence of NbFKBP13 gene:

[0035] ATGAATTCAGTTCCCTTTTCCATTGGAACATACATTCCTATAAAGCACAATACCACACTCAAATCCCCATTACTCTGTAACAAAAATCCTCAAACAACAGCTGACACAATTTCAAATGTTAAATCTGTAATTAAACTCAAAGAGAAAAGTGAATTGCCTTGTTTATTCAGAAGAAGAGAAGCACTTAGCACACTTGGGTTAAGTTTCGCTGCAACTTTTCTTGAGGCCCTTTTGCAGCCCCAGTCAAATATTGAAGCGGTTGCTGAAGAAGAAGCTACAGCTACTGGGTGTGAATTTACTGTTACTCCTTCTGGTCTGTGTTATTGTGATAAAGTTGTGGGTTATGGCCCTGAAGCTGTTAAAGGGCAGTTAATTAAGGCACATTATGTAGGTAAGCTGGAAAATGGGCAGGTCTTCGATAGCAGCTACAACCGTGGAAAGCCTCTTACGTTTCGTGTTGGCGTTGGCGAGGTTATTAAAGGTTGGGATCAGGGAATTCTAGGAGGTGATGGTTTTCCCCCAATGTTAACTGGAGGAAAGCGTAAACTGAAGATTCCTCCAGAACTTGGATATGGAATGAGAGGAGCTGGCTGCAGAGGAGGTTCATGCATTATTCCTCCGAATTCAGTTCTATTGTTCGATGTGGAGTTTGTTGGCAAGGCTTAG, SEQ ID No.1.

[0036] The specific amplification system is 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 add ddH2O to 20 μL.

[0037] The PCR amplification program is as follows: pre-denaturation at 98°C for 10 s, and the cycling parameters are denaturation at 98°C for 10 s, annealing at 57°C for 10 s, extension at 72°C for 1 min 30 s, for 34 cycles, and sufficient extension at 72°C for 10 min.

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

[0039] (3) Construction of the binary expression vector of Nicotiana benthamiana NbFKBP13

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

[0041] The ligation system was as follows: 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 made up to 10 μL with ddH2O.

[0042] For the ligation reaction conditions, incubate overnight at 4°C. Transform the ligation product into Escherichia coli (E.coli) DH5α competent cells. Pick positive clones and send them to the company for sequencing. After sequence alignment, the recombinant vector pCV-NbFKBP13-GFP was obtained.

[0043] Example 2

[0044] Agrobacterium transformation

[0045] Take 3.0 μL of the recombinant vector pCV-NbFKBP13-GFP and add it to 50 μL of Agrobacterium GV3101 competent cells. After ice-bathing for 5 min, transfer the competent cells to liquid nitrogen and let it stand for 5 min; then transfer to a 37°C water bath and let it stand for 5 min. Take out and place on ice for 5 min, then add 700 μL of antibiotic-free LB medium, culture at 28°C with constant shaking at 200 rpm for 2 h, centrifuge at 8000 rpm for 3 min, take 100 μL of the supernatant and gently pipette to resuspend the pellet, and spread it evenly on an LB solid medium containing Rif + Kana antibiotics. Culture at 28°C for 48 h until colonies grow. Pick a single colony and place it in a liquid medium containing Rif + Kana antibiotics, culture overnight at 28°C with constant shaking at 200 rpm, and perform PCR detection. All were positive, and the Agrobacterium liquid was obtained.

[0046] Example 3

[0047] Genetic transformation of Nicotiana benthamiana

[0048] Select healthy Nicotiana benthamiana leaves, cut them into pieces about 0.5 cm after disinfection 2Small pieces were placed in the pre-culture medium (MS + 1 mg / L 6-BA + 0.1 mg / L NAA) and cultured for 2 days to promote cell division. Subsequently, Nicotiana benthamiana leaves were immersed in the Agrobacterium liquid culture that had been cultured in Example 2 for 10 min, and after taking them out, the excess liquid was blotted dry. The infected leaves were placed on the co-culture medium (MS + 200 μM AS) and dark-cultured at 25 °C for 2 days. Subsequently, they were transferred to the selection medium (MS + 1 mg / L 6-BA + 0.1 mg / L NAA + 50 mg / L kanamycin), which was replaced every 2 weeks until resistant buds differentiated. The resistant buds were cut off and transferred to the rooting medium (1 / 2 MS + 0.1 mg / L NAA), and after culturing for 2 - 3 weeks, complete plants were obtained. As Figure 1 shown, there was no obvious difference in the growth phenotypes between the overexpressed NbFKBP13 transgenic and wild-type Nicotiana benthamiana. After acclimatization, they were transplanted to the greenhouse.

[0049] Example 4

[0050] Positive identification of transgenic plants

[0051] Total proteins of the positive transgenic plants in Example 3 were extracted, 10% SDS-PAGE separating gel and stacking gel were prepared, and protein electrophoresis was carried out. 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 h; subsequently, GFP monoclonal antibody was used to incubate overnight at 4 °C, and after washing with TBST, the secondary antibody labeled with HRP was incubated at room temperature for 1 h; then, it was washed 5 times thoroughly with TBST; Super ECL plus chromogenic solution was evenly dripped onto the nitrocellulose membrane, left to stand in the dark for 2 min, and chemiluminescence imaging was carried out with a BIO-RAD gel imaging system, and the image was photographed and recorded.

[0052] The results were as Figure 2 shown. The target band could be detected in the overexpressed NbFKBP13 transgenic Nicotiana benthamiana plants, indicating the stable expression of NbFKBP13 protein.

[0053] Example 5

[0054] Artificial inoculation with TYLCV

[0055] The Agrobacterium liquid culture of the TYLCV infectious clone stored in an ultra-low temperature refrigerator at -80 °C was streaked on an LB solid plate containing 100 μg / mL kanamycin sulfate and 20 μg / mL rifampicin, and cultured at a constant temperature of 28 °C for 48 hours. After single colonies formed, typical colonies were picked and inoculated into an LB liquid medium containing the same antibiotics, and cultured with shaking at 28 °C and 200 rpm for 12 hours. Subsequently, the cells were collected by centrifugation at 5000 rpm for 10 minutes, and after discarding the supernatant, the cells were fully suspended with the inoculation resuspension solution (10 mM MES, 200 μM acetosyringone, 10 mM MgCl2). The OD of the cell suspension was measured and adjusted with a spectrophotometer.600 From 0.5 to 1.0. After activation by standing at room temperature for 2 to 3 hours, inoculate Nicotiana benthamiana using a sterile syringe, 1 ml for each plant.

[0056] Example 6

[0057] Detection of virus accumulation in Nicotiana benthamiana plants

[0058] At 14 d post inoculation, observe the disease symptoms of the plants and take pictures for record. The results are as Figure 3 shown. Compared with the WT plants inoculated with TYLCV, the NbFKBP13 - OE plants inoculated with TYLCV showed milder leaf curling symptoms.

[0059] Extract the total DNA and total protein of wild - type and NbFKBP13 transgenic Nicotiana benthamiana plants inoculated with TYLCV respectively. Use TYLCV - specific quantitative detection primers (F: CCCTCAAAGCTCTATGGCAATCGG, SEQ ID No.5; R: AAGTGACGTCTGTGGAACCCTC, SEQ ID No.6), and adopt qPCR method to detect the virus accumulation in wild - type and NbFKBP13 transgenic Nicotiana benthamiana plants; use TYLCV CP protein - specific antibody and adopt Western Blot to detect the content of TYLCV CP protein.

[0060] The results are as Figure 4 , Figure 5 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] In this specification, each example is described in a progressive manner. The key point of each example is to illustrate the differences from other examples. For the same or similar parts among the examples, reference can be made to each other.

[0062] The above description of the disclosed examples enables those skilled in the art to implement or use the present invention. Various modifications to these examples will be obvious to those skilled in the art. The general principles defined herein can be implemented in other examples without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to these examples shown herein, but will conform to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. Application of NbFKBP13 gene in resistance to tomato yellow leaf curl virus, characterized in that: The nucleotide sequence of the NbFKBP13 gene is shown in SEQ ID No. 1; , SEQ ID No.

1.

2. The use according to claim 1, characterized in that Application of overexpression of NbFKBP13 gene in resistance to tomato yellow leaf curl virus.

3. Use of the biomaterial for improving the expression of the NbFKBP13 gene according to claim 1 in resistance to tomato yellow leaf curl virus, characterized in that: The biological material is any one of the following: A: an expression cassette capable of overexpressing the NbFKBP13 gene with a nucleotide sequence as shown in SEQ ID No. 1; B: a recombinant vector containing the expression cassette described in A; C: A recombinant microorganism 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: The ability to resist tomato yellow leaf curl virus is improved by overexpressing the NbFKBP13 gene shown in SEQ ID No. 1 in Nicotiana benthamiana.

5. Application of NbFKBP13 gene in breeding of Nicotiana benthamiana resistant to Tomato Yellow Leaf Curl Virus, characterized in that: The nucleotide sequence of the NbFKBP13 gene is shown in SEQ ID No.1.

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