Application of CDPK8 protein encoded by solanaceae plants in prevention and treatment of pepper mild mottle virus

Abstract

This invention relates to the fields of biotechnology and crop disease control, particularly to the application of CDPK8 protein encoded by Solanaceae plants in the control of pepper mild mottle virus. This invention discovers that the CDPK8 protein (using NbCDPK8 as an example) encoded by Solanaceae plants and its encoding gene play an important role in PMMoV infection and proliferation, and is a susceptibility gene for PMMoV encoded by the host plant. It can be used to control pepper mild mottle virus disease caused by PMMoV, and for molecular breeding of PMMoV resistance / tolerance. This invention can provide a basis for studying the role of plant proteins in virus infection, for antiviral breeding, and other fields, improve the level of research on antiviral properties of solanaceous vegetables, and ensure and promote the production safety of solanaceous vegetables.

Description

Technical Field

[0001] This invention relates to the fields of biotechnology and crop disease control technology, and in particular to the application of CDPK8 protein encoded by Solanaceae plants in the control of pepper mild mottle virus. Background Technology

[0002] Peppers and tomatoes are important solanaceous vegetable crops. In recent years, the incidence of pepper mild mottle virus (PMMoV) has shown a year-on-year increasing trend. PMMoV infection causes chlorotic mottling of leaves and stunted growth, posing a serious threat to the high and stable yields of peppers and other crops. Since there are currently no resistant / tolerant varieties and no effective control agents, it is urgent to identify disease-resistant genes and develop resistant materials to sustainably and more effectively control PMMoV and its damage, ensuring the production safety of peppers and other solanaceous vegetables.

[0003] As obligate intracellular parasites, plant viruses rely on and utilize host factors to complete life processes such as viral uncoating, genome replication, protein expression, viral particle assembly, and migration to neighboring cells during infection and proliferation. Therefore, identifying the host factors involved in plant virus infection and proliferation can help to better understand the mechanisms of viral infection and pathogenesis, thus providing new materials and theoretical basis for the prevention and control of viral diseases. Summary of the Invention

[0004] This invention provides the application of CDPK8 protein encoded by Solanaceae plants in the prevention and control of pepper mild mottle virus.

[0005] This invention provides an NbCDPK8 protein that regulates the expression of pepper mild mottle virus, the amino acid sequence of which includes any one of the following:

[0006] (1) The amino acid sequence as shown in SEQ ID No. 1;

[0007] (2) An amino acid sequence having at least 77.71% (preferably at least 99%) identity with the amino acid sequence shown in SEQ ID No. 1, wherein the protein has the function of regulating the expression of pepper mottle virus;

[0008] (3) An amino acid sequence obtained by substituting and / or deleting and / or adding one or more amino acid residues in the amino acid sequence shown in SEQ ID No.1, wherein the protein has the function of regulating the expression of pepper mottle virus.

[0009] This invention also provides an NbCDPK8 gene that regulates the expression of pepper mild mottle virus, the nucleotide sequence of which includes any one of the following:

[0010] (1) The nucleotide sequence shown in SEQ ID No. 2 or its complementary sequence;

[0011] (2) A sequence having at least 89.98% (preferably at least 99%) identity with the nucleotide sequence shown in SEQ ID No. 2, the encoded polypeptide having the function of regulating the expression of pepper mottle virus;

[0012] (3) The sequence that hybridizes with SEQ ID No. 2 under stringent conditions;

[0013] (4) A sequence obtained by substituting and / or deleting and / or adding one or more nucleotides from the nucleotide sequence shown in SEQ ID No.2, which encodes a polypeptide that has the function of regulating the expression of pepper mottle virus.

[0014] The aforementioned genes are derived from *Synthetium brevicornu*.

[0015] Given that the NbCDPK8 gene of Nicotiana spp. is highly conserved with CDPK8 in economically important Solanaceae crops such as chili peppers, this invention can also be used for dicotyledonous host plants of Solanaceae such as chili peppers.

[0016] The present invention also provides a biological material comprising the NbCDPK8 gene that regulates the expression of pepper mild mottle virus.

[0017] Preferably, the biological material is an expression cassette, vector, host cell, transgenic cell line, or recombinant microorganism.

[0018] This invention also provides the use of the NbCDPK8 protein, or its encoding gene, or biological materials containing its encoding gene in any one or more of the following:

[0019] (1) Application in regulating the expression of pepper mild mottle virus;

[0020] (2) Regulate plant resistance to pepper mild mottle virus disease;

[0021] (3) Prevention and control of pepper mild mottle virus disease;

[0022] (4) Regulating crop yield;

[0023] (5) Application in the improvement of plant germplasm resources;

[0024] (6) Application in the preparation of products for the prevention and control of pepper mild mottle virus disease;

[0025] The amino acid sequence of the NbCDPK8 protein includes any of the following:

[0026] (1) The amino acid sequence as shown in SEQ ID No. 1;

[0027] (2) An amino acid sequence having at least 99% identity with the amino acid sequence shown in SEQ ID No. 1, wherein the protein has the function of regulating the expression of pepper mottle virus;

[0028] (3) An amino acid sequence obtained by substituting and / or deleting and / or adding one or more amino acid residues in the amino acid sequence shown in SEQ ID No.1, wherein the protein has the function of regulating the expression of pepper mottle virus;

[0029] Preferably, the biological material is an expression cassette, vector, host cell, transgenic cell line, or recombinant microorganism;

[0030] Preferably, the plant or crop is a plant of the Solanaceae family, more preferably a plant of the genus *Nicotiana* or *Solanum*, and even more preferably tobacco or chili pepper.

[0031] According to the application, the resistance of plants to pepper mild mottle virus disease can be regulated, or crop yield can be controlled, or pepper mild mottle virus disease can be prevented and controlled, or germplasm resources can be improved by adjusting the content or activity of NbCDPK8 protein.

[0032] According to the application described, the content or activity of NbCDPK8 protein is regulated by genetic engineering technology.

[0033] In practice, the content or activity of the NbCDPK8 protein can be regulated by controlling substances such as cDNA and RNA. For example, it can be the RNA encoded by the nucleotide sequence shown in SEQ ID NO.2, positions 1414-1606 (silencing the NbCDPK8 gene).

[0034] In specific implementation processes, this includes, but is not limited to, using biotechnological methods such as Ti plasmids, plant virus vectors, direct DNA transformation, microinjection, and electroporation to regulate the content or activity of NbCDPK8 protein in plant cells.

[0035] According to the application described, the replication and proliferation of pepper mild mottle virus can be inhibited by reducing the content or activity of NbCDPK8 protein, thereby improving plant resistance to pepper mild mottle virus disease, increasing crop yield, or improving germplasm resources.

[0036] According to the application, the gene encoding the NbCDPK8 protein includes the sequence shown in SEQ ID No. 2;

[0037] This invention also provides a product, which is a pesticide, fertilizer, or fertilizer additive; the product achieves at least one of the following objectives by regulating the content or activity of the NbCDPK8 protein:

[0038] (1) Regulate the resistance of plants to pepper mild mottle virus disease;

[0039] (2) Regulating crop yield;

[0040] (3) Prevention and control of pepper mild mottle virus disease.

[0041] The present invention also provides a method for inhibiting the replication and proliferation of pepper mild mottle virus in plants, comprising: reducing the content or activity of NbCDPK8 protein through genetic engineering technology.

[0042] Preferably, the replication and proliferation of pepper mild mottle virus in plants is promoted by introducing a substance into the plant for overexpression of the NbCDPK8 gene.

[0043] Preferably, the replication and proliferation of pepper mild mottle virus in plants is inhibited by introducing a substance that inhibits the expression of the NbCDPK8 gene.

[0044] The pepper mild mottle virus disease described in this invention refers to a viral disease that occurs in plants such as peppers and tobacco caused by PMMoV infection.

[0045] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0046] This invention discovers that the NbCDPK8 protein and its encoding gene play a crucial role in PMMoV infection and proliferation. It is a PMMoV susceptibility gene encoded by the host plant and can be used to control mild mottle virus disease of pepper caused by PMMoV. It can also be used in molecular breeding for PMMoV resistance / tolerance. This invention provides a basis for research on the role of plant proteins in virus infection, for antiviral breeding, and other related fields, improving the level of research on antiviral properties in solanaceous vegetables and ensuring and promoting the production safety of solanaceous vegetables. Attached Figure Description

[0047] To more clearly illustrate the technical solutions in this 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 some embodiments of this invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0048] Figure 1 The results of silencing NbCDPK8 to inhibit PMMoV infection are shown in the following figures: A is an infection photograph; B is a statistical graph of green fluorescent dots; C is a graph of NbCDPK8 gene expression level; D is a graph of PMMoV RNA accumulation level; and E is a graph of PMMoV CP protein accumulation level. Detailed Implementation

[0049] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some, not all, of the embodiments of this invention. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without creative effort are within the scope of protection of this invention.

[0050] Unless otherwise specified, all examples were performed under standard experimental conditions, such as those described in Sambrook et al., Molecular Cloning: a Laboratory Manual (Sambrook J & Russell DW, 2001), or as recommended by the manufacturer’s instructions.

[0051] In the quantitative experiments in the following examples, three replicate experiments were set up, and the average value of the results was taken.

[0052] The biological materials used in the following examples are as follows:

[0053] Nicotiana benthamiana: See Goodin MM, Zaitlin D, Naidu RA, Lommel SA. 2008. Nicotianabenthamiana: its history and future as a model for plant-pathogeninteractions. Molecular Plant-Microbe Interactions 21:1015-1026.

[0054] Vectors pJM23, pJG1024, and pLoxP-Cre: See Yin, Y., Hua M., Zhao K., Wan Q., Bu S., Lu Y., Zhang, H., Rao, S., Yan F., Peng J., Chen H., and Chen J. 2022. Construction of chimeric viruses based on pepper mild mottle virus using a modified Cre / loxP system. Journal of Integrative Agriculture 21:2456-2463.

[0055] For vectors pTRV1 and pTRV2, see: Liu, Y., Schiff M and Dinesh-Kumar SP. 2002. Virus-induced gene silencing in tomato. The Plant journal 32:777-786.

[0056] Pepper mild mottle virus (PMMoV) See: Yin, Y., Hua M., Zhao K., Wan Q., Bu S., Lu Y., Zhang, H., Rao, S., Yan F., Peng J., Chen H., and Chen J. 2022. Construction of chimeric viruses based on pepper mild mottle virus using a modified Cre / loxP system. Journal of Integrative Agriculture 21:2456-2463.

[0057] The NbCDPK8 protein sequence in the following examples is shown in SEQ ID No. 1. The gene encoding the NbCDPK8 protein is named the NbCDPK8 gene, and its sequence is shown in SEQ ID No. 2.

[0058] SEQ ID No. 1:

[0059] 1mgnccvtpgkssekkknkkk nkpnpfaidy gatqssaegd gnklvvlkdptghninekyd61lgrelgrgefgvtylctdvd tgekyacksi skkklrtavd iddvrrevqi mkhlpkhpni121vtlrdtyedd savhivmelc eggelfdriv arghyteraa agimrtivevvqmchrhgvm181hrdlkpenfl fgnkketapl kaidfglsvf fkpgehfneivgspyymapevlkrnygpev 241dvwsagvilyillcgvppfwaeteqgvaqaiirsvvdfkrdpwpkvsdnakdlvkkmldp 301dptrrltaqqvlehtwlqni kkapnvslge tvkarlkqfs vmnklkkraltviaeflsae 361evagmkeafdqmdtgkkgki nlgelkiglq klghqipdad lqilmeaadvdrdgclnyae 421fvavsvhlrk mandehlhkafsffdrnqsgyieieelrsalsdeddgnseevinaimhdv 481dtdkdgrisy eefaammkag tdwrkasrqysrerfnslslklmrdgslqlgneak。

[0060] SEQ ID No. 2:

[0061]

[0062] Example 1: Construction of the recombinant vector pTRV2-NbCDPK8

[0063] 1. Total RNA was extracted from Nicotiana benthamiana and reverse transcribed into cDNA.

[0064] 2. Using the cDNA obtained in step 1 as a template, perform PCR amplification with primer pair composed of pTRV2::NbCDPK8-F and pTRV2::NbCDPK8-R to obtain the PCR amplification product of the gene fragment with nucleotide sequence shown in positions 1414-1606 of the coding region of NbCDPK8 (SEQ ID No. 2); recover the PCR product of the gene fragment shown in positions 1414-1606 of the coding region of NbCDPK8.

[0065] pTRV2::NbCDPK8-F(SEQ ID No.3)

[0066] 5'-TAAGGTTACCGAATTCTCTAGAGTCATCAATGCCATTATGCAT GA-3'

[0067] pTRV2::NbCDPK8-R(SEQ ID No.4)

[0068] 5'-GAGACGCGTG AGCTCGGTAC CATTTAGCCT CGTTTCCAAG TTGTA-3'

[0069] 3. Digest the vector pTRV2 with restriction endonucleases XbaI and KpnI, and recover the vector backbone.

[0070] 4. Connect the recovered product from step 2 with the vector backbone from step 3 to obtain the recombinant vector pTRV2-NbCDPK8.

[0071] 6. Based on the sequencing results, the structure of the recombinant vector pTRV2-NbCDPK8 is described as follows: A double-stranded DNA molecule as shown in SEQ ID No. 11 was inserted between the XbaI and KpnI restriction sites of the vector pTRV2.

[0072] SEQ ID No. 11:

[0073] GTCATCAATGCCATTATGCATGATGTTGACACTGACAAGGATGG

[0074] TCGCATTAGTTACGAAGAATTTGCTGCAATGATGAAGGCTGGAA

[0075] CGGATTGGAGAAAAGCATCGAGACAGTATTCTCGTGAACGTTT

[0076] CAATAGTCTCAGCTTAAAATTGATGAGGGACGGCTCATTACAACTTGGAAACGAGGCTAAAT.

[0077] Example 2: Silencing Experiment of NbCDPK8 Gene

[0078] 1. Extraction of PMMoV-GFP crude extract

[0079] (1) Agrobacterium species pJM23, pJG1024 and pLoxP-Cre stored at -80℃ were streaked on LB resistance plates (containing 50 μg / ml Kan and 100 μg / ml Rif) and cultured at 28℃ for about 48 h; single colonies on LB resistance plates were inoculated into 3-4 ml of LB liquid resistance medium (containing 50 μg / ml Kan and 100 μg / ml Rif) and cultured at 28℃ and 180 rpm for 14-18 h.

[0080] (2) Inoculate 100 μL of bacterial culture into 10 mL of LB liquid resistance medium (containing 50 μg / ml Kan and 100 μg / ml Rif) at a ratio of 1:100, and culture at 28℃ and 180 rpm for 10-12 h to obtain bacterial cultures of Agrobacterium pJM23, pJG1024 and pLoxP-Cre.

[0081] (3) Collect the bacterial cells by centrifugation at 4000 rpm for 10 min at room temperature, suspend the precipitate in 3 mL of soaking buffer (10 mM MES, pH 5.7, 10 mM MgCl2, 200 μM acetylsyl syringone), and determine the concentration of the bacterial suspension using a spectrophotometer.

[0082] (4) Add 3 mL of Agrobacterium pJM23 bacterial suspension (OD) 600nm 3 mL of Agrobacterium pJG1024 bacterial suspension (concentration of 0.5%) 600nm The concentration was 0.5 g / mL, and 3 mL of Agrobacterium pLoxP-Cre bacterial suspension (OD200) was used. 600nm Mix the ingredients (at a concentration of 0.25%) and let stand at room temperature for 2-3 hours to obtain a mixture. Then, use a needleless syringe (1 mL) to inject the mixture into the lower epidermis of the tobacco leaves and incubate at 22-24℃ for 6 days.

[0083] (5) Take the leaves of the tobacco plant, add 0.1M phosphate buffer and grind them, centrifuge at 4500g for 5min, and collect the virus supernatant containing the crude extract of PMMoV.

[0084] 2. Silencing the NbCDPK8 gene in tobacco using TRV VIGS

[0085] (1) The recombinant plasmid pTRV2-NbCDPK8 was transformed into Agrobacterium GV1301 to obtain recombinant Agrobacterium TRV2-NbCDPK8.

[0086] (2) Streak the pTRV1 and pTRV2-mcherry agrobacterium strain stored at -80℃ on LB resistance plates (containing 50 μg / ml Kan and 100 μg / ml Rif) and incubate at 28℃ for about 48 h; inoculate single colonies on LB resistance plates into 3-4 ml of LB liquid resistance medium (containing 50 μg / ml Kan and 100 μg / ml Rif) and incubate at 28℃ and 180 rpm for 14-18 h.

[0087] (3) Inoculate 100 μL of bacterial culture into 10 mL of LB liquid resistance medium (containing 50 μg / ml Kan and 100 μg / ml Rif) at a ratio of 1:100, and incubate at 28℃ and 180 rpm for 10-12 h to obtain the bacterial culture of Agrobacterium carrying pTRV1.

[0088] (4) Collect the bacterial cells by centrifugation at 4000 rpm for 10 min at room temperature, suspend the precipitate in 3 mL of soaking buffer (10 mM MES, pH 5.7, 10 mM MgCl2, 200 μM acetylsalicylic acid), and determine the concentration of the bacterial suspension using a spectrophotometer.

[0089] (5) Add 3 mL of Agrobacterium tumefaciens culture containing pTRV1 (OD) 600nm (Concentration 1.0) was mixed with 3 mL of recombinant Agrobacterium pTRV2-NbCDPK8 bacterial suspension (OD). 600nm A concentration of 1.0 g and 3 mL of pTRV2-mcherry agrobacterium bacterial suspension (OD200) were used. 600nm Mix the ingredients (1.0 concentration) and let stand at room temperature for 2-3 hours to obtain a mixture. Then, use a needleless syringe (1 mL) to inject the mixture into the lower epidermis of the 3rd-4th true leaves of Nicotiana benthamiana and incubate at 22-24℃ for 6 days.

[0090] 3. PMMoV-GFP challenge inoculation of Nicotiana benthamiana plants

[0091] (1) Take the NbCDPK8 silenced or mcherry silenced Nicotiana Bunseni (as control) from step 2 (5) and continue to culture in alternating light and dark (16h / 20℃ light, 8h / 18℃ darkness) for 6 days. Sprinkle a small amount of carborundum on the 5th-6th true leaves of Nicotiana Bunseni and inoculate with PMMoV-GFP by friction (inoculation amount is 50μL, concentration is 0.3g / mL). After that, continue to culture in alternating light and dark (16h / 20℃ light, 8h / 18℃ darkness) on the inoculated Nicotiana Bunseni plants.

[0092] 3. Detect the PMMoV replication and proliferation levels in Nicotiana Bunsenata plants with silenced NbCDPK8.

[0093] The disease incidence of tobacco leaves after PMMoV inoculation was continuously observed. On the 4th day after PMMoV inoculation, inoculated leaves were collected, total RNA was extracted and reverse transcribed into cDNA. Using cDNA as a template, the relative expression level of NbCDPK8 gene and the content of PMMoV genomic RNA were identified by RT-qPCR using the Kangwei Century Real-Time PCR Kit and the Actin gene as an internal control gene. At the same time, total protein was extracted from tobacco leaves to detect the accumulation of PMMoV CP protein.

[0094] The primer pairs used to identify the NbCDPK8 gene are as follows:

[0095] NbCDPK8-qRT-F: 5'-CGGAGGTTGATGTGTGGAGT-3' (SEQ ID No.5)

[0096] NbCDPK8-qRT-R: 5'-GAGCCGTCGAGTTGGATCTG-3' (SEQ ID No. 6)

[0097] The primer pairs used to identify the Actin gene are as follows:

[0098] NbActin-qRT-F: 5'-TGCATTCTCCGTCTTGACT-3' (SEQ ID No. 7)

[0099] NbActin-qRT-R: 5'-TGCAGTCTCGAGTTCCTGTT-3' (SEQ ID No. 8)

[0100] The primer pairs used to identify PMMoV CP are as follows:

[0101] PMMoV CP-qRT-F: 5'-TGGAAGACTATTCCGACCGC-3'(SEQ ID No.9)

[0102] PMMoV CP-qRT-R: 5'-CCGTGCCACGAACTAACTCA-3' (SEQ ID No.10)

[0103] The obtained data is used in 2 -ΔΔCT The analysis is performed using the following steps:

[0104] First, for the experimental group samples (test) and control group samples (calibrator), the C of the reference gene (ref) was used. T Value normalized to the C of the target gene T value:

[0105] ΔC T(test) =C T(target,test) -C T(ref,test)

[0106] ΔC T(calibrator) =C T(target,calibrator) -C T(ref,calibrator)

[0107] Secondly, using the ΔC of the control group sample T ΔC of the normalized experimental group sample T value:

[0108] ΔΔC T =ΔC T(test) -ΔC T(calibrator)

[0109] Finally, calculate the expression level ratio: 2 -ΔΔCT =Ratio of expression levels

[0110] The results showed that on day 4 after PMMoV inoculation, compared with the control group (TRV-mcherry), the experimental group with silenced NbCDPK8 gene (TRV-NbCDPK8) had fewer green fluorescent spots on the leaves infected with Nicotiana Bunsenata. Figure 1 (A, B); Compared with the control group, the expression level of NbCDPK8 in Nicotiana Bunsenata plants with silenced NbCDPK8 gene was downregulated by about 70%, see [reference needed]. Figure 1 (C); PMMoV CP accumulation was reduced by approximately 75%, see [link to relevant documentation]. Figure 1 (D)

[0111] Western blot analysis was performed using TMV CP antiserum as the primary antibody and HRP-labeled goat anti-rabbit antibody as the secondary antibody. The results showed that, compared with the control group, the accumulation of PMMoV CP protein was reduced by approximately 40% in Nicotiana Bunsenata plants with silenced NbCDPK8 gene. Figure 1 (E, quantitative values ​​were calculated using ImageJ software. Anti-actin indicates that the expression level of actin was used as an internal reference.)

[0112] The above results indicate that silencing the NbCDPK8 gene in Nicotiana Bunsenata significantly reduces PMMoV genomic RNA and CP protein levels, thus inhibiting PMMoV infection. These results suggest that the NbCDPK8 protein and its encoding gene play a crucial role in PMMoV replication and proliferation, acting as a host susceptibility gene that promotes PMMoV infection. This information could be used to control mild mottle virus disease of peppers caused by PMMoV, and for molecular breeding research aimed at PMMoV resistance.

[0113] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. The application of reducing the expression level of NbCDPK8 protein in tobacco nicotine in one or more of the following: (1) Inhibits the replication and proliferation of chili pepper mild mottle virus in tobacco; (2) Improve the resistance of tobacco to pepper mild mottle virus disease; (3) Control of mild mottle virus disease in tobacco peppers; (4) Improve the germplasm resources of Nicotiana sambac; The direction of germplasm resource improvement is resistance to pepper mild mottle virus disease; (5) Application in the preparation of products for the prevention and control of mild mottle virus disease of pepper in tobacco; The amino acid sequence of the NbCDPK8 protein is shown in SEQ ID No.

1.

2. Use according to claim 1, characterized in that, The gene encoding the NbCDPK8 protein is shown in SEQ ID No.

2.

3. The application of a product that reduces the expression level of NbCDPK8 protein in tobacco ash in any of the following: (1) Improve the resistance of tobacco to pepper mild mottle virus disease; (2) Control of mild mottle virus disease in tobacco peppers; The amino acid sequence of the NbCDPK8 protein is shown in SEQ ID No.

1.

4. A method of inhibiting replication and multiplication of Pepper mild mottle virus in Nicotiana benthamiana, characterized in that, include: The expression level of NbCDPK8 protein in Nicotiana benthamiana was reduced by genetic engineering. The amino acid sequence of the NbCDPK8 protein is shown in SEQ ID No. 1.

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