Mutants of HC-Pro protein and their application in controlling papaya malformation mosaic virus

Abstract

The present invention relates to the field of genetic engineering technology, and in particular to mutants of HC-Pro proteins and their use in preventing and treating papaya teratoma mosaic virus. The present invention identifies and finds a weak strain PLDMV-R165I on an asymptomatically infected PLDMV papaya plant through a field virus disease survey of papaya. After comparing the amino acid full-length sequence with the wild-type strong strain PLDMV, it is found that a single amino acid mutation occurs in the 165th amino acid of the PLDMV HC-Pro multifunctional protein. It is further verified that the mutation at this site affects the replication of the virus, weakens the RNA silencing inhibitory activity of HC-Pro, and reduces the accumulation of the virus in the papaya body. It is a newly discovered key amino acid that determines the occurrence of PLDMV symptoms and the accumulation of viral replication.

Description

Technical Field

[0001] The invention relates to the technical field of genetic engineering, and in particular to a mutant of HC-Pro protein and application thereof in preventing and treating papaya teratomic mosaic virus. Background Art

[0002] Papaya is a popular tropical fruit with high commercial value due to its nutritional, medicinal and industrial properties. It has become an important commercial fruit crop worldwide. The entire papaya plant, including seeds, leaves, roots and flowers, has long been used for medicinal purposes due to its anti-inflammatory, anti-skin aging, wound healing, improved gastrointestinal health and reduced cancer risk properties. Papaya is rich in vitamins A, B and C, as well as proteolytic enzymes such as papain and chymopapain. It is a good source of beta-carotene, which can prevent cancer, diabetes and heart disease. The ripe fruit is usually eaten fresh and can be processed into jams, jellies and candies. The "green" or unripe fruit can be used as a vegetable. Papaya latex, which comes from the raw fruit, is widely used in food processing, medicine and cosmetics. The market demand for papaya has steadily increased over the past decade due to rapid population growth and increasing public awareness of the health benefits of functional fruits.

[0003] Papaya leaf distortion mosaic virus (PLDMV, genusPotyvirus) belongs to the genus Potyvirus and is a major disease threatening the world's papaya planting industry. Papaya leaves infected with PLDMV will become deformed and the mosaic will appear yellow-green and mottled. In severe cases, the growth of the leaves will be greatly inhibited, becoming very small or even falling off prematurely, causing the fruit to become deformed, with an uneven surface and much smaller than normal fruit, affecting the fruit quality. At present, due to the severity of the PMDMV virus and the lack of effective disease-resistant varieties and prevention and control strategies, virus prevention and control is difficult to achieve effective implementation. Summary of the invention

[0004] In view of this, the technical problem to be solved by the present invention is to provide a mutant of HC-Pro protein and its application in preventing and controlling papaya malformation mosaic virus. The present invention provides a weak virus strain obtained by mutating the conserved amino acid sites in the HC-Pro protein of papaya malformation mosaic virus, which achieves cross-protection antiviral effect and lays a good practical foundation for subsequent field application.

[0005] The present invention provides a mutant of HC-Pro protein, characterized in that the mutant is obtained by mutating the arginine at position 165 of the HC-Pro protein of the virulent strain of papaya malformation mosaic virus to isoleucine;

[0006] In some specific embodiments, the mutant of the HC-Pro protein has an amino acid sequence as shown in SEQ ID NO:1.

[0007] The invention is based on the identification of a weak strain of PLDMV that infects papaya asymptomatically from a papaya planting base in Ledong, Hainan. Compared with the wild-type strong strain PLDMV-WT, the 165th amino acid arginine R of the virus-encoded HC-Pro multifunctional protein is mutated to isoleucine I. HC-Pro is a protein of the Potatovirus Y genus that has important functions such as protease activity, inhibiting RNA interference, participating in the formation of host symptoms and regulating viral replication during the virus infection process. Through analysis by virology and molecular biology methods, it is found that HC-Pro 165R is a new key amino acid that determines the generation of PLDMV symptoms and viral replication and accumulation.

[0008] The present invention provides a nucleic acid molecule encoding the mutant.

[0009] In some embodiments, the nucleic acid molecule has a nucleotide sequence as shown in SEQ ID NO:2.

[0010] The present invention provides a vector of the nucleic acid molecule.

[0011] The present invention provides a host cell transformed or transfected with the vector.

[0012] In some embodiments, the host cell comprises Agrobacterium, specifically Agrobacterium competent GV3101.

[0013] The present invention provides a method for preparing the mutant, comprising: culturing the host cell to obtain a culture containing the mutant.

[0014] The present invention provides the use of at least one of (1) to (5) in preventing and treating papaya teratomic virus:

[0015] (1) the mutant;

[0016] (2) the nucleic acid molecule;

[0017] (3) the carrier;

[0018] (4) the host cell;

[0019] (5) The mutant obtained by the preparation method.

[0020] The present invention provides a vaccine comprising at least one of the following ① to ⑤:

[0021] 1. the mutant;

[0022] ②, the nucleic acid molecule;

[0023] ③, the carrier;

[0024] ④, the host cell;

[0025] ⑤. The mutant obtained by the preparation method.

[0026] The invention provides a method for preventing and treating papaya freckle mosaic virus by administering the vaccine.

[0027] The present invention is based on a new HC-Pro single amino acid mutation (165R mutation I) to obtain an asymptomatic PLDMV weak strain, which lays a good practical foundation for the subsequent field papaya antiviral application. Compared with the prior art, the present invention has the following beneficial effects:

[0028] (1) The attenuated strains PLDMV-R165I and PLDMV-R165I-GFP infectious clones were successfully constructed. Virus inoculation, fluorescent virus tracing, RT-PCR, qPCR, Western blot, symptom observation and tobacco silencing suppressor experiments proved that the mutation of PLDMV HC-Pro 165R to I affected viral replication, weakened the RNA silencing inhibitory activity of HC-Pro, and reduced the accumulation of the virus in papaya. It is a newly discovered key amino acid that determines the occurrence of PLDMV symptoms and viral replication and accumulation.

[0029] (2) The strong strain of PLDMV was used to conduct a virus infection experiment on three different papaya varieties, namely, Huanong No. 1 (transgenic PRSV-resistant) Suizhonghong and Mihong, which had been inoculated with weak strains. Thirty days after the virus infection, it was found that the weak strains of PLDMV produced by HC-Pro 165R mutation I all showed good cross-protection, with a cross-protection efficiency of more than 90%. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 The figure shows the construction strategy of infectious clones of pPLDMV-R165I and pPLDMV-R165I-GFP;

[0031] Figure 2 Schematic diagram of Nicotiana benthamiana inoculation in the virus silencing suppressor activity experiment (back of the leaf);

[0032] Figure 3 The symptoms of papaya infected with the strong strain of PLDMV-WT and the weak strain of PLDMV-R165I are shown, including Mock (no virus infection), plants infected with PLDMV-R165I, and plants infected with wild-type PLDMV-WT;

[0033] Figure 4Shows the sequencing results of PLDMV-HC-Pro 165R point mutation;

[0034] Figure 5 Shown is the full-length amino acid sequence comparison analysis of the polyprotein encoded by PLDMV-WT and PLDMV-R165I;

[0035] Figure 6 The fragments required for amplification to construct PLDMV-R165I and PLDMV-R165I-GFP infectious clones are shown, including M as DL15000 DNA Marker, product fragments a, b, c, d;

[0036] Figure 7 PCR identification of pPLDMV-R165I and pPLDMV-R165I-GFP Agrobacterium, wherein, M: DL2000 DNA Marker, 1-4: pPLDMV-R165I bacterial solution, 5-8: pPLDMV-R165I-GFP bacterial solution, 9: positive control, 10: negative control;

[0037] Figure 8 Shows the symptom observation and GFP fluorescence tracing of papaya leaves infected with PLDMV-WT, PLDMV-WT-GFP, PLDMV-R165I and PLDMV-R165I-GFP;

[0038] Fig. 9 The GFP fluorescence microscope observation and analysis of papaya leaves 15 days after inoculation with PLDMV-WT-GFP and PLDMV-R165I-GFP respectively;

[0039] Fig.10 RT-PCR detection of PLDMV-R165I and PLDMV-R165I-GFP, where M: DL2000 DNAMarker, 1-8: papaya plants infected with PLDMV-R165I, 9-16: papaya plants infected with PLDMV-R165I-GFP, positive control ck+, negative control ck-;

[0040] Fig.11 The qRT-PCR analysis shows the difference in CP mRNA expression in papaya plants infected with PLDMV-R165I and PLDMV-WT, including the difference in CP mRNA expression in papaya plants infected with PLDMV-R165I for 20 d, 40 d, 60 d, and 80 d and infected with PLDMV-WT;

[0041] Fig.12Western blot analysis of the difference in GFP expression in papaya plants infected with PLDMV-WT-GFP and PLDMV-R165I-GFP, where A: Ponceau red staining, B: Western blot, M: marker (kDa), 1: total protein in healthy papaya leaves, 2-3: total protein in leaves infected with PLDMV-R165I-GFP, 4-5: total protein in leaves infected with PLDMV-WT-GFP;

[0042] Fig.13 Schematic diagram of the fragments required for PCR amplification to construct p35S-HC-Pro-WT and p35S-HC-Pro-R165I, wherein M: DL2000 DNA Marker, fragment a (HC-Pro-WT), fragment b (HC-Pro-R165I);

[0043] Fig.14 The silencing inhibition activity analysis of HC-Pro 165R RNA is shown, wherein, A: pGreenⅡ-35S, B: pGreenⅡ-35S-GFP, C: P19+pGreenⅡ-35S-GFP, D: p35S-HC-Pro-WT+pGreenⅡ-35S-GFP, E: p35S-HC-Pro-R165I+pGreenⅡ-35S-GFP;

[0044] Fig.15 Western blot analysis of the effect of HC-Pro 165R on GFP expression, where A: Ponceau S staining of the large subunit of Rubisco was used as a loading control. M: Marker (kDa), 1: pGreenⅡ-35S, 2: pGreenⅡ-35S-GFP, 3: P19 + pGreenⅡ-35S-GFP, 4: p35S-HC-Pro-WT + pGreenⅡ-35S-GFP, 5: p35S-HC-Pro-R165I + pGreenⅡ-35S-GFP, B: Western blot;

[0045] Fig.16 The cross-protection effect of PLDMV-R165I / PLDMV-R165I-GFP was observed by symptom and fluorescence 30 days after infection.

[0046] Fig.17 The phenotype and sequencing analysis results of PLDMV-R165I attenuated strain 150 days after infection are shown;

[0047] Fig.18 Shown are the results of the stability analysis of the PLDMV attenuated strain. DETAILED DESCRIPTION

[0048] The present invention provides mutants of HC-Pro protein and their use in preventing and treating papaya teratomic mosaic virus. Those skilled in the art can refer to the content of this article and appropriately improve the process parameters to achieve it. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art, and they are all considered to be included in the present invention. The methods and applications of the present invention have been described through preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the methods and applications of this article without departing from the content, spirit and scope of the present invention to implement and apply the technology of the present invention.

[0049] The present invention, through a field investigation of viral diseases in papaya, identified a weak strain PLDMV-R165I on a papaya plant asymptomatically infected with PLDMV, and compared it with the full-length amino acid sequence of the wild-type strong strain PLDMV, it was found that a single amino acid mutation occurred in the 165th amino acid of the PLDMV HC-Pro multifunctional protein. Plant virus cross-protection is that after the weak strain of the virus infects the plant, it can provide protection to the strong strain of the same or related virus. Compared with the traditional chemical pesticide control method, the virus cross-protection strategy is more effective and environmentally friendly, and the method is simple and easy to implement, and does not require complicated operating procedures or professional equipment. The test materials used in the present invention are all common commercial products, which can all be purchased on the market. The present invention is further described below in conjunction with the embodiments.

[0050] Example 1

[0051] 1. The present invention adopts a cross-protection strategy to verify the protective effect of PLDMV-R165I against virulent strains of the same or related viruses.

[0052] 2. Experimental methods

[0053] 2.1 Discovery of asymptomatic plants infected with PLDMV

[0054] A papaya plant infected with PLDMV but showing no symptoms was found at a papaya planting base in Ledong, Hainan, where PLDMV damage was severe. Total RNA of asymptomatic plants was extracted. The experimental method was referred to the instructions of Tiangen Polysaccharide and Polyphenol Plant Total RNA Kit. After total RNA was electrophoresed on 1% agarose gel, reverse transcription was performed according to the instructions of PrimeScript™ IV 1st strand cDNA SynthesisMix Reverse Transcription Kit. The cDNA after reverse transcription was used as a template. According to the instructions of PrimeSTAR® MaxDNA Polymerase (2×) high-fidelity DNA polymerase, primer pair PLDMV-1485F / PLDMV-3165R and primer pair PLDMV-8924F / PLDMV-9965R were used to amplify a segment of HC-Pro multifunctional protein and a segment of CP protein on PLDMV to identify whether asymptomatic plants were infected with PLDMV. After PCR products were electrophoresed on 1% agarose gel, the size of PCR amplification bands was consistent with that of target bands. The PCR products were sent to a biological company for sequencing. The full-length sequence of the virus was amplified using the cDNA of asymptomatic plants as a template and sent to a biological company for sequencing. Vector NTI Advance 11.5 and NCBI sequence alignment software were used to compare the full-length genome sequences of the PLDMV wild-type strong strain and the asymptomatic weak strain, to find out the base mutations in the full-length genome sequence of the asymptomatic weak strain compared with the wild-type strong strain, and to compare the full-length amino acid sequences to find out the amino acid mutation sites. Among them, the HC-Pro protein mutant of the above-mentioned PLDMV has the following amino acid sequence and nucleotide sequence:

[0055] Amino Acid Sequence:

[0056] SDAGLSFWKGFDRQFIDTREKPKNAHECKATINVEECGEMAAIVNQLLFPMWKITCTQCGELLEMLSQEEELESFRRKRNQLASKLSSLHIKFPYVDHFLNRYENSLDRMNTNFDAHKQIAQIIGGRKEIPFSNLERLNELLIKSDKLVSKDFYEMSQCLLELTIWHKNRSDSFKKGEIHHFRNKMSGKAQFNFALMCDNQLDKNGNFVWGERGYHAKRFFSNFFEKVDSTDGYKKHITRVNPNGTRQTAIGKLILSTDPSTLRQQMKGNPVTRVPVGKYCTSKRDDCYVYPACCVTMEDGTPLFSDIKMPTKNHLVIGNSGDPKYVDVPSNSSDMIVAKEGYCYLNIFLAMLLNVNESESKSFTKKVRDIIVPRLGQWPSLIDVATECYFLSAFHPETKNAELPRILVDHTSKCMHVIDSYGSLDTQFHVLKANTVSQLIKFADDDLDSEMKHYLVG (SEQ ID NO:1)

[0057] Nucleotide sequence:

[0058]

[0059] Comparison revealed that, compared with the wild-type HC-Pro protein, the 165th amino acid R of the mutant was mutated to I.

[0060] 2.2 Construction of pPLDMV-R165I and pPLDMV-R165I-GFP infectious clones

[0061] Using site-directed mutagenesis, the mutant base AT was designed into the forward and reverse primer sequences during primer design. Figure 1 As shown, primer pairs PLD-R165I-F, PLDMV-9944R; primer pairs PLDMV5F-25ov, PLD-R165I-R and primer pairs PLDMV-9920F, pGr35S-R were used to amplify target fragments a, c, and d from plasmid PLDMV-WT, respectively; and primer pairs PLD-R165I-F, PLDMV-9944R were used to amplify target fragment b from plasmid PLDMV-WT-GFP (the difference between fragment b and fragment a is that fragment b has GFP inserted between NIb / CP). After amplification, 1% agarose gel electrophoresis was used to recover the fragments for subsequent fragment connection. Using the In-Fusion connection strategy, the experimental method was referred to the instructions of the In-Fusion HD Cloning kits kit, and the target fragments a, c, and d were connected (constructing pPLDMV-R165I), and fragments b, c, and d were connected (constructing pPLDMV-R165I-GFP). Referring to the E. coli Cell-Free rapid construction of plant virus infectious cloning method constructed in this laboratory, the ligation product was directly transferred into Agrobacterium competent GV3101 (pSoup), coated with LB plates containing 50 mg / L kanamycin and 20 mg / L rifampicin, and inverted in a 28°C constant temperature incubator for 2-3 days. The single clone colonies on the plate were picked and cultured overnight at 28°C in LB liquid medium containing 50 mg / L kanamycin and 20 mg / L rifampicin. The primer pairs PLDMV-1120F, PLDMV-3165R and primer pairs PLDMV-8924F, PLDMV-9965R were used to identify the bacterial solution, respectively identifying the GFP segment inserted between the HC-Pro segment and the NIb / CP protein on PLDMV. In order to exclude random mutations introduced during the PCR process, the positive clones with correct identification were tested for the full-length genome sequence of the cloned virus, and the positive clones with correct sequencing of attenuated mutants can be used for subsequent experiments.

[0062] 2.3 Inoculation of papaya with Agrobacterium tumefaciens

[0063] 100 μL of Agrobacterium culture containing correctly sequenced pPLDMV-R165I and pPLDMV-R165I-GFP was taken respectively and put into 10 ml LB liquid culture medium with kanamycin (50 mg / L) and rifampicin (20 mg / L) resistance, and cultured overnight in a shaker at 28°C and 200 rpm for 12 h. The bacteria were collected after centrifugation on the next day, resuspended with Agrobacterium inoculation buffer to an OD value of 0.8, treated in the dark for more than 3 h, and inoculated with a needle-free syringe into 100 papaya plants of three different varieties [Huanong No. 1 (transgenic PRSV-resistant), Suizhonghong and Mihong] at the 2-month-old seedling stage as the experimental group (50 papaya plants of each variety were inoculated with pPLDMV-R165I and pPLDMV-R165I-GFP Agrobacterium infiltration buffer). At the same time, Agrobacterium containing wild-type virulent strains pPLDMV-WT and pPLDMV-WT-GFP were inoculated as virulent control groups, and five papayas of each of the three different varieties were inoculated with the two virulent bacterial solutions. The inoculated papaya plants were regularly observed and the disease status was recorded.

[0064] 2.4 Fluorescent virus tracing

[0065] After inoculation of weak and strong strains, regular observations were made under ultraviolet light (UV) and photos were taken to record the luminescence. After the strong strain began to glow 15 days after inoculation, the top tender leaves of papayas inoculated with the strong strain PLDMV-WT-GFP (the tender leaves with obvious luminescence under ultraviolet light (UV)) and the leaves inoculated with the weak strain PLDMV-R165I-GFP were taken respectively, and white light observation and green fluorescence (GFP) observation were performed under a fluorescence microscope, and photos were taken to record the green fluorescence luminescence of the top leaves of papayas.

[0066] 2.5 Detection of infectivity of attenuated strains and observation of symptoms after inoculation

[0067] 20 days after the papaya plants were inoculated with the attenuated strain infectious clones, 100 mg of leaves from the top of the plants of the three different varieties [Huanong No. 1 (transgenic PRSV-resistant), Suizhonghong and Mihong] inoculated with the attenuated strain were taken to extract the total RNA of the papaya plants, and cDNA was synthesized using TaKaRa's PrimeScript™ IV 1st strand cDNA Synthesis Mix Reverse Transcription Kit (Cat. No. 6215A). Using cDNA as a template, RT-PCR detection was performed using primers PLDMV-1485F, PLDMV-3165R and primers PLDMV-8924F, PLDMV-9965R. The GFP segment inserted between the HC-Pro segment and the NIb / CP protein on PLDMV was identified, and whether the constructed attenuated strain infectious clones were infectious to healthy papaya plants was identified, and the infection efficiency in papaya after inoculation with the attenuated strain was determined. The PCR products with positive identification results were sent to a biological company for sequencing. After sequencing, a comparative analysis was conducted to determine whether the papaya plants infected were the infectious clones of the constructed attenuated strains and whether the mutation site was still AT.

[0068] (1) Total RNA extraction

[0069] 20 days after inoculation, 100 mg of tender leaves from the top of papaya inoculated with Agrobacterium pPLDMV-R165I and pPLDMV-R165I-GFP were taken and placed in a pre-cooled 2 ml centrifuge tube without RNAse and added with small steel beads. They were quickly frozen with liquid nitrogen and ground in a tissue grinder for 1 min. The total RNA was extracted using the Polysaccharide and Polyphenol Plant Total RNA Extraction Kit of Tiangen Biochemical Technology (Beijing) Co., Ltd. The specific steps were referred to the instructions of the kit. After the total RNA was extracted, the total RNA extraction was observed by agarose gel electrophoresis.

[0070] (2) cDNA synthesis

[0071] The extracted total RNA was used as a template to synthesize cDNA using TaKaRa's PrimeScript™ IV 1st strand cDNA Synthesis Mix Reverse Transcription Kit (Cat. No. 6215A). The reverse transcription reaction system and reaction conditions are shown in Tables 1 and 2.

[0072] Table 1 cDNA synthesis reaction system

[0073]

[0074] Table 2 cDNA synthesis reaction temperature and time

[0075]

[0076] (3) RT-PCR detection

[0077] Primer pair PLDMV-1485F / PLDMV-3165R and primer pair PLDMV-8924F / PLDMV-9705R were used to identify the HC-Pro multifunctional protein segment on PLDMV and the GFP segment inserted on the NIb / CP protein, respectively. The reaction system and reaction conditions are shown in the table (Table 3, Table 4). The PCR products identified as positive were sent to a biological company for sequencing. After sequencing, the mutation position was compared with the sequence of the strong strain of PLDMV to observe the situation.

[0078] Table 3 RT-PCR reaction system

[0079]

[0080] Table 4 RT-PCR reaction conditions

[0081]

[0082] 2.6 Real-time fluorescence quantitative PCR (qRT-PCR) detection of virus accumulation

[0083] On the 20th, 40th, 60th and 80th days after the weak strain PLDMV-R165I and the strong strain PLDMV-WT infected papaya, the top tender leaves of the plants were sampled to extract total RNA, and reverse transcription was performed after agarose gel electrophoresis. (PLDMV-R165I samples were from papaya plants that were asymptomatic after being inoculated with the weak strain and were positive for identification, and PLDMV-WT samples were from plants with obvious symptoms. Each combination was sampled from three papaya plants according to the time gradient). After agarose gel electrophoresis, reverse transcription was performed using TaKaRa's PrimeScript™ RT reagent Kitwith gDNA Eraser (Perfect Real Time) quantitative PCR dedicated reverse transcription kit (RR047A). The specific experimental steps refer to the kit instructions. The primers for detecting the PLDMV internal reference gene are Pactin-289F / Pactin-289R, and the primers for the target gene are PLDMVq-F / PLDMVq-R. The reaction solution was prepared using TaKaRa's PrimeScript™ RT reagent Kit with gDNA Eraser (Perfect Real Time) quantitative PCR kit (RR820A) and the fluorescence quantitative PCR instrument (model qTOWER 3G) Real Time PCR reaction experiment (no need to add ROX for correction). Each sample was repeated 3 times (duplicate wells), and the 20 μl system and reaction conditions were shown in Tables 5 and 6. Jena software was used to analyze the results of ddCT relative quantification, amplification curve, CT / CQ value, melting curve, etc.

[0084] Table 5 qRT-PCR reaction system

[0085]

[0086] Table 6 qRT-PCR reaction conditions

[0087]

[0088] 2.7 Protein extraction by BPP method

[0089] This experimental method refers to the weighing of 0.1g of papaya top leaves infected with PLDMV-R165I, PLDMV-R165I-GFP, PLDMV-WT-GFP and PLDMV-WT, respectively, adding liquid nitrogen to the leaves and grinding them thoroughly, and then immediately adding an appropriate amount of protein extraction buffer. The protein extraction buffer system is: 100mmol / L EDTA, 50mmol / L Borax, 50mmol / L Vitamin C, 1% PVPP, 1% TritonX-100, 2% β-mercaptoethanol, 30% sucrose, 10mmol / L Tris base, pH = 8.0. After vortexing at room temperature for 10 minutes, add an equal volume of Tris-saturated phenol, vortex at room temperature, and centrifuge at 12000r / min at low temperature. Take the supernatant and add an equal volume of protein extraction buffer, vortex at room temperature for 10 minutes, centrifuge at 12000r / min at low temperature for 20 minutes, take the supernatant and add AM precipitation solution (supersaturated ammonium sulfate methanol solution (add 0.6605g ammonium sulfate to 50mL methanol solution and stir magnetically at room temperature overnight, take the supernatant when needed) and put it in a -20℃ refrigerator for precipitation overnight. After the white precipitate of AM precipitation overnight is deproteinized, add an appropriate amount of Lysis Buffer (protein lysis buffer; final concentration: 7M urea, 2M thiourea, 2% CHAPS, 1.7% (V / V) PMSF, 50mmol / L DTT) and dissolve at 22℃ for more than 2h, centrifuge at 20℃, 15000r / min for 30min, and take the supernatant for storage.

[0090] 2.8 Protein extraction using DTT method

[0091] During the sampling process, five small discs were taken from each sample with the round hole end of a 1ml pipette tip and placed in a 2ml centrifuge tube. Small steel beads were added and immediately placed in liquid nitrogen for quick freezing. After being fully ground using a tissue grinder, 120μl of DTT protein extraction buffer was immediately added. The samples were centrifuged at 4℃, 12000rpm, and the supernatant was taken and stored at -20℃ or -80℃ for later use (this method is suitable for extracting tobacco protein, but not for extracting papaya protein).

[0092] 2.9 SDS-PAGE gel electrophoresis

[0093] Refer to the SDS-PAGE gel preparation kit of Wuhan Boster Biotechnology Co., Ltd. to prepare 12% SDS-PAGE separation gel and 5% SDS-PAGE concentration gel. In the sample preparation process, add 1 microliter of protein loading buffer (5X) to every 4 microliters of protein sample, mix the protein sample and protein loading buffer (5X), and heat at 100℃ or in a boiling water bath for 5-10 minutes to fully denature the protein. When running the protein gel, first run it at 90V for 1 hour, and then run it at 130V for 40 minutes.

[0094] 2.10 Western blot

[0095] 1. After SDS-PAGE gel electrophoresis, cut out a PVDF membrane of the appropriate size for the protein gel and a filter paper of the same size as the PVDF membrane

[0096] 2. Before transferring, soak the PVDF membrane in methanol solution for 30 seconds and then put it into the transfer solution together with the filter paper for 3-5 minutes. After soaking, place it on the semi-dry transfer machine in the order of 6 layers of filter paper, PVDF membrane, protein glue, and 6 layers of filter paper from bottom to top (be careful to avoid bubbles during placement)

[0097] 3. Set the current of the transfer machine to between one and five times of the current = membrane area (length * width) * 1mA, and the transfer time is 1h-1.5h.

[0098] 4. After the transfer is completed, soak the PVDF membrane in methanol solution for 1 minute, then stain it with 1X Ponceau red staining solution for 3-5 minutes, observe the bands and take pictures, then wash the PVDF membrane with pure water on a decolorizing shaker three times, each time for 5 minutes, and then wash it three times with 1X TBST rinse solution, each time for 10 minutes.

[0099] 5. Place the PVDF membrane in a 1X TBST solution containing 5% skimmed milk powder and incubate it on a shaker at room temperature for 1-2 hours. After incubation, wash the PVDF membrane three times with 1X TBST rinse solution, each time for 10 minutes.

[0100] 6. Add the primary antibody (mouse anti-GFP monoclonal antibody) to 1X TBST solution, the ratio of primary antibody to TBST is 1:10000, place the PVDF membrane in the prepared solution and incubate on a 37℃ shaker for 1-2h, or place it in a 4℃ refrigerator and incubate overnight. After incubation, wash the PVDF membrane three times with 1X TBST rinse solution, each time for 10min.

[0101] 7. Add the secondary antibody (HRP-labeled goat anti-mouse IgG) to 1X TBST solution, the ratio of secondary antibody to TBST is 1:5000, place the PVDF membrane in the prepared solution and incubate it on a shaker at 37°C for 1-2 hours. After incubation, wash the PVDF membrane three times with 1X TBST rinse solution, each time for 10 minutes.

[0102] 8. Add the ultra-sensitive chemiluminescent ready-to-use substrate to the PVDF membrane at room temperature and incubate for 1-2 minutes. Place the PVDF membrane in a transparent plastic wrap or protective film, use an absorbent paper towel to remove excess liquid, carefully press out the bubbles, and observe the protein immunoblotting on the Tianneng chemiluminescence image analysis system.

[0103] 2.11 Silencing suppression assay activity detection

[0104] HC-Pro is a viral RNA silencing suppressor of the Potyvirus genus. The HC-Pro-WT and HC-Pro-R165I plant silencing suppressor expression vectors p35S-HC-Pro-WT and p35S-HC-Pro-R165I were constructed. The primers were designed according to the instructions of the Nimble Cloning kit. The primer pair HC-Pro-F / HC-Pro-R was used to amplify the HC-Pro segment on PLDMV from the plasmids PLDMV-WT and PLDMV-R165I, respectively. After amplification, the 1% agarose gel electrophoresis was used to recover the gel for subsequent connection experiments. The PCR recovery product was connected with the pNC-Green-35S vector (the connection product was named p35S-HC-Pro-WT and p35S-HC-Pro-R165I) and then transformed into the competent Escherichia coli DH5ɑ, coated on LB plates containing 50 mg / L kanamycin, and cultured overnight in a 37°C constant temperature incubator. After the monoclonal bacterial solution was selected and identified correctly, it was sent to a biological company for sequencing. After the correct plasmid is extracted by sequencing, it is transferred into the competent Agrobacterium GV3101 (pSoup). After culturing in a constant temperature incubator at 28°C for 48-72 hours, a single clone is selected and identified correctly and can be used for subsequent experiments. During inoculation, P19 (P19 is a tomato dwarf virus Tombusviruses; TBSVRNA silencing inhibitor), p35S-HC-Pro-WT and p35S-HC-Pro-R165I are co-infiltrated with pGreenⅡ-35S-GFP to co-express in Nicotiana benthamiana leaves. The fluorescence changes caused by the effect of HC-Pro on GFP mRNA expression are observed to evaluate the self-activity of the silencing suppressor. At the same time as inoculation, pGreenⅡ-35S empty vector and pGreenⅡ-35S-GFP are used as controls. The inoculation position is as follows: Figure 2 As shown. At 48h after inoculation, the luminescence of the inoculation site was observed with ultraviolet light and photographed and recorded. The luminescence was observed every 12h thereafter. Samples of tobacco injection sites with more severe luminescence were taken 72h after inoculation. Proteins were extracted using the DTT method (refer to 2.8 for the experimental method). Mouse anti-GFP monoclonal primary and secondary antibodies were used for Western-blot experiments to compare the GFP expression content.

[0105] 2.12 Cross-protection experiment of PLDMV attenuated strains

[0106] Using the cross-protection strategy, three different varieties of papaya plants [Huanong No. 1 (transgenic PRSV-resistant), Suizhonghong and Mihong] that had been inoculated with the weak strains PLDMV-R165I and PLDMV-R165I-GFP and identified as positive were challenged with the strong strain PLDMV-WT against the weak strain PLDMV-R165I, and the strong strain PLDMV-WT-GFP against the weak strain PLDMV-R165I-GFP 30 days after inoculation. The challenged papaya plants were set as the experimental group, and 30 papaya plants of each variety were challenged with each of the two strong strains. At the same time as the virus attack, the positive control groups PLDMV-WT and PLDMV-WT-GFP were set up with about 10 strains in each group, and 5 weak strains PLDMV-R165I and PLDMV-R165I-GFP that were not subjected to the virus attack experiment were set up as the weak virus control group (5 papayas of three different varieties inoculated with two weak viruses were left unattacked as the weak virus control group). After the virus attack, the symptoms of the experimental group and the control group were observed and photographed regularly. The papaya seedlings after the virus attack were placed in a greenhouse (26-28℃) for cultivation, and the disease conditions of the three different varieties of papaya seedlings after the virus attack were observed, and the camera was used to observe and record. On the 30th day of the virus attack, the experimental group and the strong virus control group were observed for GFP fluorescence with ultraviolet light (UV) and photographed, symptoms were observed, and the strains in the plants were detected by RT-PCR, and the cross-protection efficiency was calculated. The cross-protection efficiency calculation method is: after a period of time of attacking the weak strain with the strong strain, observe the number of diseased plants in the weak strain after the attack through symptoms, detect and sequence the types of viruses in the weak strain after the attack by RT-PCR, and count the number of diseased plants after the attack. The cross-protection efficiency is the percentage of weak strains that have not developed disease to the total number of weak strains.

[0107] 3 Results and analysis

[0108] 3.1 Identification of asymptomatic weak strains of PLDMV infected papaya

[0109] A papaya strain infected with PLDMV was identified from the papaya planting base in Ledong, Hainan Province, which showed no symptoms compared with the symptoms of the strong strain. Figure 3 ). Total RNA of the attenuated papaya was extracted and reverse transcribed to synthesize cDNA. The full-length sequence of the PLDMV attenuated strain was amplified using the cDNA of the attenuated strain as a template and sequenced. After alignment with the full-length sequence of the wild-type virulent PLDMV strain, it was found that compared with the virulent strain, the asymptomatic plant PLDMV HC-Pro multifunctional protein had a base mutation. Alignment of the full-length amino acid sequence of the encoded polyprotein revealed that the 165th amino acid arginine (Arg, abbreviated as R) on the HC-Pro multifunctional protein encoded by the virus of the asymptomatic weak strain was mutated to isoleucine (Ile, abbreviated as I) compared with the virulent strain ( Figure 4 , Figure 5), the attenuated strain was named PLDMV-R165I. The mutation of amino acid R to I at position 165 of PLDMV HC-Pro is related to the symptom expression of PLDMV in host plants. This provides a reference for constructing the full-length infectious clone pPLDMV-R165I of the attenuated strain with amino acid R mutation to I at position 165 of PLDMV HC-Pro and the infectious clone pPLDMV-R165I-GFP expressing GFP fluorescent tag using the full-length infectious clones pPLDMV-WT and pPLDMV-WT-GFP constructed in our laboratory as templates.

[0110] 3.2 Construction of infectious clones of attenuated strains

[0111] Combined with the full-length sequencing results of the asymptomatic attenuated strain, the site-directed mutagenesis method and In-Fusion splicing strategy were used according to the vector construction schematic ( Figure 1 ), using the plasmids of wild-type virulent strain infectious clones pPLDMV-WT and pPLDMV-WT-GFP constructed in our laboratory as templates, PCR amplified target fragments a, b, c, d ( Figure 6 ) (The difference between fragment b and fragment a is that fragment b has GFP inserted between NIb / CP). Fragments a, c, d (the fragments required to construct the attenuated strain PLDMV-R165I) and fragments b, c, d (the fragments required to construct the attenuated strain PLDMV-R165I-GFP) were directly transformed into Agrobacterium competent GV3101 (pSoup) after In-Fusion ligation. The bacterial solution was identified as follows Figure 7 As shown, the bacterial liquid in lanes 2 (pPLDMV-R165I) and 6 (pPLDMV-R165I-GFP) was selected for full-length sequencing to verify that the sequence was correct. The full-length cDNA infectious clones pPLDMV-R165I and pPLDMV-R165I-GFP were successfully constructed and can be further used in subsequent Agrobacterium inoculation and infection experiments.

[0112] 3.3 HC-Pro 165R mutation to I affects PLDMV symptom production and viral replication and accumulation

[0113] 3.3.1 Symptom observation and virus fluorescence tracing after infection of papaya with strong and weak virulent clones

[0114] Three different varieties of papaya were inoculated with PLDMV-R165I, PLDMV-R165I-GFP, PLDMV-WT and PLDMV-WT-GFP. After 15 days, the top leaves of the papayas in the strong virus control group showed some typical PLDMV virus symptoms such as leaf mosaic, spotting, shrinkage and deformity, while the top leaves of the papayas inoculated with the weak strains PLDMV-R165I and PLDMV-R165I-GFP showed no obvious symptoms like the uninfected papaya plant control. Under ultraviolet light (UV), the main veins of the top leaves of the plants infected with PLDMV-WT-GFP carrying the GFP tag showed obvious green fluorescence, while the top leaves of the papayas infected with the weak virus PLDMV-R165I-GFP showed no green fluorescence and were red like the negative control group ( Figure 8 ). 15 days after inoculation, the luminescence of the top leaves of papayas infected with PLDMV-WT-GFP and PLDMV-R165I-GFP carrying GFP tags was observed under a fluorescence microscope. It was found that strong green fluorescence could be clearly observed in the leaves and veins of the papayas in the strong toxic control group. The luminescence intensity of the papaya leaves infected with PLDMV-R165I-GFP was weaker than that of the strong strain, mainly on the veins ( Fig. 9 ). The PLDMV-R165I-GFP weak strain and PLDMV-WT-GFP strong strain expressing green fluorescent protein can use fluorescent tracing to detect the onset of the virus in the plant earlier, and can also observe the distribution and accumulation of the virus in the plant. Compared with the infection of PLDMV-WT-GFP, the infection of PLDMV-R165I-GFP showed no obvious fluorescence, which is related to the low accumulation of the weak strain in the virus body.

[0115] 3.3.2 RT-PCR detection of PLDMV-R165I and PLDMV-R165I-GFP in infected plants

[0116] About 20 days after the inoculation of the weak and strong strains, the strong strain PLDMV-WT-GFP was observed to have obvious luminescence under ultraviolet light (UV) and fluorescence microscope. The strong strain control group PLDMV-WT and PLDMV-WT-GFP began to develop symptoms of PLDMV. After that, the total RNA of the top leaves of the plants inoculated with the weak strains was extracted and reverse transcribed. The primer pairs PLDMV-1485F and PLDMV-3165R (identifying the HC-Pro protein segment on PLDMV) and the primer pairs PLDMV-8924F and PLDMV-9965R (identifying the GFP segment inserted by NIB / CP on PLDMV) were used for RT-PCR to detect the infectivity of the two weak strains to papaya and determine their infection efficiency. The identification results showed that most of the RT-PCR products amplified the target size band ( Fig.10), sequencing results showed that the 493rd to 494th bases of the weak strain HC-Pro were still AT, indicating that PLDMV-R165I and PLDMV-R165I-GFP can successfully infect healthy papaya plants, can be stably expressed in papaya plants, and the mutant sequence has not recovered to the wild-type virulent sequence. In three different varieties of papaya [Huanong No. 1 (transgenic PRSV resistance), Suizhonghong and Mihong], most of the healthy plants have been infected. After the two different weak strains were inoculated into three different varieties of papaya, the number of weak strains infected by each combination reached more than 45. The infectivity efficiency can reach 90%, indicating that the two weak strains have a relatively high infectivity to the three different varieties of papaya. The papaya plants that have been successfully infected can be used for subsequent symptom observation and preliminary cross-protection experiments.

[0117] 3.3.3 Comparison of virus accumulation in infected plants by qRT-PCR

[0118] Fluorescence quantitative PCR was used to compare the viral accumulation of the weak strain PLDMV-R165I and the strong strain PLDMV-WT. It was found that the viral accumulation of the weak strain PLDMV-R165I and the strong strain PLDMV-WT was much lower than that of the strong strain on the 20th, 40th, 60th and 80th days after the inoculation of papaya with the weak strain PLDMV-R165I and the strong strain PLDMV-WT ( Fig.11 ). The amount of virus accumulation after the weak strain was inoculated into papaya did not increase significantly due to the extension of the inoculation time. The expression of the virus in the host is related to the weakening of symptoms. The lack of obvious symptoms of the weak strain may be related to the low virus content.

[0119] 3.3.4 Western blot analysis of the difference in GFP protein expression between strong and weak strains in infected plants

[0120] The Bpp protein extraction method refers to 2.6, the extracted protein SDS-PAGE protein gel electrophoresis refers to 2.8, and the Western-blot experiment refers to 2.9. The results of the Western-blot experiment showed that the hybridization band of the PLDMV-R165I-GFP protein extracted from the weak strain inoculated with the GFP monoclonal antibody was weaker than that of the strong strain PLDMV-WT-GFP in the control group, which may be related to the low expression level of the virus in the plant. The extracted papaya protein inoculated with the strong strain PLDMVWT-GFP had obvious bands after Western hybridization and the band size was consistent with the target band size of 27.8kDa ( Fig.12). The results are consistent with those of symptom observation after virus inoculation, fluorescent virus tracing observation of luminescence of weak and strong strains, and qRT-PCR comparison of virus accumulation. This indicates that the mutation of PLDMV HC-Pro 165R to I affects virus replication and reduces virus accumulation in plants. HC-Pro165R is a new key amino acid that determines the occurrence of PLDMV symptoms and virus replication and accumulation.

[0121] 3.4 HC-Pro 165R mutation affects RNA silencing suppression activity

[0122] HC-Pro is a viral RNA silencing suppressor of the Potyvirus genus. Amino acid mutations on HC-Pro affect the activity of RNA silencing suppressors. In order to verify whether the 165R mutation affects the activity of HC-Pro silencing suppressors, a silencing suppressor expression vector was constructed. The required fragments for constructing the vector amplification are as follows Fig.13 , using the NC cloning method, primers were designed with reference to the instructions of the Nimble Cloning Kit, and the HC-Pro-WT and HC-Pro-R165I plant silencing suppressor expression vectors p35S-HC-Pro-WT and p35S-HC-Pro-R165I were constructed. They were co-infiltrated with pGreenⅡ-35S-GFP in Nicotiana benthamiana leaves for co-expression. The fluorescence changes caused by the effect of HC-Pro on GFP mRNA expression were observed to evaluate the self-activity of the silencing suppressor. The luminescence of pGreenⅡ-35S empty vector, pGreenⅡ-35S-GFP, P19+pGreenⅡ-35S-GFP, p35s-HC-Pro-WT+pGreenⅡ-35S-GFP, and p35s-HC-Pro-645RI+pGreenⅡ-35S-GFP were observed. The fluorescence leaf GFP observation under ultraviolet light (UV) and Western-blot results showed ( Fig.14 , Fig.15 ): HC-Pro-WT significantly promoted the expression of GFP, while HC-Pro-R165I had no effect on the expression of GFP. The R165I mutation may have affected the RNA silencing inhibitory activity of HC-Pro in PLDMV.

[0123] 3.5 Evaluation of cross-protection effect of PLDMV attenuated strains

[0124] Using the cross-protection strategy, a strong strain of PLDMV (PLDMV-WT / PLDMV-WT-GFP) was used to challenge three different varieties of papaya [Huanong No. 1 (transgenic PRSV-resistant), Suizhonghong and Mihong] 30 days after inoculation with the corresponding weak strain of PLDMV (PLDMV-R165I / PLDMV-R165I-GFP). A strong control group of PLDMV-WT and PLDMV-WT-GFP was set up and 10 trees were inoculated at the same time as the weak strain. After 30 days of challenge, no PLDMV symptoms (mosaic and deformed leaves, etc.) were observed in the experimental groups of the three different varieties [Huanong No. 1 (transgenic PRSV-resistant), Suizhonghong and Mihong] protected by the two weak strains of PLDMV-R165I and PLDMV-R165I-GFP ( Fig.16 ), RT-PCR could not detect the gene fragments of the strong strain, and no GFP fluorescence was observed on the plants under UV light ( Fig.16 ), while the strong-virulent control group showed obvious PLDMV symptoms, and obvious GFP fluorescence was observed on the plants under UV light, indicating that both weak strains showed a certain cross-protection effect. Statistics of non-diseased plants showed that the cross-protection efficiency could reach more than 90% (Table 7).

[0125] Table 7 Cross-protection efficiency of PLDMV-R165I / PLDMV-R165I-GFP in three varieties of papaya

[0126]

[0127] 150 days after the PLDMV-R165I attenuated strain was challenged, the virus types in the attenuated strain were sequenced and analyzed (e.g. Fig.17 ) found that the weak strain was stably expressed in the plant and did not revert to a strong strain.

[0128] Further passage experiments revealed that (e.g. Fig.18 ), the PLDMV attenuated strain did not revert to mutation after the HC-Pro165R amino acid mutation I after four generations of propagation and was stable in the plant.

[0129] The above are only preferred embodiments of the present invention. It should be pointed out that, for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A mutant of HC-Pro protein, characterized in that: It is obtained by mutating the arginine at position 165 of the HC-Pro protein of the virulent strain of papaya malformation mosaic virus to isoleucine; The amino acid sequence of the mutant of the HC-Pro protein is shown in SEQ ID NO:

1.

2. A nucleic acid molecule encoding the mutant according to claim 1.

3. The nucleic acid molecule according to claim 2, characterized in that The nucleic acid molecule has a nucleotide sequence as shown in SEQ ID NO:

2.

4. A vector comprising the nucleic acid molecule according to claim 2 or 3.

5. A host cell transformed or transfected with the vector according to claim 4, characterized in that: The host cell is a microorganism, an animal cell or a plant cell; The plant cells do not develop into a complete plant.

6. The host cell according to claim 5, characterized in that The host cell is Agrobacterium.

7. The method for preparing a mutant according to claim 1, characterized in that: include: Cultivate the host cell according to claim 5 or 6 to obtain a culture containing the mutant.

8. Use of at least one of (1) to (5) for preventing and treating papaya malformation mosaic virus: (1) The mutant according to claim 1; (2) The nucleic acid molecule according to claim 2 or 3; (3) The vector according to claim 4; (4) The host cell according to claim 5 or 6; (5) The mutant obtained by the preparation method according to claim 7.

9. A vaccine, characterized in that Include at least one of the following ①~⑤: ①. The mutant according to claim 1; ②, the nucleic acid molecule according to claim 2 or 3; ③. The vector according to claim 4; 4. The host cell according to claim 5 or 6; 5. The mutant obtained by the preparation method according to claim 7.

10. A method for preventing and treating papaya teratomic virus, characterized in that: Administering the vaccine of claim 9.

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