CN114686516B

CN114686516B - Rice potato leaf curl virus 2 infectious clone and construction method and application thereof

Info

Publication number: CN114686516B
Application number: CN202210418050.6A
Authority: CN
Inventors: 朱丽娟; 韩艳红; 吴建国; 李容柏; 赵珊珊; 张崇涛; 白雅妮; 解晓盈
Original assignee: Guangxi University; Fujian Agriculture and Forestry University
Current assignee: Guangxi University; Fujian Agriculture and Forestry University
Priority date: 2022-04-21
Filing date: 2022-04-21
Publication date: 2023-07-14
Anticipated expiration: 2042-04-21
Also published as: CN114686516A

Abstract

The invention discloses a Rice potato leaf curl virus 2 (RPV 2) infectious cloning vector, a construction method and application thereof, and the vector is obtained by cloning the vector into a pXT binary vector containing a cauliflower mosaic virus double 35S promoter in a homologous recombination mode on the basis of obtaining a complete sequence of an RPV2 genome. The invention prepares the infectious clone which can be successfully inoculated and stably and efficiently infest the plants of the present smoke and rice (indica rice and japonica rice) by an agrobacterium-mediated method for the first time, and lays a foundation for researching the structure and the function of the virus gene and the relation between the virus gene and a host; the forward genetics direction is utilized to research the disease-resistant mechanism of the rice, more potential disease-resistant genes in the rice are possible to be defined, theoretical and practical basis is provided for breeding new disease-resistant varieties, and finally, the yield and quality of the rice are improved.

Description

Rice potato leaf curl virus 2 infectious clone and construction method and application thereof

Technical Field

The invention belongs to the technical field of plant genetic engineering, and particularly relates to rice potato leaf curl virus 2 invasive clone, a construction method and application thereof.

Background

Rice potato leaf curl virus 2 (RPV 2) is a novel virus of the genus potato leaf curl virus found in wild Rice by this experiment, whose virion is in the form of a regular icosahedron, whose genome is a positive single stranded RNA, comprising 5822 nucleotides linked at the 5' end to VPg, encoding 7 proteins by a variety of different translation strategies. The 5 'ORFs are translated into P0, P1 and P1-P2 proteins by genome sequential translation, while the 3' ORFs are translated into P3, P4 and P3-P5 proteins by subgenomic strategy, and a non-ATG-initiated ORF exists in the region between P1-P2 and P3, encoding a small protein P3a.

The genus virus is generally unable to be inoculated mechanically by sap, is obligately transmitted by the specific mediator aphid in a persistent circulating non-proliferative manner, and exhibits phloem limitation.

Invasive cloning refers to the construction of the full-length genome sequence of a virus on a specific vector and has the function of infecting a host. Depending on the type of viral genome (DNA or RNA), it is generally classified as full-length DNA invasive clone or full-length cDNA invasive clone. The construction of virus infectious clone provides an advantageous tool for researching virus replication, gene function, pathogenesis and the like; is beneficial to the design of antiviral strategies and novel viral vectors; is beneficial to the expression of exogenous genes or the silencing of endogenous genes as vectors, and is beneficial to the research of host gene functions from a front genetic system, and the like. At present, the construction strategies mainly comprise two kinds of in vitro transcription method infectious clone controlled by prokaryotic promoters such as T7, SP6 or T3 and the like and in vivo transcription method infectious clone controlled by a cauliflower mosaic virus (Cauliflower mosaic virus, caMV) 35S promoter. The in vivo transcription method is to place the whole genome of the virus cDNA in the downstream of 35S promoter, inoculate the constructed plasmid DNA into host plant by proper method, and utilize 35S promoter to promote cDNA expression in eukaryotic organism to reach the aim of virus infection.

The genome structure of the rice viruses reported at present is complex, most of the rice viruses are in double-chain and negative-chain forms, and few researches on the discovered positive single-chain rice viruses are very few and not complete and deep enough. The construction of rice virus infectious clones has been reported only for rice yellow spot virus (Rice yellow mottle virus, RYMV) and rice Donggaru baculovirus (RTBV), and these two viruses have not occurred on Chinese rice at present, and related research of rice cannot be carried out by using the two viruses. Recently reported Rice Stripe Virus (RSV) infectious clones are only infectious in the benthamic tobacco and cannot be used for Rice, so that no virus infectious clone capable of successfully infecting Rice has been reported so far. The method comprises the steps of constructing wild rice virus invasive clone, researching a rice disease-resistant mechanism from a forward genetics direction in advance, determining more potential disease-resistant genes in rice, providing theoretical and practical basis for breeding new disease-resistant varieties, and finally realizing improvement of rice yield and quality.

Disclosure of Invention

The invention aims to provide an infectious cloning vector of rice potato leaf curl virus 2, and a construction method and application thereof. The rice potato leaf curl virus 2 infectious clone constructed by the invention not only can stabilize the plant benthonic tobacco with high-efficiency infection mode, but also can infect rice. Lays a foundation for researching the structure and the function of the virus genome and the pathogenic mechanism thereof, and makes the research on the disease-resistant mechanism of the rice from the forward genetics direction by utilizing the virus possible to determine more potential disease-resistant genes in the rice, thereby providing theoretical and practical basis for cultivating new disease-resistant varieties.

In order to achieve the above purpose, the invention adopts the following technical scheme:

the rice potato leaf curl virus 2 infectious clone carries a full-length cDNA sequence corresponding to the whole genome RNA of the rice potato leaf curl virus 2, and the full-length cDNA sequence is shown as SEQ ID No. 15.

The construction method of the rice potato leaf curl virus 2 infectious clone comprises the following steps: the full-length cDNA sequence corresponding to the whole genome RNA of the rice potato leaf curl virus 2 is cloned into a vector pXT with a double 35S promoter and ribozyme RZ to prepare the rice potato leaf curl virus.

The construction method of the rice potato leaf curl virus 2 infectious clone comprises the following specific steps:

(1) Amplifying the full-length cDNA sequence of the RPV2 in two sections by using a primer pair XT001F/RPV2-3267R and a primer pair RPV2-3248F/XT3R respectively; primer sequences of the primers XT001F, RPV2-3267R, RPV2-3248F, XT R are respectively shown in SEQ ID No. 1-4;

(2) Combining the purified target fragment with a purified peptideStuI andBamcarrying out recombination reaction on the H I linearized pXT vector, transferring a connection product into escherichia coli, screening positive clones for sequencing, and further obtaining clones with the full-length cDNA sequence of RPV2;

(3) Extracting plasmid containing full-length cDNA sequence clone of RPV2 to obtain its infectious clone carrier XT-RPV2.

The system for double enzyme digestion of the pXT carrier in the step (2) is as follows: 2.5 Mu.g of pXT plasmid, 10 Xcutmart buffer,Stui andBamh I2.5. Mu.L each and ddH ₂ O was made up to 50. Mu.L.

The recombination system in the step (2) is 10 mu L: 5 XCE MultiS Buffer 2. Mu.L, viaStuI andBamh I linearized cloning vector pXT 90 ng, target fragment 1 obtained by amplification of primer XT001F/RPV2-3267R and target fragment 2 obtained by amplification of RPV2-3248F/pXT3R 65 ng and 50 ng,Exnase MultiS 1. Mu.L, ddH, respectively ₂ O was adjusted to 10. Mu.L.

A recombinant bacterium comprising an infectious clone of rice potato leaf curl virus 2. Furthermore, the recombinant bacteria are agrobacterium containing rice potato leaf curl virus 2 infectious clone. Further, the Agrobacterium is obtained by introducing an Agrobacterium strain GV3101 with a pXT-RPV2 vector.

The application of the rice potato leaf curl virus 2 infectious clone in preparing a single virus infection plant model, wherein the single virus is the rice potato leaf curl virus 2.

The application of the recombinant bacteria containing rice potato leaf curl virus 2 infectious clone in preparing a single virus infection plant model.

The rice potato leaf curl virus 2 infectious clone or the recombinant bacteria containing the rice potato leaf curl virus 2 infectious clone are applied to researching the pathogenic mechanism of the rice potato leaf curl virus 2.

The rice potato leaf curl virus 2 infectious clone or the recombinant bacteria containing the rice potato leaf curl virus 2 infectious clone are applied to screening of rice resistance genes.

The invention has the beneficial effects that:

the invention prepares the RPV2 infectious clone which can be inoculated and stably and efficiently infects the host plants of the present smoke and rice by the agrobacterium method for the first time, is favorable for researching the structure and the function of the virus genome and the pathogenic mechanism thereof, and has important significance for researching the disease-resistant mechanism of the rice from the forward genetics direction and defining more potential disease-resistant genes in the rice.

Drawings

FIG. 1 is a strategy for constructing the infectious cloning vector pXT-RPV2.

FIG. 2 is a graph showing the identification of symptoms and infectivity of the same by inoculating RPV2 Agrobacterium. a: infiltrating the symptoms of primary smoke 15 dpi, EV: infiltrating empty carrier pXT, RPV2: infiltrating RPV2; b: the accumulation of 8, 14, 18, 22, 30 and 38 dpi of the leaf RPV2 of the present smoke generating system is detected by using Northern blot, EV: infiltration of empty vector pXT,8, 14, 18, 22, 30, 38 dpi:

day

8, 14, 18, 22, 30, 38 after infiltration of RPV2;

FIG. 3 shows the symptoms and detection of RPV2 Agrobacterium-inoculated rice. a: northern blot detection of accumulation of rice system leaf RPV2, EV: infection of empty vector pXT,15, 22, 30, 37, 44 dpi:

day

15, 22, 30, 37, 44 after infection with RPV2; b: sequencing and analyzing small RNA infected with RPV2 rice; c: distribution characteristics of virus-derived small RNAs along RPV2 genome; d: northern blot detection of virus small RNA accumulation of RPV2 rice infection; e: western blot detects the expression of RPV2 in rice; f: symptoms of rice after infection with RPV2.

Detailed Description

The experimental methods used in the following examples are conventional methods unless otherwise specified.

Materials, reagents and the like used in the examples described below are commercially available unless otherwise specified.

The primer sequences used in the examples of the present invention are shown in Table 1:

TABLE 1

Example 1 determination of the complete sequence of the genome of Rice Potato leaf curl Virus 2 (RPV 2)

1. Extraction of total RNA from plants

The TRIzol method is used for extracting total RNA of wild rice leaves with toxicity, and the extraction method is described with reference to a plant RNA extraction kit.

Placing about 0.1 g leaf into 2.0 centrifuge tube containing steel balls, grinding into powder under liquid nitrogen with plant tissue grinder, rapidly adding 1 mL TRIzol, shaking vigorously for 30 s, incubating at room temperature for 5 min, adding 0.2 mL chloroform, shaking vigorously for 30 s, incubating at room temperature for 5 min,4 deg.C, centrifuging 12000g for 15 min, collecting upper water phase in new centrifuge tube, adding equal volume isopropanol, mixing upside down, standing at-20deg.C for 10-30 min,4 deg.C, centrifuging 12000g for 10 min, removing supernatant, washing precipitate with 75vol% ethanol twice, drying, adding appropriate amount of ddH ₂ O was dissolved and stored at-20 ℃.

2. Determination of RPV 25 ', 3' sequence and Whole genome sequence

Designing primers 5'RACE-RPV2-GSP1, 5' RACE-RPV2-GSP2, 3'RACE-RPV2-GSP1, 3' RACE-RPV2-GSP2 and commonly used AUAP, UAP, R1 and R2 based on the reference sequence assembled by deep sequencing of small RNAs, wherein the sequences are shown as SEQ ID No. 5-12; performing first round amplification of 5', 3' terminal with a 5'RACE-RPV2-GSP1/AUAP primer pair and a 3' RACE-RPV2-GSP1/R1 primer pair, respectively; using the diluted first round PCR product as a template, and respectively using a 5'RACE-RPV2-GSP2/UAP primer pair and a 3' RACE-RPV2-GSP2/R2 primer pair to carry out second round amplification of 5', 3' terminal ends; the PCR product obtained was purified and ligated to pTOPO-Blunt vector is transformed into escherichia coli, positive clones are obtained, and 5 'and 3' terminal sequences of RPV2 are determined through sequencing; based on the determined 5', 3' terminal and reference sequence, primer pair XT001F/RPV2-3267R, RPV2-3248F/XT3R is designed to carry out segmented amplification on the whole genome, first, primers RPV2-3267R and XT3R are respectively used for carrying out reverse transcription on total RNA, and the first strand cDNA of two fragments is respectively synthesized, wherein the reaction system is as follows: 1. mu.g total RNA, 2. Mu.L 2.5 mmol/L dNTPs, 0.5. Mu.L 3' -end downstream primer (10. Mu. Mol/L), 2. Mu.L 5 XM-MLV Buffer, and finally 0.5. Mu. L M-MLV RT enzyme, ddH were added ₂ O was made up to 10. Mu.L. After mixing, incubating at 42 ℃ for 30 min, and finally heating at 85 ℃ for 5s inactivation. Then, using cDNA as a template, respectively amplifying with primer pairs XT001F/RPV2-3267R and RPV2-3248F/XT3R to obtain two DNA fragments A1 and A2, wherein a PCR amplification system is as follows: 31. mu L ddH ₂ O, 10. Mu.L of 5 XQ 5 Reaction Buffer Buffer, 1. Mu.L of 10mM dNTPs, 2.5. Mu.L of each of the upstream primer and the downstream primer, 2.5. Mu.L of cDNA template, 0.5. Mu. L Q5.5 High-Fidelity DNA Polymerase, and the total reaction system was 50. Mu.L. The reaction conditions of each fragment are: 30 s at 98 ℃;98 ℃ for 10 s,60 ℃ for 30 s,72 ℃ for 1.5 min,30 cycles; and at 72℃for 10 min. After the product is recovered and purified by glue, the product is cloned to pTOPO-Blunt vector to obtain recombinant plasmids pTOPO-A1 and pTOPO-A2, at least three positive clones are selected from each fragment to sequence, the result is analyzed and compared by DNAMAN 10.0 software, the similarity between each clone of the same fragment is ensured to be more than 99%, and the DNAMAN software is utilized to splice the same fragment to obtain the full-length cDNA sequence of RPV2 (the specific nucleotide sequence is shown as SEQ ID No. 15).

EXAMPLE 2 construction of wild Rice Virus-invasive clones

The complete sequence of RPV2 nucleotides was amplified in two steps using the plasmids pTOPO-A1 and pTOPO-A2 sequenced correctly as in example 1 as templates, using primer pairs XT001F/RPV2-3267R and RPV2-3248F/XT 3R; subsequently, the purified amplified fragment of interest was amplified with a Cloneexpress MultiS One Step Cloning Kit recombinant cloning kit (Nanjinouzan Biotechnology Co., ltd.)StuI andBamh I linearized pXT vector was subjected to recombination reactions using a 10. Mu.L system in which 5 XCE MultiS Buffer 2. Mu.L, exnase MultiS 1. Mu.L, linearizedThe cloning vector pXT 90 ng of (1) the target fragment obtained by amplification of primer XT001F/RPV2-3267R and the target fragment 2 obtained by amplification of RPV2-3248F/XT3R are about 65 ng and 50 ng, ddH, respectively ₂ The mixture was stirred to 10. Mu.L with O, and the components were gently mixed and reacted at 37℃for 30 minutes, immediately after the completion of the reaction, on ice. Then transferring the connection product into escherichia coli JM109, selecting positive clone for cloning and sequencing, and sequencing to obtain the RPV2 cDNA sequence shown in SEQ ID No. 15. The plasmid from which the correct positive clone was sequenced was extracted and designated pXT-RPV2 (FIG. 1). Subsequently, it was introduced into Agrobacterium strain GV3101 to obtain pXT-RPV2 (GV 3101) strain having infectivity.

EXAMPLE 3 PXT-RPV2 invasive cloning inoculation

1. The smoke generation inoculation

Firstly, a single colony of agrobacterium GV3101 containing plasmid pXT-RPV2 was picked and inoculated in LB medium containing kanamycin (Kan, 50 mg/L) and rifampicin (Rif, 50 mg/L) resistance, and shake culture activated overnight at 28 ℃; the following day the activated bacterial liquid was transferred to the desired liquid LB (in addition to 50 mg/L Kan+Rif, 10mM MES pH 5.6, 45 mM acetosyringone) at 1:100, and OD was cultivated at 28 ℃ ₆₀₀ To 0.8-1.0; finally, 4000 rpm/min, room temperature, centrifuging for 10 min, and collecting thalli; with MgCl at a final concentration of 10mM ₂ 10mM MES (pH 5.6), 150 mu M acetosyringone (As) buffer was resuspended and the bacterial concentration was adjusted to OD ₆₀₀ For infestation, 0.8-1.0 is left at room temperature in the absence of light for at least 3-4 h. The agrobacteria liquid to be used is sucked by a1 mL injector, the needle is removed, the injector is lightly propped against the back of the blade, the blade is slightly forcefully used but not damaged, the injection is slowly performed, and the blade is filled with the impregnating liquid as much as possible.

2. Rice inoculation

The procedure from culturing to collecting the cells was consistent with the above-mentioned raw tobacco, and the cells were resuspended to OD ₆₀₀ After 2-3, the plates were plated on LB plates containing kanamycin (Kan, 50 mg/L) and rifampicin (Rif, 50 mg/L) resistance and incubated for 24-48 hours. The above-mentioned cultured Agrobacterium is picked up and spread on the midvein (10-15 days old) of the rice leaf and stem, and then scraped (transversely) and pricked with an extremely fine glass needle, causing slight injury.

Example 4 detection of infectivity of pXT-RPV2 infectious clones

1. Identification of infectivity in Bensheng cigarette

To test the biological activity of the RPV2 invasive cDNA clone, GV3101 containing pXT-RPV2 was inoculated into 4-5 week old benthamiana (wild type and dcl2/4 i), empty vector pXT (EV) was used as a negative control, and virus was detected on the 5 th day (5 dpi) after inoculation of the infiltrated leaf. Subsequently, we collected 6 systematic leaves at different times (8, 14, 18, 22, 30, 38 dpi) for RT-PCR detection, and we found that the wild-type benthamiana virus started to accumulate later than the RNA interference related mutant DCL2/4i (mutant interfering with DCL2 and DCL4 gene expression) and also accumulated much less. RNA with low viral content is not suitable for hybridization due to limited RNA hybridization sensitivity. Therefore, we selected dcl2/4i samples for further characterization by RNA hybridization, found that the virus accumulation trend increased and then decreased, and eventually tended to stabilize (fig. 2 b), and periodically observed symptoms of inoculated plants to find no apparent symptoms of infiltrated benthamic smoke (fig. 2 a).

2. Identification of infectivity in Rice

In order to determine whether the RPV2 invasive clone can infect rice plants, we inoculated two rice varieties Japanese sunny @ respectively with Agrobacterium containing pXT-RPV2Oryza sativa L.spp.japonica，var niponbare) And 9311（O. sativa L1 spp1 indica，var1 9311）The empty vector pXT (EV) was used as a negative control. According to the rule of one week interval, 5 system leaves with different time (15, 22, 30, 37 and 44 dpi) are collected, firstly, RT-PCR detection is carried out on 15 dpi samples, and the virus can be detected in 2 kinds of rice.

To further understand the replication process of the virus in rice, samples at different time points were examined by RNA hybridization, we found that the virus accumulated in Japanese was increasing and then decreasing, and eventually tended to stabilize, with the highest virus content at 30 dpi, but the virus accumulated relatively stably and at a higher level at 9311 (FIG. 3 a). Symptom observation revealed a more pronounced tillering reduction after one month of virus infection to rice (fig. 3 f). These results indicate that the RPV2 invasive clone can be transferred to the non-natural host rice by agrobacterium inoculation, allowing it to replicate and move systematically. pXT-RPV2 is the first infectious clone in China to successfully infect rice systematically. To verify the viral infectivity from different levels, we performed protein hybridization (FIG. 3 d) and small RNA hybridization (FIG. 3 e) on samples of 44 dpi, both positive. The results of the small RNA hybridization confirm that there are small RNAs of viral origin triggered by RPV2 and mediated by plant RNA silencing in japan and 9311, to further understand the small RNAs of viral origin, we constructed two different small RNA libraries for deep sequencing, one from the sunny leaf infected with RPV2 and the other from the 9311 leaf infected with RPV2. Both cases indicate that most small RNAs have two polarities, with a large distribution at 20, 21 and 22 nt,21 nt having a major peak (fig. 3 b). And it was found that RPV2 derived small RNAs cover almost the entire viral genome, with no apparent regional preference. Unlike most other positive single stranded RNA viruses, the viral micrornas are mainly derived from the positive strand of the virus, and the proportion of RPV2 viral micrornas derived from the positive and negative strands is close and biased very little (fig. 3 c).

The specific experimental steps are as follows:

RT-PCR detection:

the primers RPV2-5301F and RPV2-5805R are designed, the sequences are shown as SEQ ID No. 13-14, total RNA of the system leaves is extracted, genome removal is carried out, RT-PCR detection is carried out (DNA pollution of the sample is avoided), and the genome removal method is as follows: the system is as follows: 1. mu.g total RNA, 1. Mu.L DNase I enzyme (2U/. Mu.L), 1. Mu.L 10 XDNase I Buffer, add ddH ₂ O is added to 10 mu L, the reaction is carried out for 20-30 min at 37 ℃,1 mu L of protective agent is added, and the reaction is inactivated for 10 min at 75 ℃. The tobacco RNA extraction method was referred to as the hot phenol method, and the rice was referred to as the TRIzol method of example 1.

High molecular weight Northern blot detection:

total RNA (10. Mu.g) was separated by electrophoresis on a 1.2% (w/v) agarose-formaldehyde gel containing formaldehyde (37%, 12.3 mol/L) and transferred to a Hybond N+ membrane by capillary; an ultraviolet crosslinking film;methylene blue staining was used to monitor loading. Adding appropriate amount of Church Buffer (1% BSA,1mmol/L EDTA,7% SDS,0.5mol/L Na) ₂ HPO ₄ ) Prehybridization was performed at 65℃for at least 1 h, followed by addition of the prepared specific DNA probe (nts 3451-4120), hybridization at 65℃for 12-16 h. The probe preparation 20 mu L system is: 250 mu M dATP+dCTP+dGTP,67.5 mu M dTTP,32.5 mu M Biotin-11-dUTP,1 mu g DNA template, 2 mu L10 xLA Taq Buffer, 0.2 mu L TaKaRa LA Taq, and 0.8 mu L of each of the upstream and downstream primers RPV2-3451F and RPR2-4120R, wherein the sequences are shown as SEQ ID No. 16-17. The reaction conditions are as follows: 3 min at 95 ℃; 30 s at 95 ℃,30 s at 57 ℃,30 s at 72 ℃ and 30 cycles; and at 72℃for 10 min. The washing was performed twice with 1 XSSC and 0.2 XSSC (containing 0.1% SDS) at 65℃for 10 min each, and then with 0.1 XSSC at 65℃for 5 min. Then washed with chemiluminescent nucleic acid detection module kit (Thermo ScientificTM, no. 89880) according to the manufacturer's instructions. The signals were collected in image 600 (GE Amersham).

Low molecular weight Northern blot detection:

separating low molecular weight RNA by 15% polyacrylamide denaturing gel electrophoresis; removing and cutting glue after bromophenol blue is out, transferring the part below the xylenonitrile to a membrane, dyeing the part above the xylenonitrile blue with nucleic acid dye for 10 min, and shooting and preserving with a BIO-RAD gel imaging instrument to serve as loading control; transferring the small molecular weight RNA to a nylon membrane by a semi-dry transfer method, and sequentially assembling a membrane transfer structure from the bottom (positive electrode) to the upper layer: three filters, film (right side up), glue (note bubble removal), three filters. The transfer conditions are 10V-5 min and 15-V-35 min (Trans-Blot SD Semi-Dry transfer Cell) (Bio-Rad)]The method comprises the steps of carrying out a first treatment on the surface of the After completion of transfer, ddH for membrane ₂ O flushing and blow drying, pouring the prepared EDC solution into a tray paved with filter paper until the liquid does not flow, putting the film on the upper side of the filter paper, sealing the preservative film, and crosslinking the EDC at 55 ℃ in a dark place for 1.5-2.5 h, wherein the EDC is specifically prepared as follows: 82. mu L methylimidazole, 1 mol/L HCl 100 mu L, ddH ₂ O8 mL, EDC (final addition) 0.251 g. After the crosslinking is finished, the membrane is washed and dried, the front surface is rolled inwards into a hybridization tube, a proper amount of prehybridization buffer solution is added, and the hybridization is carried out at 42 ℃ for at least 1 h; addingAn appropriate amount of probe (1. Mu.L diluted to 49. Mu.L ddH) was added ₂ O) hybridization overnight at 42 ℃, 20. Mu.L of the probe preparation system is: 100 ng purified RPV2 genome full-length PCR product as template, 2 [ mu ] L10×T7 polymerase Buffer, ATP, CTP, GTP mmol/L2 [ mu ] L, TTP 10 mmol/L1 [ mu ] L,100 [ mu ] mol/L UTP 1 [ mu ] L, T7 polymelae 2 [ mu ] L, ddH ₂ O was fixed to a volume of 20. Mu.L. The labeled mixture was reacted in a water bath at 37℃for 1-3 h (the reaction time was prolonged when the fragment was short). Hydrolyzing the probe: DNaseI (RNase free) 1 [ mu ] L of water was added to the mixture and the mixture was reacted in a 37℃water bath for 30 minutes to digest the DNA template. Adding 300 mu L200 mmol/L carbonic acid buffer solution [80mmol/L NaHCO ] ₃ (6.72 mg/mL)+120 mmol/L Na ₂ CO ₃ (12.77 mg/mL)]The probe was hydrolyzed to 50-100 nucleotides at 60℃with about 1-3 h. And adding 20 mu L of 3mol/L NaAc (pH 5.2) to terminate the reaction, and completing the preparation of the probe. The wash membrane was consistent with Northern blot of high molecular weight RNA.

Western blot detection:

adding 100-200 μl of protein extraction buffer (0.25M Tri-HCl,8% beta-mercaptoethanol, 20% glycerol, 8% SDS) into 0.1-g leaves ground into powder, steaming at 95deg.C for 10 min, 4deg.C, 12000 rpm/min, centrifuging for 10 min to obtain supernatant, transferring protein onto nitrocellulose membrane after electrophoresis of appropriate amount of supernatant by SDS-PAGE, transferring into appropriate amount of sealing solution (TBST contains 3% BSA), sealing at 37deg.C for at least 1 h; adding antiserum into a sealing solution according to the volume ratio of 1:1000, reacting for 1 h at 37 ℃, rinsing the membrane for 3 times by using TBST buffer solution, and 10 min each time; then adding diluted secondary antibody (1:40000) for reaction for 30 min, and rinsing with TBST for 3 times for 10 min each time; and (5) developing.

The above examples are only illustrative of the preferred embodiments of the present invention and are not intended to limit the present application, and various modifications and improvements made by those skilled in the art to the technical solutions of the present invention should fall within the scope of the present application without departing from the spirit and principles of the present application.

SEQUENCE LISTING

<110> Fujian university of agriculture and forestry, university of Guangxi

<120> rice potato leaf curl virus 2 infectious clone, construction method and application thereof

<130> 17

<160> 17

<170> PatentIn version 3.3

<210> 1

<211> 35

<212> DNA

<213> XT001F

<400> 1

catttcattt ggagaggaca aaagatatcc aggca 35

<210> 2

<211> 20

<212> DNA

<213> RPV2-3267R

<400> 2

tcaccggaat ggcgaggtca 20

<210> 3

<211> 20

<212> DNA

<213> RPV2-3248F

<400> 3

tgacctcgcc attccggtga 20

<210> 4

<211> 32

<212> DNA

<213> XT3R

<400> 4

gccatgccga cccgggacca gagttccgaa ga 32

<210> 5

<211> 24

<212> DNA

<213> 5’RACE-RPV2-GSP1

<400> 5

catcatcttc ttagatgggg gctg 24

<210> 6

<211> 20

<212> DNA

<213> 5’RACE-RPV2-GSP2

<400> 6

agcgtttgca gcatacacta 20

<210> 7

<211> 21

<212> DNA

<213> 3’RACE-RPV2-GSP1

<400> 7

gtctcgacaa ggaatagtcg t 21

<210> 8

<211> 19

<212> DNA

<213> 3’RACE-RPV2-GSP2

<400> 8

atcagtagca ctggccaga 19

<210> 9

<211> 38

<212> DNA

<213> AUAP

<400> 9

ggccacgcgt cgactagtac gggggggggg gggggggg 38

<210> 10

<211> 32

<212> DNA

<213> UAP

<400> 10

ctactactac taggccacgc gtcgactagt ac 32

<210> 11

<211> 47

<212> DNA

<213> R1

<400> 11

taccgtcgtt ccactagtga tttcactata ggtttttttt ttttttt 47

<210> 12

<211> 23

<212> DNA

<213> R2

<400> 12

taccgtcgtt ccactagtga ttt 23

<210> 13

<211> 23

<212> DNA

<213> RPV2-5301F

<400> 13

aataggggcc caagtcctcc tgt 23

<210> 14

<211> 25

<212> DNA

<213> RPV2-5805R

<400> 14

gactacaggt gcaccaatcc tgtga 25

<210> 15

<211> 5822

<212> DNA

<213> full-length cDNA sequence of RPV2

<400> 15

acaaaagata tccaggcagg tcctgtgata tcttccactc agagcagctt acgcgcctga 60

gagctttcag agcagcttta gcgcctgaac gcaaactgaa tagcagcttt aagcgctttc 120

agaacgagcc agccatacac actcgttttt agtgtatgct gcaaacgctc gagagtgggc 180

aactagtcgt ttcatacgat tttgttgctg agattagcac gactgcggcg gtctacgtcc 240

tttcgttgct cccagacttc tttcaaatct cacttttaac ttaccattat ggagttccac 300

agctacgcag ttttcttgct agctgttttc accttgctcc ccttctcatc tctgggaagg 360

ctcgaagcat cggggggagt cgacttattt ttcgacgtag ggaagcccat catcttctta 420

gatgggggct ggcaacaggc tacactcccc gcttggaact caataagcga tccgctatca 480

tgcacttgcg agaaatgtcc ggaacttaca gagagcagct actcagagtt gagtcaagcg 540

ctctttcaaa aagtctccgc gcaaacccaa cagctcttct cagaggctcg tggtacttcc 600

gcgcgtgcat ggaactcaat ttgcgcgaac ttgaaagtaa cgttccatct tgtgttggga 660

acattcctct ggctgaccgc ctacttttgg gcgtacatat tgctggcggt gttgtcaact 720

atttggacgc ttttggcgca acattatgtt ccggtcgcat tactggtatc gctcggtatt 780

ataactgtgc ttatttgccg ggcgttcaag tggatttttg ggactgggcc aacatctcta 840

ttcatagcca gcctgaagta tattttcaag actcttacct tcagaaaata cttcgatgaa 900

aaggctatag agggctacaa agcttactct atcccgcaga agcctccaaa gcacagcgtg 960

gttcaaatca gacggtctga taaatctcac atcggatatg ccgtgagcgt ttcattgtac 1020

aatggcaaaa gcgctctggt aactgcaaac cacaatatgg aggagggctg tgaatttcac 1080

tcattccgca cgggtcgcgc cattaaggct gctgacttca ggcttctttt cctttcaaag 1140

gagcttgatg taatccttgt tgaaggtccg cccaactatg aaagtgtgtt gggatgcgga 1200

agtgtccact tcactacctc tgatttgctt gcaaagtgtc cagcagcact ttacgcttat 1260

gaggatggtg aatggttaca taaatctgcg tcggtggtcg ggcaccatga ccactttgca 1320

actgttcttt cccagaccca aaagggccac tccggtggcg gttactttca tgggaagact 1380

cttgtaggcc tgcacaaagg ccaccctggg gtggaataca attataacct tatgatcaca 1440

attcccccca ttcccggctt gactagtcct ctctactcgg ttgaatcgga tcctccgcag 1500

ggtcgcgtct tctcggatgg cgaagtcgac agaatcgaac aaatcgccga ggagactgct 1560

gaacgcctcg aaaaagaggc gcaaaggatt ctcgagtaca aacctgtgag cggtcgcctt 1620

tgggctgact acgaggacga gttgggaaac gagatggcgg ccgcgcccgc ctcaacaacc 1680

gcaagcgccc tacccgccgg caccccttcc gtgcccattg cccgcgaggt aggtgcagct 1740

ttggaagcgc cctctccacc agcagctatt gcaccatccc tggacattcc tgtgggttct 1800

ggcctacagg acataacgaa tcaagtgatc cagcgattgg tgaacgcggt ggacatctca 1860

agagtggaga aatctgtgat cgatcaaatc gcacagagcg ccctcaagaa gccccgtccg 1920

caatctcgcg ggaagcgggg ctctggcagc aagcagaaga ctggaaacag tacttcgcct 1980

ccctctacac ctggaaagag gagcagcaac cagaagccat cccaggcttc aagacagtcg 2040

gcaaactcaa atccccccaa tattttccgc gtgccaaaag cagctcagaa tgggggaaaa 2100

gggtatgtgc cgaacaccct gagctggcga ccaaaacagc cggtttcggg tggccccagg 2160

tcggggccaa cgcagaactg agatctctca gactgcaagc agcacgctgg cttgaacgct 2220

ccaagtcagc caaacgtccg tctgatgctg ctagacagag cgtgatcaat cgcaccgtca 2280

gggcctactc aaaatgtaaa actaacatgc ccaggtgcac gcaagccgca ttaagctgga 2340

gatcatttct tgaagatttt aatgaagcaa ttcattcact tcagcttgat gcaggggttg 2400

gagtacccat gatagctgcc ggatggccca cacacagggg gtgggtcgaa gatccagagc 2460

gccgtggtgt actcgcgcgc cttacctacg accgcttact taagatgtcg caggcaagag 2520

cagccgggac cccgatagag cttgtaaagg aaggtctctg tgatccaatt cgtctcttcg 2580

taaaacaaga gccacataaa cagagcaagc ttgatgaagg tcgctaccgt ctcatcatgt 2640

ccatttcctt ggttgatcaa ctggtagccc gggttctatt tcaagctcag aacaaatccg 2700

agattgcctt gtggagggcg atcccgtcga gacccggttt tggcttatcc accgaggacc 2760

aagttcacga tttcatggaa gtcctcgcca gcactgttgg atgcagtgct gaggaggttt 2820

gcagccagtg gcgcaacctc ctcattccca cagactgttc cggttttgac tggtcagtct 2880

ccgactggat gctcgaagat gatatggcgg tgcgcaatgc gttaaccatc aattgcaacg 2940

agctcacgaa gcacatgaga gctgtatggc tacattgttt atccaattca gtcatgtgtt 3000

tgtcggacgg gaccatgcta tcccaggaga accctggtgt gcaaaagagc ggctcgtaca 3060

acacgagctc gaccaactcg cgcatcaggg tcatggcagc ctatcactgc ggcgcctcct 3120

gggcaatggc aatgggggat gatgccctcg aagccccaga tactgatctg agcaagtata 3180

aagaactggg gtttaaagtc gaagtcagtg gagagttgga attttgctct catattttta 3240

aagcccctga cctcgccatt ccggtgaacg ccaacaaaat gttgtaccgc ctcatccacg 3300

gttacaaccc ggaatgtggc aaccatgagg tacttaacaa ctacctcaat gcagctgtct 3360

ccgtgctgca tgagctccgc catgacaaag agctggttgc caaactcttt gagtggttga 3420

tttccgacgt caccacaaaa caaagctaag gaggagtcgg agtgagccag acatacgtaa 3480

gctgcaagta ctggacactt agccttacac atataccacc acaattgatt ttcttgcagg 3540

tttctgttta ggttttctaa caggaattcc tatcactgtg gtgtgcactt attttatcta 3600

cctaaaagtc tccagccacg tgcgttcaat cgtgaatgag tacggtcgtg gttagaggca 3660

atggcagtgg ttcgcgcccc cgaggacgaa ggggtcggcg ggctcctcgt agagctcagc 3720

cagtggttgt ggtcgccccc ccccaacagg gaggacaacc tcgacgacgt cgagcgagac 3780

gaagaagtac tcgtaggaac agaagaaatg ctgttggagg gacaagtcgt tatgaaacac 3840

tcgtattcac aaaggacaac ctcaatggcg acgcccaagg gtatctcacc tttgggccgt 3900

cgttatctga ttacccggca ttccaaaatg gacttcttaa agcctaccat gagtataaga 3960

tcacaatggt cacaattcag ttcgtctccg aagccgcctc cacggctgca gggtcaattg 4020

cttacgagct ggacccccac tgcacatcaa cggcgctcac ttcaacgata aacaagttca 4080

gtatcaccaa gaatggtact cggacatttc aagcgtcgaa gataaacggg ttacaatggc 4140

atgacacttc aactgatcag ttccgcgtcc tttataaggg cagcggagcg aagaacgcgc 4200

ttgctggttc tttccgagtc accatgcgcg ttaatatgca aaacccgaaa taggtagaag 4260

cagagcctgg gcctagtcca agcccagccc catcacccag cccccaaccc cagccaactc 4320

cgcgagaggc ccgatttatc gtgtactccg gggtggctca agttcggatt cgagctcaag 4380

ggacggatga cgccataggc gtttacgacc cccccataca aactttcagg tacgtggagg 4440

atgaaaaatt ctataaattc gaccttaacg ctggttggta ttcaaataca cagctaaagg 4500

ccgtttttat gctgatagtg ccagtgccgc aaggcatttg gtctgtgcag atttctgcag 4560

aggggtatca acccacttcc agcaccacag atcctaatat ggggaaaatc gatggcttga 4620

tagcctacga tgactcatct gaggggtgga atatcggcat gggaaagaat gtttccatca 4680

caaataatag agctgataac acgtggaagt atgggcatcc agatttggaa gtcaatgcat 4740

gccacttcaa tcagaagcaa tgtttggaac gtgatggaat tatctccttt catattgaaa 4800

ctaccggtga agacgccaat ttctttttgg tggctccacc ggtacagaag ctctcgaagt 4860

acaattatgc tgtatcttat ggtgcgtgga ccgatcgaga catggagata gggttgatta 4920

cgatcacttt ggatcaaaaa ttaggctctg cccttacgag agaagccagg aaaggacacc 4980

ctggagcgca cacaacaaac actctcgttg aacctgcgcc ggagaaggaa aactccgata 5040

acgaaccctc acctgcgggt gatgacgagg agcagcttga tagcgccctc ccaacccagg 5100

aaggtacggt tgttggcgtg cccgttggta tgactgttag ttcggacagc tccgacgatg 5160

acaaaccggg aattacattc cgacctgggg accgcatggc tgttgatgaa cccatgagaa 5220

aacctgacac gagcgcacga gaccggcgtg agtatttgct ccgagaccgg gaccccttag 5280

cctacacgat cgagaaggaa aataggggcc caagtcctcc tgttgaggag gatccgtgga 5340

aacatgttag gtcattccaa aagcagcagt cagcccccac tgaatctgga cgttcagagg 5400

gggcattgcg ggggggttct cttagaccga gagaaacccc ggcgcctagt gctaaacccc 5460

gaagcgtgag ccctcccaaa acagtgaagt ccagtacttc ctttatgggg gtgcttcgaa 5520

agaaagagga cactaggtta gctaagttga cgactgaaca agcgttgact cataggcgaa 5580

tcaagaatca aagcgggtta gtcgccgctg agaattattt gaaatcgcta ggtctcgaca 5640

aggaatagtc gttaaaaccc tgacaaacac gagccctttg ctggccgggg gaactgctcg 5700

tgttaatcag tagcactggc cagaacgaaa atcccacagg gatgagtagg cctgagtaag 5760

tatcccaagt taggggcctt cacaggattg gtgcacctgt agtcccttct tcggaactct 5820

gg 5822

<210> 16

<211> 18

<212> DNA

<213> RPV2-3451F

<400> 16

ggaggagtcg gagtgagc 18

<210> 17

<211> 22

<212> DNA

<213> RPV2-4120R

<400> 17

gacgcttgaa atgtccgagt ac 22

Claims

1. A rice potato leaf curl virus 2 infectious clone, characterized in that: the infectious clone carries a full-length cDNA sequence corresponding to the rice potato leaf curl virus 2 whole genome RNA, and the full-length cDNA sequence is shown as SEQ ID No. 15.

2. The method for constructing rice potato leaf curl virus 2 infectious clone according to claim 1, wherein: the full-length cDNA sequence corresponding to the whole genome RNA of the rice potato leaf curl virus 2 is cloned into a vector pXT with a double 35S promoter and ribozyme RZ to prepare the rice potato leaf curl virus.

3. The method for constructing rice potato leaf curl virus 2 infectious clone according to claim 2, wherein: the method comprises the following specific steps:

(1) Amplifying the full-length cDNA sequence of the RPV2 in two sections by using a primer pair pXT001F/RPV2-3267R and a primer pair RPV2-3248F/pXT3R respectively; the sequences of the primers pXT001F, RPV2-3267R, RPV2-3248F, pXT R are respectively shown in SEQ ID No. 1-4;

(2) Combining the purified target fragment with a purified peptideStu I andBamh I linearized double 35S promoter andcarrying out recombination reaction on a pXT vector of ribozyme RZ, transferring a connection product into escherichia coli, screening positive clones for sequencing, and further obtaining clones with the full-length cDNA sequence of RPV2;

(3) Extracting plasmid containing RPV2 full-length cDNA sequence clone to obtain its infectivity clone carrier pXT-RPV2.

4. A method of construction according to claim 3, wherein: the system for double enzyme digestion of the pXT carrier in the step (2) is as follows: 2.5 Mu.g of pXT plasmid, 10 Xcutmart buffer 5. Mu.L,Stu i andBamh I2.5. Mu.L each and ddH ₂ O was made up to 50. Mu.L.

5. A method of construction according to claim 3, wherein: the recombination system in the step (2) is 10 mu L: 5 XCE MultiS Buffer 2. Mu.L, viaStu I andBamh I linearized cloning vector pXT 90 ng, target fragment 1 obtained by amplification of primer pXT001F/RPV2-3267R and target fragment 2 obtained by amplification of RPV2-3248F/pXT3R 65 ng and 50 ng,Exnase MultiS 1. Mu.L, ddH, respectively ₂ O was adjusted to 10. Mu.L.

6. A recombinant bacterium comprising the rice potato leaf curl virus 2 infectious clone of claim 1.

7. The recombinant bacterium according to claim 6, wherein: the recombinant bacteria are agrobacterium containing rice potato leaf curl virus 2 infectious clone.

8. The use of the rice potato leaf curl virus 2-infecting clone of claim 1 or the recombinant bacterium containing the rice potato leaf curl virus 2-infecting clone of claim 6 in the preparation of a single virus-infecting plant model, characterized in that: the single virus is rice potato leaf curl virus 2.

9. Use of the rice potato leaf curl virus 2 infectious clone of claim 1 or the recombinant bacterium containing the rice potato leaf curl virus 2 infectious clone of claim 6 for studying the pathogenic mechanism of rice potato leaf curl virus 2.

10. The use of the rice potato leaf curl virus 2-infectious clone of claim 1 or the recombinant bacterium containing the rice potato leaf curl virus 2-infectious clone of claim 6 for screening rice resistance genes.