Industrial hemp bifid virus gene and application thereof

Abstract

The invention belongs to the technical field of plant virus detection, and particularly relates to an industrial hemp bifid virus gene and application thereof, the virus gene comprises two dsRNAs, the dsRNA1 sequence is shown as SEQ ID NO: 1, and the dsRNA2 sequence is shown as SEQ ID NO: 2, the invention provides an industrial hemp bifid virus gene sequence, and the industrial hemp bifid virus gene is obtained by utilizing the sequence provided by the invention and adopting the existing technical means. The prepared primer, kit or antibody protein and the like for detecting the virus can be used for culturing virus-free industrial cannabis sativa seedlings, detecting high-quality industrial cannabis sativa seeds, detecting industrial cannabis sativa diseases and the like, and the diseases can be quickly diagnosed and timely prevented and controlled.

Description

Technical Field

[0001] The invention belongs to the technical field of plant virus detection, and specifically relates to an industrial hemp dichotomous virus gene and an application thereof. Background Art

[0002] Industrial hemp refers to the type of hemp variety with a tetrahydrocannabinol (THC) content of less than 0.3% in the dry matter of the hemp plant. Industrial hemp is a "treasure" all over. The stems can be made into plant fibers, the flowers and leaves can be used to extract CBD, the seeds can be used to extract edible oil and protein powder, and can be made into Chinese medicinal materials for use in cosmetics. The wide application of industrial hemp in many fields is gradually being recognized, developed and utilized by people. Yunnan is one of the two provinces in my country that allows the cultivation of industrial hemp. It can be legally promoted and utilized under the supervision of the public security. Industrial hemp is planted in 38 counties in 13 states. During the cultivation, it was found that industrial hemp showed symptoms such as yellowing of mosaics and leaves, chlorosis of leaves, small leaflets with clear veins, dwarfed leaves, and plant death, which caused certain losses to the quality and yield of industrial hemp. After research, it was found that a dipartite virus caused the disease.

[0003] Partitiviridae is a type of virus with a double-stranded RNA (dsRNA) genome. The genome fragment is about 1.3-2.5 kb in length. Each of the two dsRNAs has only one open reading frame. dsRNA1 is responsible for encoding RNA-dependent RNA polymerase (RdRp), while dsRNA2 is mainly responsible for encoding capsid protein (cp). Dipartite viruses have been shown to infect a wide range of hosts, including plants, fungi, and protozoa. The yield and quality of infected plants and crops will decrease. Dipartite viruses are inherited from mothers to offspring through vertical transmission, and exist in both asexual and sexual reproduction. In asexual reproduction, the mother's roots, stems, leaves, callus, etc. contain viruses. After cultivation with roots, stems, leaves, and callus, the new generation of plants will carry the virus. Most higher plants reproduce sexually. During this process, if the mother carries the virus, the virus will be spread with the seeds. The prevention and control of viruses is extremely difficult, and there is no completely effective method so far. Therefore, isolating and identifying the industrial hemp dipartite virus and its genome is an important means to quickly diagnose the disease, strengthen early detection of the virus, cultivate non-toxic industrial hemp seedlings, and prevent and control industrial hemp virus diseases. Summary of the invention

[0004] The purpose of the present invention is to provide an industrial hemp bipartite virus gene and an application of the industrial hemp bipartite virus gene. Specifically, the present invention provides the following technical solutions: On the one hand, the present invention provides an industrial hemp bipartite virus gene, wherein the virus gene has two dsRNAs, the dsRNA1 sequence is shown in SEQ ID NO:1, and the dsRNA2 sequence is shown in SEQ ID NO:2.

[0005] Furthermore, the present invention provides a product for detecting a bipartite viral gene, wherein the viral gene has two dsRNAs, the dsRNA1 sequence is shown in SEQ ID NO:1, and the dsRNA2 sequence is shown in SEQ ID NO:2.

[0006] Further, the product is a specific primer. It should be understood that those skilled in the art can design primers that conform to the detection principle by using online tools such as Primer3, SnapGene, Benchling, etc. according to the viral gene sequence provided by the present invention and the corresponding detection method.

[0007] The full-length genome sequence or its fragments of the present invention can usually be obtained by PCR amplification, recombination or artificial synthesis. For PCR amplification, primers can be designed based on the relevant nucleotide sequences disclosed in the present invention, especially the open reading frame sequences, and the cDNA library prepared by conventional methods known to those skilled in the art is used as a template to amplify the relevant sequence. When the sequence is long, it is often necessary to perform two or more PCR amplifications, and then splice the fragments amplified in each time together in the correct order.

[0008] In a specific embodiment, in the product, the specific primer sequence for detecting dsRNA1 is shown as SEQ ID NO: 3, 4, 9 or 11; the specific primer sequence for detecting dsRNA2 is shown as SEQ ID NO: 5, 6, 7, 8, 10, or 12.

[0009] Further, the product is a kit. The kit contains any one of the primer sequences shown in SEQ ID NO: 3 to 12. In a specific embodiment, the kit is an RT reaction kit, including reagents such as primers, reverse transcriptase, RT reaction buffer and substrate. In another specific embodiment, the kit is a PCR reaction kit, including reagents such as primers, Tag DNA polymerase, PCR reaction buffer and substrate.

[0010] Furthermore, the present invention provides the use of the product in cultivating detoxified industrial hemp seedlings, detecting high-quality industrial hemp seeds, or detecting industrial hemp diseases.

[0011] Further, the present invention provides the use of a bipartite virus gene in the preparation of primers, probes or kits for detecting industrial hemp bipartite virus, or for expression vector construction, gene editing, or for the preparation of anti-industrial hemp bipartite virus antibodies, wherein the virus gene has two dsRNAs, the dsRNA1 sequence is shown in SEQ ID NO: 1, and the dsRNA2 sequence is shown in SEQ ID NO: 2. In some specific embodiments, the industrial hemp bipartite virus gene provided by the present invention, i.e., a viral antigen or an antigenic fragment thereof, can be used in an immunoassay to detect antibody levels (or conversely, an antiviral antibody can be used to detect antigen levels). Based on a clear immunoassay, a recombinant antigen can be developed to replace an invasive diagnostic method. Antibodies to viral proteins or fragments thereof can be detected in industrial hemp samples. The design of the immunoassay can vary greatly, and its various schemes are known in the art. The scheme of the immunoassay can be based on, for example, a competitive, direct reaction or sandwich-type assay. The scheme can also use a solid support, or an immunoprecipitation method can be used, for example. Most tests involve the use of labeled antibodies or polypeptides; the label can be, for example, a fluorescent label, a chemiluminescent label, a radioactive label or a dye molecule. Assays which amplify the probe signal are also known; examples are assays using biotin and avidin, enzyme-labeled and mediated immunoassays such as ELISA assays.

[0012] Furthermore, the present invention provides a method for detecting industrial hemp bipartite virus genes, comprising the following steps: (1) Extracting total RNA from the sample to be tested; (2) Using reverse transcriptase to reverse transcribe the extracted RNA into cDNA; using cDNA as a template, PCR amplification, cloning, and sequencing are performed under the action of DNA polymerase to obtain specific fragments; (3) Analyze the PCR products by electrophoresis to see whether there are bands of the expected size, so as to determine whether the sample contains the target viral RNA; or use qRT-PCR, that is, add fluorescent probes or dyes during the PCR process and determine the presence and relative amount of viral RNA in the sample by analyzing the changes in the fluorescent signal.

[0013] The viral gene sequence is shown in SEQ ID NO: 1 and / or SEQ ID NO: 2.

[0014] Furthermore, the present invention provides an isolated nucleic acid molecule, wherein the nucleic acid molecule is a cDNA obtained by reverse transcription of a viral gene, and the viral gene sequence is shown in SEQ ID NO:1 and / or SEQ ID NO:2.

[0015] The technical effects achieved by the present invention are: Industrial hemp virus causes yellowing of industrial hemp mosaics, chlorosis of leaves, dwarfing of leaves, poor development of mosaics and death of hemp on industrial hemp, and other symptoms, which bring serious economic losses to industrial hemp planting enterprises. The present invention obtains industrial hemp dipartite virus that causes industrial hemp disease by sequencing the macro-genome of diseased plants and comparing and analyzing the genome sequence, and the virus is the first virus isolated from diseased industrial hemp by the inventor, and the genome sequence information and functional genes of the virus have not been reported at home and abroad. The acquisition of the viral genome sequence is of great significance to understanding the source of the virus, the evolutionary process and molecular epidemiology, and also lays the foundation for the differential diagnosis of the disease and the development of disease-resistant genes, with huge economic and social benefits.

[0016] The present invention provides an industrial hemp dipartite virus gene sequence. The sequence provided by the present invention is used to prepare primers, kits or antibody proteins for detecting the virus by adopting existing technical means. The primers, kits or antibody proteins for detecting the virus can be used in multiple scenarios such as cultivating detoxified industrial hemp seedlings, detecting high-quality hemp seeds, and detecting hemp diseases, so as to quickly diagnose and promptly prevent and control the disease. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 The trait diagram of industrial hemp plants infected with the bipartite virus collected from the planting field of the present invention; Figure 2 This is a diagram of the characteristics of industrial hemp plants after being inoculated with the dipartite virus in the laboratory of the present invention. DETAILED DESCRIPTION

[0018] The present invention is further described below in conjunction with the embodiments, but the present invention is not limited in any way. Any changes or substitutions made based on the teachings of the present invention belong to the protection scope of the present invention.

[0019] The industrial hemp bipartite virus gene sequence provided by the present invention is two dsRNAs, the dsRNA1 sequence is shown in SEQ ID NO: 1, and the dsRNA2 sequence is shown in SEQ ID NO: 2. According to the high-throughput sequencing analysis results, the contigs sequences related to each virus are obtained, and these contigs are BLAST-aligned by NCBI. From the results, the homologous virus with the highest sequence consistency is selected as the reference sequence, and the software Premier 5 is used to specifically design several pairs of specific primers based on the viral contigs genome sequence to amplify the cannabis bipartite virus and verify the correctness of the de novo assembled viral sequence (Table 1). According to the viral gene sequence and primers of the present invention, the industrial hemp sample to be tested can be selectively detected by reverse transcription polymerase chain reaction (RT-PCR), multiple real-time fluorescence quantitative PCR (RT-qPCR) and other technologies.

[0020] The present invention is further described below with specific embodiments: Example 1

[0021] Cannabis divaricata virus detection and validation 1. Materials

[0022] Two cannabis samples were collected at the Xiaoshao Base in Yunnan Province. One sample showed chlorotic leaves, small leaves with bright veins, and dwarfing; the other showed yellowing of mosaic leaves and chlorosis ( Figure 1 ).

[0023] 2. RT-PCR, RACE and Sequencing 2.1 RT-PCR (1) Total RNA extraction and reverse transcription of industrial hemp samples The total RNA of cannabis samples was extracted using the Eastep® Super total RNA extraction kit (Promega, Shanghai), and the reverse cDNA was generated using the Hifair® III Ist Strand cDNA Synthesis SuperMix for qPCR (gDNA digesterplus) (Yisheng, Shanghai, China).

[0024] (2) PCR reaction PCR reaction system (50uL): 2×Hiffer® Robust PCR master Mix (Yisheng, Shanghai, China) 25uL, VCV-RNA1F / VCV-RNA2-1F / VCV-RNA2-2F (10 pmol / L) 1uL, VCV-RNA1R / VCV-RNA2-1R / VCV-RNA2-2R (10 mol / L) 1 uL each, cDNA template 5 uL, ddH20 18 uL.

[0025] Amplification conditions: 94℃ pre-denaturation for 3 min; 94℃ denaturation for 10s, 56℃ annealing for 20s, 72℃ extension for 30s, 33 cycles; 72℃ extension for 5min. The amplified product was purified and recovered after 1% agarose gel electrophoresis and sent to Beijing Qingke Biotechnology Co., Ltd. Kunming Branch for sequencing. The sequence returned by sequencing was processed, analyzed and spliced ​​using DNAMAN 5.0, thereby obtaining two cannabis dipartite virus dsRNA1 sequences as shown in SEQ ID NO:1 and dsRNA2 sequences as shown in SEQ ID NO:2.

[0026] 2.2 RACE Verification According to the manufacturer's instructions, the SMARTerRACE5′ / 3′ kit (Aicore, Hunan, China) was used to amplify the 5′ and 3′ terminal sequences of Cannabis bipartite virus based on the designed 5′ RACE-specific primers (SEQ ID NO: 11 or 12) and 3′ RACE-specific primers (SEQ ID NO: 9 or 10), the specific sequences of which are shown in Table 1 .

[0027] First, the first-strand cDNA was synthesized by reverse transcription using the Evo M-MLV RTase for RACE kit from Aikerui Biotechnology (Hunan) Co., Ltd. Total RNA was converted into cDNA. The reaction system of 3′RACE cDNA 20uL included 2uL total RNA, 1uL 3'RACE RT Primer, and 8.5uL Nuclease free water; the reaction system of 5′RACE cDNA 20uL included 2uL total RNA, 1uL 5'RACE RT Primer, and 7.5uL Nuclease free water, followed by incubation at 72°C for 3 minutes and standing on ice for two minutes. 4uL 5X RACE RT Buffer, 2uL dNTP Mix, 0.5uL RNase Inhibitor, and 2uL Evo M-MLVRTase for RACE were added to 11.5uL 3′RACE cDNA and 10.5uL 5′RACE cDNA systems, respectively. 1uL Template Switching Oligo was additionally added to 5′RACE cDNA to form a 20ul system, which was incubated at 42°C for 90min and 72°C for 15min.

[0028] PCR was then performed using cDNA and 5′ and 3′ specific primers designed for the cannabis divirus. A 50uL reaction contained 3uL cDNA, 1uL Universal Short Primer, 1uL 5′ specific primer (SEQ ID NO: 11 or 12) and 3′ specific primer (SEQ ID NO: 9 or 10), 20uL 5X L-Exp Taq PCR Buffer, 0.5uL L-Exp Taq HS DNA Polymerase, 2 uL dNTP Mix, and 20.5uL ddH20.

[0029] Amplification conditions: 94℃ pre-denaturation for 1 min; 94℃ denaturation for 30s, 60℃ annealing for 30s, 72℃ extension for 3min, 30 cycles; 72℃ extension for 5min. The amplified product was purified and recovered after 1% agarose gel electrophoresis, and connected to the pMD18-T cloning vector (TaKaRa), and then heat-shocked and transformed into Escherichia coli DH5a (TaKaRa). After screening on MacConkey medium containing ampicillin, it was sent to the Kunming branch of Beijing Qingke Biotechnology Co., Ltd. for sequencing. DNAMAN5.0 was used for sequence processing and analysis to obtain complete 5' and 3' sequences. Through sequence alignment and analysis, the results showed that the sequences of SEQ ID NO:1 and SEQ ID NO:2 provided by the present invention are correct.

[0030] Table 1 Primers for RT-PCR amplification of cannabis dipartite virus Example 2

[0031] Industrial hemp dichotomous virus detection method

[0032] A method for detecting industrial hemp bipartite virus genes comprises the following steps: (1) Extraction of total RNA from samples: Use Eastep® Super Total RNA Extraction Kit (Promega, Shanghai) to extract total RNA from cannabis samples. (2) The extracted RNA was reverse transcribed into cDNA using reverse transcriptase, and the cDNA was reversed using Hifair® III Ist StrandcDNA Synthesis SuperMix for qPCR (gDNA digester plus) (Yisheng, Shanghai, China); Continue to use cDNA as a template, perform PCR amplification, cloning and sequencing under the action of DNA polymerase to obtain specific fragments; PCR reaction system (50uL): 2×Hiffer® Robust PCR master Mix (Yisheng, Shanghai, China) 25uL, VCV-RNA1F / VCV-RNA2-2F (10 pmol / L) 1uL, VCV-RNA1R / VCV-RNA2-2R (10 mol / L) 1 uL, cDNA template 5 uL, ddH20 18 uL.

[0033] Amplification conditions: pre-denaturation at 94°C for 3 min; denaturation at 94°C for 10 s, annealing at 56°C for 20 s, extension at 72°C for 30 s, 33 cycles; extension at 72°C for 5 min.

[0034] (3) Analyze the PCR product by 1% agarose gel electrophoresis to see whether there is a band of the expected size, so as to determine whether the sample contains the target viral RNA.

[0035] Using the above method, 20 industrial hemp plants inoculated with the virus ( Figure 2 ) samples were tested, and the test results were consistent with expectations, indicating that the established RT-PCR detection technology system can be used for virus detection in industrial hemp field samples.

[0036] The above-described embodiment is only a preferred solution of the present invention, but it is not intended to limit the present invention. Those skilled in the relevant art can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, any technical solution obtained by equivalent replacement or equivalent transformation falls within the protection scope of the present invention.

Claims

1. An industrial hemp bipartite virus gene, wherein the virus gene has two dsRNAs, the dsRNA1 sequence is shown in SEQ ID NO: 1, and the dsRNA2 sequence is shown in SEQ ID NO:

2.

2. A product for detecting bipartite virus genes, characterized in that: The viral gene has two dsRNAs, the dsRNA1 sequence is shown in SEQ ID NO:1, and the dsRNA2 sequence is shown in SEQ ID NO:

2.

3. The product according to claim 2, characterized in that The product is a specific primer.

4. The product according to claim 3, characterized in that The specific primer sequence for detecting dsRNA1 is shown in SEQ ID NO: 3, 4, 9 or 11; the specific primer sequence for detecting dsRNA2 is shown in SEQ ID NO: 5, 6, 7, 8, 10 or 12.

5. The product according to claim 2, characterized in that The product is a test kit.

6. The product according to claim 5, characterized in that The kit contains any one of the primer sequences shown in SEQ ID NOs: 3 to 12.

7. Use of the product of any one of claims 2-6 in cultivating detoxified industrial hemp seedlings, detecting high-quality industrial hemp seeds, or detecting industrial hemp diseases.

8. Use of the bipartite virus gene in preparing primers, probes or kits for detecting industrial hemp bipartite virus, or in constructing expression vectors, gene editing, or in preparing antibodies against industrial hemp bipartite virus, characterized in that: The viral gene has two dsRNAs, the dsRNA1 sequence is shown in SEQ ID NO:1, and the dsRNA2 sequence is shown in SEQ ID NO:

2.

9. A method for detecting industrial hemp bipartite virus genes, characterized in that: The following steps are involved: (1) Extracting total RNA from the sample to be tested; (2) Using reverse transcriptase to reverse transcribe the extracted RNA into cDNA; using cDNA as a template, PCR amplification, cloning, and sequencing are performed under the action of DNA polymerase to obtain specific fragments; (3) Analyze PCR products by electrophoresis or add fluorescent probes or dyes during the PCR process to determine the presence and relative amount of viral RNA in the sample by analyzing the changes in the fluorescent signal; The viral gene sequence is shown in SEQ ID NO: 1 and / or SEQ ID NO:

2.

10. An isolated nucleic acid molecule, characterized in that The nucleic acid molecule is cDNA obtained by reverse transcription of viral genes, and the viral gene sequence is shown in SEQ ID NO: 1 and / or SEQ ID NO: 2.

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