Industrial hemp double-split virus gene and application thereofTechnical Field
The invention belongs to the technical field of plant virus detection, and particularly relates to an industrial hemp double-split virus gene and application thereof.
Background
Industrial hemp (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. The whole body of industrial hemp is 'Bao', stems can be made into plant fibers, flowers and leaves can extract CBD, seeds can extract edible oil and protein powder, and can be made into traditional Chinese medicinal materials for use in cosmetics, and the industrial hemp is widely applied in various fields and is gradually recognized, developed and utilized by people. Yunnan is one of two provinces which allow industrial hemp to be planted in China, the industrial hemp can be legally popularized and utilized under the supervision of public security, the industrial hemp is planted in 38 counties of 13 states, the symptoms of yellow flowers and leaves, green leaves, bright vein lobules, dwarf leaves, dead plants and the like of the industrial hemp are found in the planting, certain loss is brought to the quality and the yield of the industrial hemp, and the research shows that the disease is caused to be a double split virus.
The geminivirus (PARTITIVIRIDAE) is a class of viruses with a double-stranded RNA (dsRNA) genome having a genome segment length of about 1.3-2.5 kb, two dsRNA each with and only 1 open reading frame, dsRNA1 being responsible for encoding RNA-dependent RNA polymerase (RdRp), while dsRNA2 is mainly responsible for encoding capsid protein (cp). The geminiviruses have been shown to infect a wide range of hosts including plants, fungi and protozoa, and the infected plants, crops have reduced yield, quality and the geminiviruses inherit the virus from the parent to the offspring by vertical transmission, both in asexual and sexual reproduction. In asexual propagation, the root, stem, leaf, callus and the like of the parent body contain viruses, and after the root, stem, leaf, callus and the like are adopted for culture, the new generation plant can carry viruses. Most higher plants are sexually propagated, and in the process, if the parent plant carries a virus, the virus will propagate along with the seed. The prevention and control of the viruses and the difficulty thereof are not completely effective until now, so that the separation and identification of the industrial cannabis bisegregant viruses and the genomes thereof are an important means for rapidly diagnosing the diseases, enhancing the early detection of the viruses, cultivating the nontoxic industrial cannabis seedlings and preventing and controlling the industrial cannabis virus diseases.
Disclosure of Invention
The invention aims to provide an industrial hemp double-split virus gene and an application thereof, in particular to the following technical scheme:
in one aspect, the invention provides an industrial hemp bisegregant virus gene, which comprises two dsRNAs, wherein the sequence of the dsRNA1 is shown as SEQ ID NO. 1, and the sequence of the dsRNA2 is shown as SEQ ID NO. 2.
Furthermore, the invention provides a product for detecting the double-split virus gene, the virus gene is provided with two dsRNAs, the sequence of the dsRNA1 is shown as SEQ ID NO. 1, and the sequence of the dsRNA2 is shown as SEQ ID NO. 2.
Further, the product is a specific primer. It should be understood that, by the viral gene sequences provided by the present invention, a person skilled in the art performs Primer design conforming to the detection principle using an on-line tool such as Primer3, snapGene, benchling, etc., according to the corresponding detection method.
The full-length genomic sequence of the present invention or a fragment thereof can be usually obtained by a PCR amplification method, a recombinant method or an artificial synthesis method. For the PCR amplification method, primers can be designed based on the nucleotide sequences disclosed in the present invention, particularly the open reading frame sequences, and amplified to obtain the sequences according to a conventional method known to those skilled in the art using cDNA library as a template. When the sequence is longer, it is often necessary to perform two or more PCR amplifications, and then splice the amplified fragments together in the correct order.
In a specific embodiment, the specific primer sequence for detecting the dsRNA1 is shown as SEQ ID NO. 3,4, 9 or 11, and the specific primer sequence for detecting the dsRNA2 is shown as SEQ ID NO. 5, 6, 7, 8, 10 or 12.
Further, the product is a kit. The kit contains any primer sequence shown as SEQ ID NO. 3-12, in one specific embodiment, the kit is an RT reaction kit comprising primers, reverse transcriptase, RT reaction buffer solution, substrate and other reagents, and in another specific embodiment, the kit is a PCR reaction kit comprising primers, tag DNA polymerase, PCR reaction buffer solution, substrate and other reagents.
Further, the invention provides application of the product in cultivation of detoxified industrial hemp seedlings, detection of high-quality industrial hemp seeds or detection of industrial hemp diseases.
Furthermore, the invention provides application of the double-split virus gene in preparing a primer, a probe or a kit for detecting industrial hemp double-split virus, or in constructing an expression vector, editing genes or preparing an anti-industrial hemp double-split virus antibody, wherein the virus gene is provided with two dsRNAs, the dsRNA1 sequence is shown as SEQ ID NO. 1, and the dsRNA2 sequence is shown as SEQ ID NO. 2. In some embodiments, the industrial cannabis bisexsomatized viral genes provided herein, i.e., viral antigens or antigenic fragments thereof, can be used in immunoassays to detect antibody levels (or conversely, antiviral antibodies can be used to detect antigen levels). Based on the definitive immunoassay, recombinant antigens can be developed to replace invasive diagnostic methods. Antibodies to viral proteins or fragments thereof in industrial cannabis samples can be detected. The design of the immunoassay may vary widely, and various protocols are known in the art. The protocol for the immunoassay may be based on, for example, a competitive, or direct reaction or sandwich type assay. The protocol may also employ, for example, a solid support, or may employ immunoprecipitation. Most assays involve the use of labeled antibodies or polypeptides, and the label may be, for example, a fluorescent label, a chemiluminescent label, a radioactive label, or a dye molecule. Assays for amplifying probe signals are also known, examples of which are assays employing biotin and avidin, enzyme-labelled and mediated immunoassays, such as ELISA assays.
Further, the invention provides a detection method of industrial hemp double-split virus genes, which comprises the following steps:
(1) Extracting total RNA of a sample to be detected;
(2) Reverse transcribing the extracted RNA into cDNA by using reverse transcriptase, continuing to use the cDNA as a template, and performing PCR amplification cloning and sequencing under the action of DNA polymerase to obtain a specific fragment;
(3) Analyzing the PCR product by electrophoresis to see if a band with the expected size appears, thereby judging whether the sample contains the target viral RNA, or using qRT-PCR, namely adding fluorescent probes or dyes in the PCR process, and determining the existence and relative quantity of the viral RNA in the sample by analyzing the change of fluorescent signals.
The viral gene sequence is shown as SEQ ID NO. 1 and/or SEQ ID NO. 2.
Furthermore, the invention provides an isolated nucleic acid molecule which is cDNA obtained by reverse transcription of a viral gene, wherein the sequence of the viral gene is shown as SEQ ID NO. 1 and/or SEQ ID NO. 2.
The invention achieves the technical effects that:
The invention relates to an industrial hemp double split virus which causes industrial hemp diseases and is obtained by sequencing macroscopic genome of disease plants, comparing and analyzing genome sequences, identifying and separating, wherein the virus is obtained by separating industrial hemp from disease for the first time by an inventor, and genome sequence information and functional genes of the virus are not reported at home and abroad. The acquisition of the viral genome sequence has important significance for virus source resolution, evolution process and molecular epidemiology, lays a foundation for differential diagnosis of diseases and development of disease-resistant genes, and has great economic and social benefits.
The invention provides an industrial hemp double-split virus gene sequence, which is used for preparing a primer, a kit or an antibody protein for detecting the virus by adopting the sequence provided by the invention and adopting the prior art means, and can be used for cultivating detoxified industrial hemp seedlings, detecting high-quality industrial hemp seeds, detecting industrial hemp diseases and the like, and rapidly diagnosing and timely preventing and controlling the diseases.
Drawings
FIG. 1 is a diagram of the characteristics of an industrial cannabis plant infected with a geminivirus collected from a planting field in accordance with the present invention;
FIG. 2 is a diagram of the traits of industrial cannabis plants after laboratory inoculation with a geminivirus according to the present invention.
Detailed Description
The invention is further illustrated, but is not limited in any way, by the following examples, and any alterations or substitutions based on the teachings of the invention are within the scope of the invention.
The gene sequence of the industrial hemp double-split virus provided by the invention is two dsRNAs, the dsRNA1 sequence is shown as SEQ ID NO. 1, the dsRNA2 sequence is shown as SEQ ID NO. 2, the contigs sequences related to each virus are obtained according to the high throughput sequencing analysis result, BLAST comparison is carried out on the contigs through NCBI, the homologous virus with the highest sequence consistency is selected from the result as a reference sequence, and several pairs of specific primers are designed in a targeted manner based on the virus contigs genome sequence by using software Premier 5 to amplify the industrial hemp double-split virus and verify the correctness of the virus sequences assembled from scratch (Table 1). According to the viral gene sequences and the primers, the industrial hemp samples to be detected can be selectively detected by adopting reverse transcription polymerase chain reaction (RT-PCR), multiplex real-time fluorescent quantitative PCR (RT-qPCR) and other technologies.
The invention is further illustrated by the following examples:
Example 1
Industrial hemp double-split virus detection and verification
1. Material
2 Hemp samples were collected at whistle base of Yunnan province, one showing leaf chlorosis, vein lobule, dwarf and the other showing yellow flower leaf and chlorosis (FIG. 1).
RT-PCR, RACE and sequencing
2.1 RT-PCR
(1) Total RNA extraction and reverse transcription of industrial hemp samples
The total RNA of industrial hemp samples was extracted using Eastep% Super total RNA extraction kit (Promega, shanghai), and the inverted cDNA was performed using Hifair% III IST STRAND CDNA SYNTHESIS SuperMix for qPCR (GDNA DIGESTER plus) (next holy, shanghai, china).
(2) PCR reaction
PCR reaction System (50 uL):
2X Hiffer cubic Robust PCR MASTER Mix (next san. Shanghai, china) 25uL,
VCV-RNA1F/VCV-RNA2-1F/VCV-RNA2-2F each (10 pmol/L) 1uL,
VCV-RNA1R/VCV-RNA2-1R/VCV-RNA2-2R each (10 mol/L) 1-uL,
The cDNA template 5 uL was used as a template,
ddH20 18 uL。
The amplification conditions were 94℃for 3 min, 94℃for 10s,56℃for 20s,72℃for 30s,33 cycles, and 72℃for 5min. The amplified product was purified and recovered after 1% agarose gel electrophoresis detection, and was sent to Kunming division of Beijing Qinke Biotech Co. The sequencing return sequence is subjected to sequence processing, analysis and sequence splicing by DNAMAN 5.0, so that 2 industrial hemp double-split virus dsRNA1 sequences are shown in SEQ ID NO.1, and dsRNA2 sequences are shown in SEQ ID NO. 2.
2.2 RACE verification
The 5' and 3' terminal sequences of the industrial cannabis bipartite virus were amplified according to the manufacturer's instructions using SMARTERRACE '/3' kit (Ai Kerui, hunan, china) 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), see Table 1 for specific sequences.
First, first strand cDNA was synthesized by reverse transcription using Evo M-MLV RTase for RACE, a kit of Ai Kerui Bio-engineering (Hunan) Inc. The reaction system for converting total RNA into cDNA,3'RACE cDNA 20uL includes 2uL total RNA, 1uL 3'RACE RT Primer and 8.5. 8.5uL Nuclease free water, and the reaction system for 5'RACE cDNA 20uL includes 2uL total RNA, 1uL 5'RACE RT Primer and 7.5. 7.5uL Nuclease free water, and then the reaction system is kept at 72 ℃ for 3min, and kept on ice for two minutes. 4uL 5X RACE RT Buffer, 2uL dNTP Mix, 0.5uL RNase Inhibitor and 2uL Evo M-MLV RTase for RACE are respectively added into 11.5uL 3'RACE cDNA and 10.5uL 5'RACE cDNA systems, 1uL Template Switching Oligo is additionally added into 5' RACE cDNA to form a 20uL system, and the temperature is kept at 42 ℃ for 90min and 72 ℃ for 15min.
Subsequently, PCR was performed using cDNA and industrial hemp double virus designed 5 'and 3' specific primers, and 50uL reactions containing 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 12) 10）、20uL 5X L-Exp Taq PCR Buffer、0.5uL L-Exp Taq HS DNA Polymerase、2 uL dNTP Mix、20.5uL ddH20.
The amplification conditions were 94℃for 1 min, 94℃for 30s, 60℃for 30s,72℃for 3min and 30 cycles, and 72℃for 5min. The amplified product was purified and recovered by 1% agarose gel electrophoresis, and ligated to pMD18-T cloning vector (TaKaRa), followed by heat shock transformation into E.coli DH5a (TaKaRa). Screening on a medium containing ampicillin, and sequencing the medium in Kunming division of Biotechnology Co., ltd. And (3) performing sequence processing and analysis by using DNAMAN 5.0 to obtain complete 5 'and 3' sequences. The results of sequence alignment and analysis show that the sequences of SEQ ID NO. 1 and SEQ ID NO. 2 provided by the invention are correct.
TABLE 1 RT PCR amplification of Industrial Cannabis sativa Di-split Virus primers
| Sequence number | Primer name | Primer sequence (5 'to 3') | Primer starting position |
| SEQ ID NO:3 | VCV-RNA1F | TGTTATAGACGTTGAGAACGGGT | 447 |
| SEQ ID NO:4 | VCV-RNA1R | GATGTTCGTCCAAGGAAACTGAT | 1342 |
| SEQ ID NO:5 | VCV-RNA2-1F | AACAGCAGACCCGCACAGGAATC | 222 |
| SEQ ID NO:6 | VCV-RNA2-1R | TCGGCCAGTGTAGCTTGAGGAAA | 816 |
| SEQ ID NO:7 | VCV-RNA2-2F | TAACAGCAATGAAACACCTCAAAGT | 774 |
| SEQ ID NO:8 | VCV-RNA2-2R | CTTCCTTAACGAAGATGAACTGTG | 1646 |
| SEQ ID NO:9 | 3′RACEVCV-RNA1F | TCCCGAATACCCTGTCGAAAC | 1419 |
| SEQ ID NO:10 | 3′RACEVCV-RNA2F | ACAGACATTCACACCCAGTCAGCCT | 1412 |
| SEQ ID NO:11 | 5′RACEVCV-RNA1R | ACCTCCTTTAGGTCCTTTCTCG | 567 |
| SEQ ID NO:12 | 5′RACEVCV-RNA2R | CATGTCGAGACGTAGATTCTAGCCG | 465 |
| Universal Short Primer、3’RACE RT Primer、5’RACE RT Primer | Kit provision | |
Example 2
Industrial hemp double split virus detection method
The method for detecting the industrial hemp double-split virus gene comprises the following steps:
(1) Extracting total RNA of a sample to be detected, namely extracting total RNA of an industrial hemp sample by adopting Eastep cube Super total RNA extraction kit (Promega, shanghai),
(2) Reverse transcribing the extracted RNA into cDNA using reverse transcriptase, reversing the cDNA using Hifair < 3 > cube III IST STRAND CDNA SYNTHESIS SuperMix for qPCR (GDNA DIGESTER plus) (next san. Shanghai, china);
Continuing to use cDNA as a template, and performing PCR amplification cloning and sequencing under the action of DNA polymerase to obtain a specific fragment;
PCR reaction System (50 uL):
2X Hiffer cubic Robust PCR MASTER Mix (next san. Shanghai, china) 25uL,
VCV-RNA 1F/VCV-RNA 2-2F (10 pmol/L) 1uL each,
VCV-RNA 1R/VCV-RNA 2-2R each (10 mol/L) 1-uL,
The cDNA template 5 uL was used as a template,
ddH20 18 uL。
The amplification conditions were 94℃for 3 min, 94℃for 10s,56℃for 20s,72℃for 30s,33 cycles, and 72℃for 5min.
(3) The PCR product was analyzed by 1% agarose gel electrophoresis to see if bands corresponding to the expected size appeared, thereby judging whether the sample contained the target viral RNA.
By adopting the method, the detection results of the 20 industrial hemp (figure 2) samples inoculated with viruses are consistent with the expected results, which shows that the established RT-PCR detection technology system can be used for detecting the viruses of the industrial hemp field samples.
The above embodiment is only a preferred embodiment of the present invention, but it is not intended to limit the present invention. Various changes and modifications may be made by one skilled in the relevant art without departing from the spirit and scope of the invention. Therefore, all the technical schemes obtained by adopting the equivalent substitution or equivalent transformation are within the protection scope of the invention.