CN103484480A - Papaya ringspot virus genes and method for constructing RNA interference (RNAi) expression vectors of papaya ringspot virus genes - Google Patents

Abstract

The invention relates to papaya ringspot virus (PRSV) genes and a method for constructing RNA interference (RNAi) expression vectors of the papaya ringspot virus genes. The PRSV genes are CP, Hc-Pro and NIb genes, and the nucleotide sequences of the CP, Hc-Pro and NIb genes are as shown in SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:3 respectively. The method for constructing the RNAi expression vectors of the PRSV genes comprises the following steps: extracting total RNA from papaya leaves which are infected with papaya ringspot viruses and serve as materials; reversely transcribing the RNA into cDNA; constructing the CP, Hc-Pro and NIb into high-efficiency stable anti-virus plant expression vectors respectively through genetic transformation by taking the CP, Hc-Pro and NIb as conserved regions of target interference genes so as to obtain anti-virus plants. The RNAi expression vectors are constructed by utilizing the conserved sequences of the CP, NIb and Hc-Pro gene fragments, and the high-efficiency anti-disease design concept of the RNAi is introduced into the anti-disease genetic engineering breeding of the papaya, so the invention can be used for creating high-efficiency broad-spectrum new varieties capable of resisting the PRSV disease.

Description

Construction method of papaya ringspot virus gene and its RNAi expression vector

technical field

The invention belongs to the fields of molecular biology and genetic engineering, and relates to an analysis of coat protein gene (CP), auxiliary component protein (HC-Pro), replicase gene (NIb) gene and conserved sequence in papaya ringspot virus (PRSV) And the RNAi expression vector construction method, the RNAi plant expression vector can be used for gene gun or Agrobacterium-mediated genetic transformation, to create a new germplasm with high efficiency and broad-spectrum resistance to PRSV virus disease.

Background technique

The main uses of papaya (Carica papaya L.) are the production of fruit and commercially valuable proteolytic enzymes. It is one of the few crops that can bear fruit all year round and can bear mature fruits after 9 months of cultivation. Papaya trees can live for 25 years or more, and each leaf axil can continuously pump one or more fruits, each containing about 1000 more seeds. Pollen collected from male or hermaphrodite flowers can easily be artificially pollinated to female or hermaphrodite flowers. The habit of annual flowering and fruiting, together with the ease of artificial hybridization, allows papaya to produce a large number of offspring and obtain fairly reliable genetic transformation results, so papaya is an attractive model for genetic/genomic research and crop improvement.

The commercialization of rainbow and sunrise varieties of Hawaiian genetically modified papaya in 1998 is well known as a scientific breakthrough, representing the first practical application of genetically modified fruit crops. The transgenic varieties screened from the CP gene transgenic lines initially transformed with the Hawaiian mild strain HA5-1 chain by gene gun method have the ability to resist Hawaiian PRSV. Afterwards, many research groups used the Agrobacterium-mediated method to carry out a large amount of transformation work. The explants used included leaves, petioles, etc., but the most commonly used regeneration system was somatic embryogenesis. The highest transformation efficiency of papaya is obtained by treating the embryogenic callus with silicon carbide or tungsten, causing the callus to be injured and then co-cultured. To complete within 13 months, regenerated plants generally have only one insertion site. In recent years, papaya germplasm resistant to Phytophthora palmi with anti-fungal gene has been obtained by gene gun method. When the regulation of transgenic breeding technology is relaxed, transgenic papaya lines will become an important factor in the growth of papaya industry worldwide.

The resistance mediated by the PRSV-CP gene has a certain broad-spectrum, especially the plants transfected with the read-through part of the CP gene of each strain show a certain resistance to the virus of the same genus, which is the advantage of using the CP gene . However, there are still great limitations in application: the resistance mediated by PRSV-CP is mainly manifested in the early stage of virus infection, delaying the onset and relieving symptoms, and the resistance level is low; the CP expressed by transgene can be coated with heterologous viral RNA , so that viruses that could not be transmitted by aphids can be transmitted by aphids (Dougherty, 1994), or the plant expression product of a virus CP gene may package the genome of another virus or other pathogenic factors, thus forming a new pathogenic Disease factor (Gao Yanmei et al., 2009).

Using PRSV replicase (NIb) gene to construct artificial resistance gene to transform plants to obtain virus-resistant plants is another important progress in the biotechnology breeding of PRSV-resistant papaya. As early as 1996 and 1998, researchers from my country's Sun Yat-sen University and Hong Kong University cloned and transformed the replicase gene of papaya PRSV (Ye Changming et al., 1996; Chen Gu, 1998). Researchers from South China Agricultural University in my country transformed the replicase gene of papaya PRSV-Ys strain into papaya, and the transformed plants showed high resistance to PRSV Ys, Vb, and Sm strains. During the 9 months of planting in the field, None of the transgenic plants became diseased, while all of the control plants became diseased. This genetically modified variety, Huanong 1, was approved by the government in 2006 for commercial cultivation in Guangdong Province. The replicase gene contains a 35S promoter and a NOS terminator, and has the function of screening kanamycin resistance. Huanong 1 was reported to be resistant to papaya ringspot virus strains from four different regions in China (Ruan Xiaolei et al., 2004; Ruan Xiaolei et al., 2009; Ruan Xiaolei et al., 2010). Previously, researchers from South China Agricultural University tried to use the binary vector pBI121 to transfer the untranslatable replicase gene and coat protein gene into papaya, but the transformed plants did not acquire disease resistance. The resistance characteristics of plants transformed with PRSV-NIb gene are: strong resistance, specificity, long duration, but narrow resistance range, generally have strong resistance to parental virus strains that provide replicase genes, and those Virus strains with poor homology to the parental strain do not exhibit resistance.

Papaya transgenic researchers in Taiwan found that when the Hc-Pro gene of the PRSV-YK strain was changed, the transgenic germplasm resistant to the PRSV-YK strain would lose disease resistance (Ying-Huey Cheng, 1996), which indicated that the Hc-Pro Genes play an important role in the process of PRSV infection, and silencing the expression of the Hc-Pro gene of the virus is also a transgenic breeding strategy worth trying.

In addition, the research found that the resistance mediated by the CP gene is not only related to coat protein, but also related to RNA-mediated resistance, that is, high-level or broad-spectrum plant virus resistance is caused by coat protein and RNA interference. caused jointly. RNA-mediated resistance is significantly different from protein-mediated resistance. It only relies on the RNA transcription of the transgene, and does not require the protein encoded by the viral gene, so it has high biological safety and is easily accepted by the public; At the same time, its resistance phenotype has nothing to do with the dose of the inoculum, similar to immunization, it is more efficient; RNA-mediated resistance only needs to be homologous to the viral RNA sequence and the target sequence, allowing mutations or even deletions of some sites, And it is produced along with the production of dsRNA, which is highly homologous to the sense or antisense RNA of the silent gene, but when the non-homology between the transgene sequence and the viral gene sequence is higher than 10%, the transgene will be It is difficult to endow plants with disease resistance (Bau HJ2003), so the specificity is stronger. In order to obtain broad-spectrum virus resistance, by adding more virus-related sequences to expand the transgenic structure, transgenic plants can obtain broader-spectrum resistance. sex (Bucher EC2006).

RNAi (RNA interference), or RNA interference, is an ancient biological phenomenon that has been discovered in recent years and is ubiquitous in organisms. It is a specific gene silencing phenomenon mediated by double-stranded RNA (dsRNA) and participated by specific enzymes. , it blocks the expression of genes at the transcription level, post-transcription level and translation level. Simply put, RNAi refers to the efficient gene silencing phenomenon mediated by RNA. Utilize RNAi technology to design short-segment inverted repeating viral gene-related sequences of hairpin structure. After the sequence is expressed, it can directly produce dsRNA, and produce RNA with related genes such as CP or RP and other related genes of traditional transviruses, and further cut and produce in plants. Compared with dsRNA, which mediates RNA resistance, the anti-virus efficiency of RNAi technology is much higher. Generally speaking, the virus resistance conferred by the single-stranded sense or antisense gene of the virus is only about 20%, but the transfer can produce Plants with the IR sequence of dsRNA are as high as 90% resistant to viruses (Waterhouse PM2003). Therefore, it is theoretically feasible to use RNAi technology to cultivate new papaya strains with broad spectrum and high resistance to PRSV.

At present, there is no research in the world that uses RNAi technology to transform papaya to obtain transgenic germplasm and varieties (lines).

Contents of the invention

The object of the invention is a kind of construction method of papaya ringspot virus gene and RNAi expression carrier thereof, utilize anti-PRSV effective virus CP, NIb, Hc-Pro gene segment conservative sequence to construct RNAi expression vector and carry out papaya transformation, solved Respectively use CP and NIb gene transformation to make papaya resistant to PRSV, and introduce the design concept of high-efficiency disease resistance of RNAi into the disease-resistant genetic engineering breeding of papaya, which can be used to create high-efficiency, broad-spectrum resistance to PRSV virus disease of new germplasm.

The technical scheme adopted in the present invention:

A kind of papaya ringspot virus gene is CP, Hc-Pro, NIb gene, is extracted from the papaya leaf that infects papaya ringspot virus, and its nucleotide sequence is respectively as SEQ ID NO: 1, SEQ ID NO: 2. The nucleotide sequence shown in SEQ ID NO:3.

Using papaya leaves infected with papaya ringspot virus as materials, total RNA was extracted, reverse-transcribed into cDNA, and CP, Hc-Pro, and NIb were used as targets to interfere with the conserved regions of the genes, and introduced into papaya's disease resistance through genetic transformation In genetic engineering breeding, they are respectively constructed into high-efficiency and stable anti-virus plant expression vectors, so as to obtain anti-virus plants.

Another object of the present invention is to provide a method for constructing the RNAi expression vector of papaya ringspot virus CP, NIb, Hc-Pro gene, which is to infect papaya ringspot virus papaya leaves as material, extract total RNA, and reverse Transcribed into cDNA, using the CP gene, Hc-Pro gene, and NIb gene (the nucleotide sequences of which are as SEQ ID NO.1, SEQ ID NO.2, and SEQ ID NO.3) as templates, design specific primers, and use Perform PCR reaction with high-fidelity Taq enzyme, introduce XhoI and BglII restriction sites into the forward fragments of CP, Hc-Pro and NIb genes respectively, and introduce BamHI and SalI enzymes into the reverse fragments of CP, Hc-Pro and NIb genes respectively Cutting site, the primer sequence is as follows:

CP forward sequence: restriction sites XhoI and BglII

PRSV-CP-RNAi-P1:5'-CCCTCGAGTCAACGCCGGAACTAGTGGAACTT-3'

PRSV-CP-RNAi-P2:5'-GAAGATCTTCACGAGCCCTATCAGGTGTCTTT-3'

Gene size 544bp

CP reverse sequence: Restriction sites SalI and BamHI

PRSV-CP-RNAi-P3:5'-GCGTCGACTCAACGCCGGAACTAGTGGAACTT-3'

PRSV-CP-RNAi-P4:5'-CGGGATCCTCACGAGCCCTATCAGGTGTCTTT-3'

Hc-Pro Forward Sequence: Restriction sites XhoI and BglII

PRSV-Hcpro-RNAi-P1:5'-CCCTCGAGTGAATGCACGTAACATGAACGA-3'

PRSV-Hcpro-RNAi-P2:5'-GAAGATCTACCATTTGCTGCCGAAACCTCT-3'

Gene size 484bp

Hc-Pro reverse sequence: Restriction sites SalI and BamHI

PRSV-Hcpro-RNAi-P3:5'-GCGTCGACTGAATGCACGTAACATGAACGA-3'

PRSV-Hcpro-RNAi-P4:5'-CGGGATCCACCATTTGCTGCCGAAACCTCT-3'

NIb Forward Sequence: Restriction sites XhoI and BglII

PRSV-NIb-RNAi-P1:5'-CCCTCGAGCTTTGTATTGCCATTCACCCAGAT-3'

PRSV-NIb-RNAi-P2:5'-GAAGATCTACTCATCCATAGAACCACGCTCAC-3'

Gene size 424bp

NIb reverse sequence: Restriction sites SalI and BamHI

PRSV-NIb-RNAi-P3:5'-GCGTCGACCTTTGTATTGCCATTCACCCAGAT-3'

PRSV-NIb-RNAi-P4:5'-CGGGATCCACTCATCCATAGAACCACGCTCAC-3'

Connect the PCR product to the PMD18-T Simple vector, transform Escherichia coli DH5α competent cells, screen the positive clones, extract the bacteria solution and PCR to identify the correct recombinant plasmid, and obtain the CP, Hc-Pro and NIb obtained by double digestion with XhoI and BglII The forward fragment of the gene was connected with pRNAi1017 which was digested with XhoI and BglII, transformed, positive clones were screened, and the correct recombinant plasmid was identified by PCR of the extracted bacteria solution, and then combined with CP, Hc-Pro and NIb genes by double digestion with BamHI and SalI The reverse fragments were ligated, transformed, and positive clones were screened, and the correct recombinant plasmid was identified by PCR of the extracted bacterial solution, which was digested with PstI and SalI and pCAMBIA2300-35S-OCS plant expression vector. Positive clones were screened, detected by electrophoresis and verified by sequencing. The plant expression vectors pCAMBIA2300-35S-CP front and back-OCS, pCAMBIA2300-35S-Hc-Pro front and back-OCS, and pCAMBIA2300-35S-NIb front and back-OCS were successfully constructed.

Technical advantage and beneficial effect of the present invention:

1. The present invention adopts RNAi technology to design papaya ringspot virus CP, Hc-Pro, NIb conservative region reverse repeat hairpin structure, so that it can efficiently, quickly and stably produce dsRNA, which is different from traditional full-length CP or RP of transviruses, etc. Compared with dsRNA produced by genes, RNAi technology has higher antiviral efficiency.

2. The coat protein gene (CP), auxiliary component protein gene (Hc-Pro) and replicase gene (NIb) in the papaya ringspot virus (PRSV) provided by the present invention are the self-reproduction, replication, The important coding genes of infection-related proteins were constructed into high-efficiency and stable anti-virus plant expression vectors, and introduced into the disease-resistant genetic engineering breeding of papaya through gene gun or Agrobacterium-mediated genetic transformation. The protein is blocked at the mRNA level, which can effectively inhibit the reproduction, replication and spread of the virus, so that the foreign virus cannot infect the transgenic anti-virus plants, and achieve the effect of anti-virus.

3. The papaya anti-PRSV virus plant expression vectors pCAMBIA2300-35S-CP front and back-OCS, pCAMBIA2300-35S-Hc-Pro front and back-OCS, and pCAMBIA2300-35S-NIb front and back-OCS are reported for the first time. Gene gun or Agrobacterium-mediated genetic transformation can create new germplasm with high efficiency and broad-spectrum resistance to PRSV virus disease.

Description of drawings

Fig. 1 is the common conserved region analysis map of papaya ringspot virus PRSV functional gene CP, Hc-Pro, NIb;

Figure 2 is the forward sequence and reverse sequence PCR amplification of CP, Hc-Pro, NIb gene interference fragments;

Figure 3 shows the recombinant plasmids pRNAi1017-CP positive, pRNAi1017-Hc-pro positive, pRNAi1017-NIb positive, which were digested by XhoⅠ and BglⅡ;

Figure 4 shows the positive and negative sides of recombinant plasmids pRNAi1017-CP, pRNAi1017-Hc-pro positive and negative, and pRNAi1017-NIb positive and negative sides cut by SalI and PstI;

Figure 5 shows the double enzyme digestion of the anti-PRSV virus plant transgene vector.

Detailed ways

Below in conjunction with the examples, the specific implementation of the present invention will be further described in detail. The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention. The experimental methods that do not indicate specific conditions in the following examples are generally in accordance with conventional conditions, or in accordance with the conditions suggested by the manufacturer.

Embodiment one: Papaya ringspot virus CP, Hc-Pro and NIb gene conservative region analysis

In June 2011, the leaves of suspected virus samples of papaya were collected in Changjiang, Ledong, Chengmai, Wenchang and Sanya, the main papaya planting areas of Hainan Island (mainly based on the obvious mottled deformity on the young leaves) Diseased leaves), a total of 76 samples to be tested were collected (the samples were collected from different locations or within 500m of the same location, all sample locations were located by GPS, and the samples were stored at -80°C for later use). The total RNA of the leaves of the samples was extracted respectively, after reverse transcription by Random primers, PCR amplification was carried out with the primers listed in Table 1 respectively, and molecular detection (detection of the genetic diversity of papaya ringspot virus CP, Hc-Pro and NIb genes) ), papaya ringspot virus PRSV was detected in 59 samples, that is, the detection rate of papaya ringspot virus disease was 59/76=77.63%. Therefore, papaya ringspot virus PRSV is affecting the health of papaya industry in Hainan The main viral disease that develops.

The inventors carried out molecular cloning and sequencing of the detected PRSV samples, using the PRSV virus genome CP, Hc-Pro and NIb as target genes, Blast compared the PRSV nucleotide information in the GenBank database, and analyzed the sequence by using Clustalx software , the representative samples with the most typical PRSV virus CP, Hc-Pro and NIb genes were selected, and the sequence analysis map is shown in Figure 1, (a) analysis of the common conserved region of papaya ringspot virus PRSV functional gene CP, ( b) Analysis of the common conserved region of papaya ringspot virus PRSV functional gene Hc-Pro, (c) analysis of common conserved region of papaya ringspot virus PRSV functional gene NIb. It can be seen from the figure that the representative samples have the common conserved region characteristics of Hainan papaya ringspot virus PRSV functional genes, which can be used as templates for designing broad-spectrum antiviral plant expression vectors of papaya ringspot virus CP, Hc-Pro and NIb genes.

Table 1 PCR primers for papaya mosaic disease detection

Embodiment two: the cloning method of papaya ringspot virus CP, Hc-Pro and NIb gene conserved region interference fragment

1. Primer design

The RNA virus genome sequence of PRSV infecting papaya was selected from GenBank as the data background. The nucleotide sequences of CP, Hc-Pro and NIb were the target genes for PRSV detection, and PCR detection primers were designed by comparing the nucleotide sequences within and between species (Table 1).

2. Total RNA extraction and reverse transcription

The total RNA of papaya suspected virus samples was extracted by TransGen Trizol one-step method. The integrity of total RNA was checked by agarose gel electrophoresis. The extracted total RNA was reverse-transcribed with an RNA reverse transcription kit from Fermantas to obtain total cDNA, which was used as a template for PCR reactions.

3. Pathogen detection RT-PCR

94，℃30s，50，℃30s，72，℃1min，35个循环；72，℃10min。PCR结束后，取8μL产物进行1.5%琼脂糖凝胶电泳。The reverse transcription products were amplified by PCR using the primers in Table 1, respectively. Taq plus DNA polymerase (5U/μL) was purchased from Beijing TransGen Company. Reaction system 20μL. PCR amplification conditions are: 94

94, ℃ 30s, 50, ℃ 30s, 72, ℃ 1min, 35 cycles; 72, ℃ 10min. After PCR, 8 μL of the product was taken for 1.5% agarose gel electrophoresis.

4. Sequence determination and homology comparison

Under ultraviolet light, the electrophoresis band of RT-PCR product was cut, and the target gene fragment was recovered using a DNA recovery kit (Omega Company, USA). Connect it to the pMD18-T vector, transform Escherichia coli DH5α, pick positive clones and send them to Shanghai Invitrogen Company for sequencing. The sequencing results were compared by Blast on the Internet (http://www.ncbi.nlm.nih.gov/blast/Blast.cgi), and the results showed that the CP, Hc-Pro, and NIb genes (other Nucleotide sequences such as SEQ ID NO.1, SEQ ID NO.2 and SEQ ID NO.3), the homology with the sequence of the American Hawaiian transgenic papaya virus HA strain is 90.20%, 84.57%, 84.25%, respectively, The homology with the Taiwan transgenic papaya virus YK strain sequence was 92.65%, 89.88%, 92.18%, respectively.

Embodiment 3: Construction of papaya ringspot virus CP, Hc-Pro and NIb gene conservative region interference fragment RNAi plant expression vector

Using papaya ringspot virus-infected papaya representative sample leaves as materials, total RNA was extracted and reverse-transcribed into cDNA. According to the sequencing results of papaya ringspot virus samples, the conservation of CP, Hc-Pro and NIb as target interference genes were found. region, design specific primers, perform PCR reaction with high-fidelity Taq enzyme, introduce XhoI and BglII restriction sites in the forward fragments of CP, Hc-Pro and NIb genes respectively, and introduce restriction sites in the reverse of CP, Hc-Pro and NIb genes Introduce BamHI and SalI restriction sites to the fragments respectively, and the primer sequences are as follows:

CP forward sequence: restriction sites XhoI and BglII

PRSV-CP-RNAi-P1:5'-CCCTCGAGTCAACGCCGGAACTAGTGGAACTT-3'

PRSV-CP-RNAi-P2:5'-GAAGATCTTCACGAGCCCTATCAGGTGTCTTT-3'

Gene size 544bp

CP reverse sequence: Restriction sites SalI and BamHI

PRSV-CP-RNAi-P3:5'-GCGTCGACTCAACGCCGGAACTAGTGGAACTT-3'

PRSV-CP-RNAi-P4:5'-CGGGATCCTCACGAGCCCTATCAGGTGTCTTT-3'

Hc-Pro Forward Sequence: Restriction sites XhoI and BglII

PRSV-Hcpro-RNAi-P1:5'-CCCTCGAGTGAATGCACGTAACATGAACGA-3'

PRSV-Hcpro-RNAi-P2:5'-GAAGATCTACCATTTGCTGCCGAAACCTCT-3'

Gene size 484bp

Hc-Pro reverse sequence: Restriction sites SalI and BamHI

PRSV-Hcpro-RNAi-P3:5'-GCGTCGACTGAATGCACGTAACATGAACGA-3'

PRSV-Hcpro-RNAi-P4:5'-CGGGATCCACCATTTGCTGCCGAAACCTCT-3'

NIb Forward Sequence: Restriction sites XhoI and BglII

PRSV-NIb-RNAi-P1:5'-CCCTCGAGCTTTGTATTGCCATTCACCCAGAT-3'

PRSV-NIb-RNAi-P2:5'-GAAGATCTACTCATCCATAGAACCACGCTCAC-3'

Gene size 424bp

NIb reverse sequence: Restriction sites SalI and BamHI

PRSV-NIb-RNAi-P3:5'-GCGTCGACCTTTGTATTGCCATTCACCCAGAT-3'

PRSV-NIb-RNAi-P4:5'-CGGGATCCACTCATCCATAGAACCACGCTCAC-3'

Perform PCR amplification to obtain the forward and reverse interference fragments of the CP, Hc-Pro, and NIb genes of the PRSV virus (Figure 2), and then the obtained CP, Hc-Pro, and The PCR product of the NIb interference fragment was subcloned into pMD18-T simple, and double-digested with XhoI and BglII, and the excised fragment was connected to the pRNAi1017 interference vector that had been cut by the same double enzymes, and the recombinant plasmids pRNAi1017-CP positive and pRNAi1017 were constructed. -Hc-Pro positive, pRNAi1017-NIb positive. The three recombinant plasmids were digested with XhoI and BglII, and the target bands with the expected size were excised respectively (Figure 3), which indicated that the CP, Hc-Pro and NIb gene interference fragments were forward connected to the pRNAi1017 vector.

Then, the reverse interference fragments of CP, Hc-Pro and NIb genes were digested with SalI and BamH, and the excised fragments were connected to the recombinant plasmids pRNAi1017-CP positive and pRNAi1017 with the same double digestion with T4 ligase -Hc-pro positive, pRNAi1017-NIb positive, construct the RNA interference vector containing CP, Hc-Pro and NIb forward and reverse sequences. The above-mentioned recombinant RNA interference vector containing forward and reverse sequences of CP, Hc-Pro and NIb was identified by double enzyme digestion with SalI and PstI (Figure 4). After assembly, it was constructed into the plant expression vector 2300-35s-OCS, and finally the papaya anti-virus plant transgenic vectors pCAMBIA2300-35S-CP front-back-OCS, pCAMBIA2300-35S-Hc-Pro front-back-OCS, pCAMBIA2300- 35S-NIb pros and cons-OCS, identified by double enzyme digestion with Sal and IPst (Figure 5), the finally constructed pCAMBIA2300-35S-CP pros and cons-OCS, pCAMBIA2300-35S-Hc-Pro pros and cons-OCS, Sequencing verification of pCAMBIA2300-35S-NIb front and back-OCS, the results showed that 3 constructed plant expression vectors pCAMBIA2300-35S-CP front and back-OCS, pCAMBIA2300-35S-Hc-Pro front and back-OCS, pCAMBIA2300-35S-NIb The homology of CP, Hc-Pro, NIb genes in representative samples of Hainan papaya ringspot virus in front-back-OCS and molecular detection is 100%, 100%, 100%, respectively, that is, to construct the vector The CP, Hc-Pro, and NIb genes were completely consistent before and after constructing the vector.

The above is only a preferred embodiment of the present invention, it should be pointed out that for those of ordinary skill in the art, without departing from the technical principle of the present invention, some improvements and modifications can also be made. These improvements and modifications It should also be regarded as the protection scope of the present invention.

Claims (2)

1. a papaya ringspot virus gene is characterized in that: the papaya ringspot virus gene is CP, Hc-Pro, NIb gene, and its nucleotide sequence is respectively as SEQ ID NO: 1, SEQ ID NO: 2. The nucleotide sequence shown in SEQ ID NO:3. 2. a construction method of the RNAi expression vector of the papaya ringspot virus gene described in claim 1, is characterized in that: be material with infecting papaya ringspot virus papaya leaf, extract total RNA, reverse transcription is cDNA, using the CP gene, Hc-Pro gene, and NIb gene whose nucleotide sequences are shown in SEQ ID NO.1, SEQ ID NO.2, and SEQ ID NO.3 as templates, design specific primers, and use high-fidelity Perform PCR reaction with Taq enzyme, introduce XhoI and BglII restriction sites into the forward fragments of CP, Hc-Pro and NIb genes respectively, and introduce BamHI and SalI restriction sites into the reverse fragments of CP, Hc-Pro and NIb genes respectively point, the primer sequences are as follows: CP forward sequence: restriction sites XhoI and BglII PRSV-CP-RNAi-P1:5'-CCCTCGAGTCAACGCCGGAACTAGTGGAACTT-3' PRSV-CP-RNAi-P2:5'-GAAGATCTTCACGAGCCCTATCAGGTGTCTTT-3' Gene size 544bp CP reverse sequence: Restriction sites SalI and BamHI PRSV-CP-RNAi-P3:5'-GCGTCGACTCAACGCCGGAACTAGTGGAACTT-3' PRSV-CP-RNAi-P4:5'-CGGGATCCTCACGAGCCCTATCAGGTGTCTTT-3' Hc-Pro Forward Sequence: Restriction sites XhoI and BglII PRSV-Hcpro-RNAi-P1:5'-CCCTCGAGTGAATGCACGTAACATGAACGA-3' PRSV-Hcpro-RNAi-P2:5'-GAAGATCTACCATTTGCTGCCGAAACCTCT-3' Gene size 484bp Hc-Pro reverse sequence: Restriction sites SalI and BamHI PRSV-Hcpro-RNAi-P3:5'-GCGTCGACTGAATGCACGTAACATGAACGA-3' PRSV-Hcpro-RNAi-P4:5'-CGGGATCCACCATTTGCTGCCGAAACCTCT-3' NIb Forward Sequence: Restriction sites XhoI and BglII PRSV-NIb-RNAi-P1:5'-CCCTCGAGCTTTGTATTGCCATTCACCCAGAT-3' PRSV-NIb-RNAi-P2:5'-GAAGATCTACTCATCCATAGAACCACGCTCAC-3' Gene size 424bp NIb reverse sequence: Restriction sites SalI and BamHI PRSV-NIb-RNAi-P3:5'-GCGTCGACCTTTGTATTGCCATTCACCCAGAT-3' PRSV-NIb-RNAi-P4:5'-CGGGATCCACTCATCCATAGAACCACGCTCAC-3' Connect the PCR product to the PMD18-T Simple vector, transform Escherichia coli DH5α competent cells, screen the positive clones, extract the bacteria solution and PCR to identify the correct recombinant plasmid, and obtain the CP, Hc-Pro and NIb obtained by double digestion with XhoI and BglII The forward fragment of the gene was connected with pRNAi1017 which was digested with XhoI and BglII, transformed, positive clones were screened, and the correct recombinant plasmid was identified by PCR of the extracted bacteria solution, and then combined with CP, Hc-Pro and NIb genes by double digestion with BamHI and SalI The reverse fragments were ligated, transformed, and positive clones were screened, and the correct recombinant plasmid was identified by PCR of the extracted bacterial solution, which was digested with PstI and SalI and pCAMBIA2300-35S-OCS plant expression vector. Positive clones were screened, detected by electrophoresis and verified by sequencing. The plant expression vectors pCAMBIA2300-35S-CP front and back-OCS, pCAMBIA2300-35S-Hc-Pro front and back-OCS, and pCAMBIA2300-35S-NIb front and back-OCS were successfully constructed.

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