CN103484480A - Papaya ringspot virus genes and method for constructing RNA interference (RNAi) expression vectors of papaya ringspot virus genes - Google Patents
Papaya ringspot virus genes and method for constructing RNA interference (RNAi) expression vectors of papaya ringspot virus genes Download PDFInfo
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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
Technical field
The invention belongs to molecular biology and genetically engineered field, relate to a kind of prv (PRSV) housing protein gene (CP), ancillary component albumen (HC-Pro), rdrp gene (NIb) gene, conserved sequence analysis and RNAi expression vector establishment method, the RNAi plant expression vector can be used for particle gun or agriculture bacillus mediated genetic transformation, creates new germ plasm efficient, the anti-PRSV virus disease of wide spectrum.
Background technology
The main application of papaya (Carica papaya L.) is the proteolytic ferment of producing fruit and having commercial value.It is rare can bearing fruit the whole year, and just can bear the crop of mature fruit in cultivation after 9 months.Papaya can survive 25 years or the longer time, and each axil is the one or more fruits of pumping constantly, and each fruit is containing having an appointment more than 1000 seed.The pollen of adopting from male flower or hermaphrodite flower, be easy to artificial pollination to female flower or hermaphrodite flower.The habit of yielding positive results in the anniversary, and be easy to artificial hybridization, make papaya can produce a large amount of offsprings and obtain genetic transformation result quite reliably, so papaya is an attractive model of heredity/genome research and crop improvement.
Hawaii transgenic papaya rainbows in 1998 rise kind with day and are started commercial applications, are well-known scientific breakthroughs, have represented the transgenic fruit crop of practical application for the first time.The transformed variety that utilizes particle bombardment to filter out from the CP gene transgenic strain of initial conversion Hawaii gentle strain HA5-1 chain has the ability of anti-Hawaii PRSV.Afterwards, many research groups utilize agrobacterium-mediated transformation to carry out a large amount of transformations, and the explant of utilization comprises blade, petiole etc., but the most frequently used be the regeneration system that the body embryo occurs.The transformation efficiency that papaya is the highest is to utilize silicon carbide or tungsten to process embryo callus subculture, carry out again after making callus injured that common cultivation obtains, the screening that kalamycin resistance transforms system's (generally in 150 mg/litre) completed in 6 to 13 months, and regeneration plant generally only has an insertion point.In recent years, the anti-palm mould germplasm of papaya that turns anti-fungal gene obtains by the method for particle gun.After the control of transgenic breeding technology is relaxed, transgenic papaya system will become the important factor of papaya industrial growth in world wide.
Resistance by the PRSV-CP gene mediated has certain broad spectrum, especially turns the plant of reading over part of each strain CP gene, to belonging to virus together, all shows certain resistance, and this is the advantage place that utilizes the CP gene.But the resistance that also has significant limitation: PRSV-CP mediation in application is mainly manifested in, virus infection is early stage, postponement is fallen ill and mitigation symptoms, and resistance level is lower; The CP of transgene expression can be coated with the allos viral RNA, thereby make originally the virus can not aphid passed can be by aphis propagation (Dougherty, 1994), perhaps a kind of expression of plants product of viral CP gene may be packed the genome of another kind of virus or other virulence factors, thereby form a kind of new virulence factor (Gao Yanmei etc., 2009).
Utilizing the gene constructed artificial resistant gene conversion of plant of replicative enzyme (NIb) of PRSV to obtain antiviral plant, is the another impressive progress of anti-PRSV papaya biotechnology breeding.As far back as the researchist of China Zhongshan University in 1996,1998 and Hong Kong University just to the rdrp gene of papaya PRSV carried out clone and Transformation In Papaya research (leaf is kept burning day and night etc., 1996; Chen Gu, 1998).The researchist of China Agricultural University Of South China is transformed into the rdrp gene of papaya PRSV-Ys strain in papaya, Ys, Vb, the Sm strain performance high resistance of transformed plant to PRSV, in 9 middle of the month in field planting field, none morbidity of transfer-gen plant, contrast is all morbidities.This transformed variety " No. 1, magnificent agriculture " obtained government permission and plants in Guangdong Province's commercialization in 2006.This rdrp gene contains 35S promoter and a NOS terminator, and tool kalamycin resistance screening function.It is reported, magnificent agriculture can resist Strains of Papaya Rilgspot Virus (Ruan little Lei etc., 2004 from 4 different areas of China for No. 1; Ruan little Lei etc., 2009; Ruan little Lei etc., 2010).Before, the investigators of Agricultural University Of South China attempt utilizing rdrp gene and the coat protein gene that binary vector pBI121 can not translate to proceed in papaya, but transformed plant does not obtain disease resistance.The resistance characteristics that turn the plant of PRSV-NIb gene are: strong resistance, single-minded, time length is long but the resistance scope is narrow, generally the parental virus strain that rdrp gene is provided is had to very strong resistance, and poor virus strain does not show resistance to those and parent's strain homology.
Taiwan papaya transgenic research person finds after the Hc-Pro of PRSV-YK strain gene alteration, the transgenosis germplasm of anti-PRSV-YK strain will lose resistance against diseases (Ying-Huey Cheng, 1996), this explanation Hc-Pro gene plays an important role in the PRSV course of infection, and the Hc-Pro genetic expression of silencing virus is also to be worth the transgenic breeding strategy of attempting.
In addition, find in research that the resistance mediated by Transgenic cp is not only relevant with coat protein, also relevant with the resistance of RNA mediation, the plant virus resistance of high level or wide spectrum is disturbed and is jointly caused by coat protein and RNA.It is significantly different that the resistance of RNA mediation and the resistance of protein mediation have, and it only relies on genetically modified rna transcription, and does not need the protein of virogene coding expression, thus there is higher biological safety, easily accepted by the public, simultaneously, the dosage indifference of its resistance-phenotype and inoculum, be similar to immunity, and it is more efficient, the resistance of RNA mediation only needs viral RNA sequence and target sequence homology, allow the sudden change in part site, even lack, and be accompanied by the generation of dsRNA and produce, this dsRNA is with the justice of silencer or sense-rna high homology, but when the non-homology of transgenic sequence and virus gene sequence higher than 10% the time, transgenosis just is difficult to conferring disease resistance in plants (Bau HJ2003), thereby specificity is stronger, in order to obtain the virus resistance of wide spectrum, expand transgenic structure by additional more viral correlated series, can obtain the more resistance of wide spectrum (Bucher EC2006) by the render transgenic plant.
RNAi (RNA interference) is that RNA disturbs, it is ubiquitous a kind of ancient biological phenomena in vivo of discovered in recent years, by double-stranded RNA (dsRNA) mediation, by the reticent phenomenon of the specific gene of certain enzyme participation, the expression of its blocking gene on transcriptional level, post-transcriptional level and translation skill, briefly, RNAi just refers to by the reticent phenomenon of the high efficiency gene of RNA mediation.Utilize RNAi technical project short-movie section inverted repeat hairpin structure virogene correlated series, sequence can directly produce dsRNA after expressing, turn viral total length with tradition and produce RNA as genes involveds such as CP or RP, and further shear in plant materials and produce dsRNA, thereby the mediate rna resistance is compared, the antiviral efficiency far of RNAi technology is higher, in general turning viral strand justice or inverted defined gene gives the virus resistance of plant and only has 20% left and right, but the plant that proceeds to the IR sequence that can produce dsRNA to viral resistance but up to 90% (Waterhouse PM2003).Therefore utilizing the RNAi technology to cultivate wide spectrum, high resistance PRSV papaya new lines, is feasible in theory.
At present, also do not utilize in the world RNAi technical transform papaya to obtain the research of transgenosis germplasm and kind (being).
Summary of the invention
The construction process that the objective of the invention is a kind of prv gene and RNAi expression vector thereof, utilize the effective viral CP of antagonism PRSV, NIb, Hc-Pro gene segment conserved sequence to build the RNAi expression vector and carry out the papaya conversion, solved and utilized respectively CP and NIb gene transformation to make papaya obtain the existing deficiency of anti-PRSV, and the efficient disease-resistance design concept of RNAi is incorporated in the disease-resistant gene Engineering Breeding of papaya, can be used for creating new germ plasm efficient, the anti-PRSV virus disease of wide spectrum.
The technical solution adopted in the present invention:
A kind of prv gene, for CP, Hc-Pro, NIb gene, extract in the papaya blade of self-infection prv, its nucleotide sequence is the nucleotide sequence as shown in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3 respectively.
The infection prv papaya blade of take is material, extract total RNA, reverse transcription is cDNA, take CP, Hc-Pro, NIb as the target interference base because of conservative region, pass through genetic transformation, be incorporated in the disease-resistant gene Engineering Breeding of papaya, it be built into efficiently respectively, stablize antivirus plant expression vector, thereby obtain having antiviral plant.
Another object of the present invention is to provide a kind of prv CP, NIb, the construction process of the RNAi expression vector of Hc-Pro gene, that to infect prv papaya blade be material, extract total RNA, reverse transcription is cDNA, with the CP gene, the Hc-Pro gene, (its nucleotide sequence is respectively as SEQID NO.1 for the NIb gene, SEQ ID NO.2 and SEQ ID NO.3) be template, the design Auele Specific Primer, carry out the PCR reaction with high-fidelity Taq enzyme, at CP, the forward fragment of Hc-Pro and NIb gene is introduced respectively XhoI and BglII restriction enzyme site, at CP, the reverse fragment of Hc-Pro and NIb gene is introduced respectively BamHI and SalI restriction enzyme site, primer sequence is as follows:
CP forward sequence: restriction enzyme site XhoI and BglII
PRSV-CP-RNAi-P1:5’-CCCTCGAGTCAACGCCGGAACTAGTGGAACTT-3’
PRSV-CP-RNAi-P2:5’-GAAGATCTTCACGAGCCCTATCAGGTGTCTTT-3’
Gene size 544bp
CP reverse sequence: restriction enzyme site SalI and BamHI
PRSV-CP-RNAi-P3:5’-GCGTCGACTCAACGCCGGAACTAGTGGAACTT-3’
PRSV-CP-RNAi-P4:5’-CGGGATCCTCACGAGCCCTATCAGGTGTCTTT-3’
Hc-Pro forward sequence: restriction enzyme site 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 enzyme site SalI and BamHI
PRSV-Hcpro-RNAi-P3:5’-GCGTCGACTGAATGCACGTAACATGAACGA-3’
PRSV-Hcpro-RNAi-P4:5’-CGGGATCCACCATTTGCTGCCGAAACCTCT-3’
NIb forward sequence: restriction enzyme site XhoI and BglII
PRSV-NIb-RNAi-P1:5’-CCCTCGAGCTTTGTATTGCCATTCACCCAGAT-3’
PRSV-NIb-RNAi-P2:5’-GAAGATCTACTCATCCATAGAACCACGCTCAC-3’
Gene size 424bp
NIb reverse sequence: restriction enzyme site SalI and BamHI
PRSV-NIb-RNAi-P3:5’-GCGTCGACCTTTGTATTGCCATTCACCCAGAT-3’
PRSV-NIb-RNAi-P4:5’-CGGGATCCACTCATCCATAGAACCACGCTCAC-3’
The PCR product is connected to PMD18-T Simple carrier, transform the bacillus coli DH 5 alpha competent cell, screening positive clone, extract bacterium liquid PCR and identify correct recombinant plasmid, the CP obtained by XhoI and BglII double digestion, the forward fragment of Hc-Pro and NIb gene, with XhoI, with the pRNAi1017 of BglII double digestion, be connected, transform, screening positive clone, extract bacterium liquid PCR and identify correct recombinant plasmid, through BamHI and SalI double digestion and CP, the reverse fragment of Hc-Pro and NIb gene is connected, transform, screening positive clone, extract bacterium liquid PCR and identify correct recombinant plasmid, through PstI, with the SalI double digestion, with after the same double digestion of pCAMBIA2300-35S-OCS plant expression vector, be connected, transform, screening positive clone, electrophoresis detection sequence verification, the just trans-OCS of plant expression vector pCAMBIA2300-35S-CP, the just trans-OCS of pCAMBIA2300-35S-Hc-Pro, the just trans-OCS of pCAMBIA2300-35S-NIb successfully constructs.
Technical superiority of the present invention and beneficial effect:
1, the present invention adopts the RNAi technology, design prv CP, Hc-Pro, NIb conservative region inverted repeat hairpin structure, enable the generation dsRNA of efficient fast and stable, turn the genes generation dsRNA such as viral total length CP or RP with tradition and compare, the antiviral efficiency of RNAi technology is higher.
2, prv provided by the invention (PRSV) housing protein gene (CP), ancillary component protein gene (Hc-Pro), rdrp gene (NIb), it is the prv self-reproduction, copy, infect the important encoding gene of associated protein, it is built into efficiently respectively, stablize antivirus plant expression vector, by particle gun or agriculture bacillus mediated genetic transformation, be incorporated in the disease-resistant gene Engineering Breeding of papaya, its functional protein is blocked in the mRNA level, can effectively suppress viral breeding, copy and spread, make the virus of external source can not infect the antiviral plant of transgenosis, reach antiviral effect.
3, the just trans-OCS of the anti-PRSV virus plant expression vector pCAMBIA2300-35S-CP of the papaya that the present invention builds, the just trans-OCS of pCAMBIA2300-35S-Hc-Pro, the just trans-OCS of pCAMBIA2300-35S-NIb are reported first, by particle gun or agriculture bacillus mediated genetic transformation, can create new germ plasm efficient, the anti-PRSV virus disease of wide spectrum.
The accompanying drawing explanation
The common conservative region that Fig. 1 is prv PRSV functional gene CP, Hc-Pro, NIb is analyzed collection of illustrative plates;
Fig. 2 is CP, Hc-Pro, NIb gene interference fragment forward sequence and reverse sequence pcr amplification;
Fig. 3 be recombinant plasmid pRNAi1017-CP just, pRNAi1017-Hc-pro just, pRNAi1017-NIb just, through Xho I and Bgl II double digestion;
Fig. 4 is that recombinant plasmid pRNAi1017-CP is positive and negative, pRNAi1017-Hc-pro is positive and negative, pRNAi1017-NIb is positive and negative in Sal I and Pst I double digestion;
Fig. 5 is anti-PRSV virus plant transgene carrier double digestion.
Embodiment
Below in conjunction with embodiment, the specific embodiment of the present invention is described in further detail.Following examples are used for the present invention is described, but are not used for limiting the scope of the invention.The experimental technique of unreceipted actual conditions in the following example, usually according to normal condition, or the condition of advising according to manufacturer.
Embodiment mono-: prv CP, Hc-Pro and NIb gene conservative region are analyzed
Gather the doubtful viral sample blade of papaya (mainly take on young leaflet tablet the symptom that can see obvious mottled deformity gather the disease leaf for benchmark) on the main growing area Chang River of Hainan Island papaya, Le Dong, Chengmai, Wenchang and Sanya and other places in June, 2011, (collected specimens all is separated by 500m from different location or same place to gather altogether 76 parts of testing samples, the all samples place adopts the GPS location, and sample is adopted back with rearmounted-80 ℃ and saved backup).Extract respectively total RNA of sample blade, after the reverse transcription of Random primer, carry out pcr amplification with the listed primer of table 1 respectively, Molecular Detection (detecting prv CP, the Hc-Pro of existence and the genetic diversity of NIb gene), the sample that prv PRSV detected has 59, be that the Resisting Ringspot Virus of Papaya recall rate is 59/76=77.63%, so prv PRSV affect the main diseases viral disease that Hainan Region papaya industry develops in a healthy way.
The contriver carries out the molecular cloning order-checking by the PRSV sample detected, with PRSV viral genome CP, Hc-Pro and NIb are as target gene, PRSV Nucleotide information in Blast comparison GenBank database, by sequence alignment and utilization Clustalx software analysis, pick out and there is typical PRSV virus CP most, the representative sample of Hc-Pro and NIb gene, the sequential analysis collection of illustrative plates as shown in Figure 1, (a) the common conservative region analysis of prv PRSV functional gene CP, (b) the common conservative region analysis of prv PRSV functional gene Hc-Pro, (c) the common conservative region analysis of prv PRSV functional gene NIb.Find out that in figure representative sample has the common conservative region characteristics of Hainan prv PRSV functional gene, can be used as the template of design prv CP, Hc-Pro and NIb gene broad-spectrum antiviral plant expression vector.
Table 1 Mosaic Disease of Papaya detects the PCR primer
Embodiment bis-: the cloning process of prv CP, Hc-Pro and NIb gene conservative region interference fragment
1, design of primers
Choose PRSV and infect the rna virus cdna group sequence of papaya as data background from GenBank.CP, Hc-Pro and NIb nucleotides sequence are classified PRSV as and are detected target gene, and by planting nucleotide sequence comparison between interior and kind, design PCR detects primer (table 1).
2, total RNA extracts and reverse transcription
The total RNA of the doubtful viral sample of papaya adopts TransGen company's T rizol single stage method to be extracted.Agarose gel electrophoresis is checked the integrity of total RNA.Extract the total RNA obtained and carry out obtaining total cDNA after reverse transcription with the RNA reverse transcription test kit of Fermantas company, as the template of PCR reaction.
3, cause of disease detects RT-PCR
Carry out pcr amplification with the primer pair reverse transcription product in table 1 respectively.Taq plus archaeal dna polymerase (5U/ μ L) is purchased from Beijing TransGen company.Reaction system 20 μ L.The pcr amplification condition is: 94
94, ℃ 30s, 50, ℃ 30s, 72, ℃ 1min, 35 circulations; 72, ℃ 10min.PCR gets 8 μ L products and carries out 1.5% agarose gel electrophoresis after finishing.
4, sequencing and homology are relatively
Under ultraviolet lamp, cutting RT-PCR product electrophoretic band, adopt DNA to reclaim test kit (U.S. Omega company) and reclaim the goal gene fragment.Be connected to the pMD18-T carrier, transform bacillus coli DH 5 alpha, the picking positive colony is served the order-checking of extra large Invitrogen company.Sequencing result carries out Blast comparison (http://www.ncbi.nlm.nih.gov/blast/Blast.cgi) by internet, as a result in Hainan prv representative sample CP, Hc-Pro, NIb gene (its nucleotide sequence is respectively as SEQ ID NO.1, SEQ ID NO.2 and SEQ ID NO.3), be respectively 90.20%, 84.57%, 84.25% with the homology of Hawaii, America transgenosis pawpaw virus HA strain sequence, with the homology of Taiwan transgenosis pawpaw virus YK strain sequence, be respectively 92.65%, 89.88%, 92.18%.
Embodiment 3: the structure of prv CP, Hc-Pro and NIb gene conservative region interference fragment RNAi plant expression vector
The papaya representative sample blade that infects prv of take is material, extract total RNA, reverse transcription is cDNA, according to prv sample sequencing result, to find CP, Hc-Pro and NIb be the target interference base because of conservative region, the design Auele Specific Primer, carry out the PCR reaction with high-fidelity Taq enzyme, introduce respectively XhoI and BglII restriction enzyme site in the forward fragment of CP, Hc-Pro and NIb gene, introduce respectively BamHI and SalI restriction enzyme site in the reverse fragment of CP, Hc-Pro and NIb gene, primer sequence is as follows:
CP forward sequence: restriction enzyme site XhoI and BglII
PRSV-CP-RNAi-P1:5’-CCCTCGAGTCAACGCCGGAACTAGTGGAACTT-3’
PRSV-CP-RNAi-P2:5’-GAAGATCTTCACGAGCCCTATCAGGTGTCTTT-3’
Gene size 544bp
CP reverse sequence: restriction enzyme site SalI and BamHI
PRSV-CP-RNAi-P3:5’-GCGTCGACTCAACGCCGGAACTAGTGGAACTT-3’
PRSV-CP-RNAi-P4:5’-CGGGATCCTCACGAGCCCTATCAGGTGTCTTT-3’
Hc-Pro forward sequence: restriction enzyme site 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 enzyme site SalI and BamHI
PRSV-Hcpro-RNAi-P3:5’-GCGTCGACTGAATGCACGTAACATGAACGA-3’
PRSV-Hcpro-RNAi-P4:5’-CGGGATCCACCATTTGCTGCCGAAACCTCT-3’
NIb forward sequence: restriction enzyme site XhoI and BglII
PRSV-NIb-RNAi-P1:5’-CCCTCGAGCTTTGTATTGCCATTCACCCAGAT-3’
PRSV-NIb-RNAi-P2:5’-GAAGATCTACTCATCCATAGAACCACGCTCAC-3’
Gene size 424bp
NIb reverse sequence: restriction enzyme site SalI and BamHI
PRSV-NIb-RNAi-P3:5’-GCGTCGACCTTTGTATTGCCATTCACCCAGAT-3’
PRSV-NIb-RNAi-P4:5’-CGGGATCCACTCATCCATAGAACCACGCTCAC-3’
Carry out pcr amplification, obtain CP, the Hc-Pro of PRSV virus, forward and the reverse interference fragment (Fig. 2) of NIb gene, by the two ends of acquisition, CP, Hc-Pro and the NIb interference fragment PCR product with Xho I and Bgl II site is subcloned on pMD18-T simple again, carry out double digestion by Xho I and Bgl II, the fragment cut is connected on the pRNAi1017 of same double digestion interference carrier, be built into recombinant plasmid pRNAi1017-CP just, pRNAi1017-Hc-Pro just, pRNAi1017-NIb just.These 3 recombinant plasmids are carried out to Xho I and Bgl II double digestion, cut out respectively the consistent purpose band (Fig. 3) of expection size, show that CP, Hc-Pro and NIb gene interference fragment forward link on the pRNAi1017 carrier.
Again by CP, Hc-Pro and NIb gene reverse interference fragment, advance I row double digestion with Sal I and BamH, the fragment cut with the T4 ligase enzyme be connected to through the recombinant plasmid pRNAi1017-CP of same double digestion just, pRNAi1017-Hc-pro just, pRNAi1017-NIb just goes up, and is built into the rna interference vector that contains CP, Hc-Pro and the forward and reverse sequence of NIb.The rna interference vector that above-mentioned restructuring is contained to CP, Hc-Pro and the forward and reverse sequence of NIb, carry out double digestion evaluation (Fig. 4) by Sal I and Pst I.After group, it is building up in plant expression vector 2300-35s-OCS, finally successfully build the just trans-OCS of papaya antivirus plant transgene carrier pCAMBIA2300-35S-CP, the just trans-OCS of pCAMBIA2300-35S-Hc-Pro, the just trans-OCS of pCAMBIA2300-35S-NIb, advance I row double digestion with Sal and I Pst and identify (Fig. 5), to finally building the just trans-OCS of pCAMBIA2300-35S-CP, the just trans-OCS of pCAMBIA2300-35S-Hc-Pro, the just trans-OCS of pCAMBIA2300-35S-NIb carries out sequence verification, result shows 3 just trans-OCS of the plant expression vector pCAMBIA2300-35S-CP built, the just trans-OCS of pCAMBIA2300-35S-Hc-Pro, CP in the just trans-OCS of pCAMBIA2300-35S-NIb, Hc-Pro, CP in NIb sequence and Molecular Detection Hainan prv representative sample, Hc-Pro, the NIb DNA homolog is respectively 100%, 100%, 100%, be before carrier construction and carrier construction after CP, Hc-Pro, the NIb gene is in full accord.
The above is only the preferred embodiment of the present invention; it should be pointed out that for those skilled in the art, under the prerequisite that does not break away from the technology of the present invention principle; can also make some improvements and modifications, these improvements and modifications also should be considered as protection scope of the present invention.
Claims (2)
1. a prv gene, it is characterized in that: described prv gene is CP, Hc-Pro, NIb gene, its nucleotide sequence is the nucleotide sequence as shown in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3 respectively.
2. the construction process of the RNAi expression vector of a prv gene claimed in claim 1, it is characterized in that: be that to infect prv papaya blade be material, extract total RNA, reverse transcription is cDNA, with nucleotide sequence respectively as SEQ ID NO.1, CP gene shown in SEQ ID NO.2 and SEQ ID NO.3, the Hc-Pro gene, the NIb gene is template, the design Auele Specific Primer, carry out the PCR reaction with high-fidelity Taq enzyme, at CP, the forward fragment of Hc-Pro and NIb gene is introduced respectively XhoI and BglII restriction enzyme site, at CP, the reverse fragment of Hc-Pro and NIb gene is introduced respectively BamHI and SalI restriction enzyme site, primer sequence is as follows:
CP forward sequence: restriction enzyme site XhoI and BglII
PRSV-CP-RNAi-P1:5’-CCCTCGAGTCAACGCCGGAACTAGTGGAACTT-3’
PRSV-CP-RNAi-P2:5’-GAAGATCTTCACGAGCCCTATCAGGTGTCTTT-3’
Gene size 544bp
CP reverse sequence: restriction enzyme site SalI and BamHI
PRSV-CP-RNAi-P3:5’-GCGTCGACTCAACGCCGGAACTAGTGGAACTT-3’
PRSV-CP-RNAi-P4:5’-CGGGATCCTCACGAGCCCTATCAGGTGTCTTT-3’
Hc-Pro forward sequence: restriction enzyme site 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 enzyme site SalI and BamHI
PRSV-Hcpro-RNAi-P3:5’-GCGTCGACTGAATGCACGTAACATGAACGA-3’
PRSV-Hcpro-RNAi-P4:5’-CGGGATCCACCATTTGCTGCCGAAACCTCT-3’
NIb forward sequence: restriction enzyme site XhoI and BglII
PRSV-NIb-RNAi-P1:5’-CCCTCGAGCTTTGTATTGCCATTCACCCAGAT-3’
PRSV-NIb-RNAi-P2:5’-GAAGATCTACTCATCCATAGAACCACGCTCAC-3’
Gene size 424bp
NIb reverse sequence: restriction enzyme site SalI and BamHI
PRSV-NIb-RNAi-P3:5’-GCGTCGACCTTTGTATTGCCATTCACCCAGAT-3’
PRSV-NIb-RNAi-P4:5’-CGGGATCCACTCATCCATAGAACCACGCTCAC-3’
The PCR product is connected to PMD18-T Simple carrier, transform the bacillus coli DH 5 alpha competent cell, screening positive clone, extract bacterium liquid PCR and identify correct recombinant plasmid, the CP obtained by XhoI and BglII double digestion, the forward fragment of Hc-Pro and NIb gene, with XhoI, with the pRNAi1017 of BglII double digestion, be connected, transform, screening positive clone, extract bacterium liquid PCR and identify correct recombinant plasmid, through BamHI and SalI double digestion and CP, the reverse fragment of Hc-Pro and NIb gene is connected, transform, screening positive clone, extract bacterium liquid PCR and identify correct recombinant plasmid, through PstI, with the SalI double digestion, with after the same double digestion of pCAMBIA2300-35S-OCS plant expression vector, be connected, transform, screening positive clone, electrophoresis detection sequence verification, the just trans-OCS of plant expression vector pCAMBIA2300-35S-CP, the just trans-OCS of pCAMBIA2300-35S-Hc-Pro, the just trans-OCS of pCAMBIA2300-35S-NIb successfully constructs.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN103952423A (en) * | 2014-04-11 | 2014-07-30 | 中国热带农业科学院热带生物技术研究所 | Chimeric gene capable of simultaneously targeting carica papaya viruses PRSV and PLDMV, plant expression vector and construction method of vector |
| CN103952423B (en) * | 2014-04-11 | 2016-06-29 | 中国热带农业科学院热带生物技术研究所 | The mosaic gene of target Fructus Caricae virus PRSV and PLDMV, plant expression vector and construction method thereof the while of a kind of |
| CN110484557A (en) * | 2018-05-14 | 2019-11-22 | 浙江省农业科学院 | A kind of instantaneous silent carrier of Atg5 |
| CN109468335A (en) * | 2018-11-09 | 2019-03-15 | 中国热带农业科学院热带生物技术研究所 | Improve the gene and edit methods of PRSV breeding for disease resistance high efficiency and broad spectrum activity |
| CN117778620A (en) * | 2024-02-27 | 2024-03-29 | 中国热带农业科学院三亚研究院 | DNA probe, kit and method for distinguishing resistant varieties of papaya ringspot viruses |
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