CN102071208A - Nucleic acid molecule of transgenic pawpaw strain 16-0-1 as well as detection method and applications thereof - Google Patents
Nucleic acid molecule of transgenic pawpaw strain 16-0-1 as well as detection method and applications thereof Download PDFInfo
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Abstract
The invention relates to a nucleic acid molecule of a transgenic pawpaw strain 16-0-1 as well as a detection method and applications thereof. The invention provides a method for detecting the transgenic pawpaw strain 16-0-1, comprising the following steps of: providing a pawpaw nucleic acid sample and a primer pair; forming a polymerase chain mixture from the pawpaw nucleic acid sample and the primer pair and carrying out an amplification reaction to obtain an amplification reaction product; and detecting the amplification reaction product, wherein if an amplification segment in a preset size exists, the sample contains the DNA (Deoxyribonucleic Acid) of the transgenic pawpaw strain 16-0-1. The invention also provides a primer for detecting the transgenic pawpaw strain 16-0-1, a probe and a kit. The sequence, the method, the primer and the kit are favorable to meeting the regulatory requirements and intellectual property protection and rapidly sieving homozygous transgenic offspring.
Description
Technical field
The present invention relates to a kind of nucleic acid molecule with transgenosis pawpaw strain 16-0-1 of the anti-pawpaw ring spot virus of popularity characteristic.What the present invention also relates to the application of aforementioned sequence a kind ofly is used to detect the method that has the transgenosis pawpaw strain 16-0-1 of extensive resistance for the pawpaw ring spot virus, and according to designed primer, probe and the test kit that goes out of aforementioned sequence.
Background technology
Pawpaw (Papaya) (Carica papaya L.) grows in the torrid zone and subtropical zone.The greatest problem that the volume production pawpaw is faced is to be pawpaw ring spot virus (papaya ringspot virus, the destructive disease that PRSV) causes (Purcifull et al.1984).1975, PRSV found (Wang et al.1978) in area, South Platform gulf first, had destroyed the most pawpaw in commercial orchard so far and had produced.PRSV is a member that belongs to potato Y group virus (Potyvirus), and this group's virus is the hugest and influences the very huge plant virus group of economy (Fauquetet al.2005).PRSV can transmit and bring out the generation of leaf portion spot and distortion, cane striped and cessation of growth cessation by aphid, and causes die-off (the Purcifull et al.1984) of fruit quality and output.
Be used for protecting pawpaw to avoid the measure of control that PRSV infects at present, mainly comprise: select implantation time, to avoid the aphid summit of growth phase; Use silver color coverture (silver mulch), to expel aphid away from seedling; And cross-protection (cross protection) (the Yeh et al.1988 that uses the PRSV virus strain that weakens; Yehand Gonsalves, 1994).Yet these methods all can't provide the permanently effective protection of antagonism PRSV.At present in Taiwan, pawpaw planted in the solarium, become the most effective control method that the PRSV that is used to avoid pawpaw to suffer to transmit through aphid infects.Yet the solarium is constructed needs higher cost, and employed plastic material is difficult to decompose in nature, and this will work the mischief to environment, and the also major issue (Bau et al.2003) for considering of the hurtful height risk of tropical storm.
In recent years, resistance (pathogen-derived resistance with the pathogeny syntaxy, PDR) idea (Sanfordand Johnson, 1985) be the basis, the research and development that contain the transgenic plant of plant virus genomic fragment have been used widely as the viral countermeasure (Beachy, 1997) of control.In most example, resistance (posttranscriptional resistance) mechanism of transcribing property after can producing, this is to regulate path due to (English et al.1996 in the sequence-specific mode at the RNA of the RNA of virus and transgenosis mRNA degraded because of a kind of; Lindboet al.1993; Siien and Kooter 2000; Vaucheret et al.1998).Coat protein (the coatprotein of PRSV, CP) gene is transferred to (Fitch etal.1990) in the pawpaw by particle bombing pattern (particle bombardment), and filters out transgenic strain (the Fitch et al.1992 that infection has the height resistance to PRSV; Lius et al.1997).
The transgenosis pawpaw from 1998 successfully in Hawaii commercialization (Gonsalves, 2002; Tripathi etal.2007).In Taiwan, the transgenosis pawpaw strain that has the CP gene of the serious virus strain PRSV YK in Taiwan then goes out (Cheng et al.1996) by successful manufactured of the method for transformation (Agrobacterium-mediated transformation) of Agrobacterium media.When aforementioned transgenosis pawpaw was given PRSV YK by inoculation, they dropped on height susceptibility (high susceptibility) to the scope with complete resistance (complete resistance) interior (Bau et al.2003) to the reaction of virus.Under greenhouse experiment, transgenic strain 16-0-1,17-0-1,17-0-5 and 18-2-4 have resistance (Bau et.al. widely for the PRSV that is derived from different geographic origin, 2003), the resistance (Bau et al.2004) that height is then arranged in the test of field shows that these strains have the ability of the PRSV in the control different geographic regions.
Experimental observation finds that 16-0-1,17-0-5 and 18-2-4 are when plant strain growth to 5 centimeter height, the transgenosis of meeting apparent altitude, yet then in same phase not performance fully, the transgenosis in the transgenosis pawpaw strains such as visible 16-0-1,17-0-5 and 18-2-4 is inserted in genomic site must its singularity such as 18-0-9,19-0-1 for other.Prior art still can't be learnt aforementioned transgenosis pawpaw strain, the genome sequence of the transgenosis on position side of transgenosis pawpaw strain 16-0-1 particularly, insert the genome position if can understand it, can directly carry out at the pawpaw of various kinds such as the transgenosis pawpaw strain that has anti-pawpaw ring spot virus characteristic with the efficient generation of the gene recombination of homology, and need not via long screening and field test, to obtain the pawpaw strain of high antiviral.
On the other hand, in recent years, (genetically modified organism, security subject under discussion GMO) has caused sizable attention (Singh et al., 2006) to the genetic modification biology.Various countries have implemented various rules about authentication of GM food and sign (food apporval and labeling).European Union's rules the 1830/2003rd [European Union (EU) regulation (EC) No.1830/2003] are defined in each stage of producing to the market of supply chain and must be able to follow the trail of genetic modification biology and biologically-derived product (the EuropeanParliament and Council of European Union of genetic modification, 2003, p.1-5).
Same, Taiwan also requires to provide information about the genome sequence of the transgenosis side of specific transgenic strain for the rules of the field test of genetic modification crop (GM crop) and the kind power that continues.Therefore, be necessary for meeting statutory regulations in order to tool specificity and the reliable method of identifying specific gene transformation biology, and for cultivating, have importance for the sale of the undelegated genetic modification biology of monitoring.
Therefore, in this technical field, have urgent demand for a kind of in order to the method for identifying specific gene transformation biology, wherein this method for aforesaid for the transgenosis pawpaw strain of 16-0-1, must be to have specificity, reproducibility, high sensitive and reliable.
Summary of the invention
Brief summary of the invention
Because have the demand of the transgenosis pawpaw strain of the anti-pawpaw ring spot virus of popularity characteristic for efficient generation in the prior art field, and the prior art shortage has specific authentication method for transgenosis pawpaw strain, purpose of the present invention promptly is to solve prior art must be via long screening and field test, just can obtain the problem of the pawpaw strain of high antiviral, and the various related art scheme that the problem of identifying at transgenosis pawpaw strain 16-0-1 is provided, the nucleic acid molecule that for example has the transgenosis pawpaw strain of the anti-pawpaw ring spot virus of popularity characteristic, the detection method of relevant transgenosis pawpaw strain, primer, probe, test kit, and aforesaid technical scheme must be to have specificity for the transgenosis pawpaw strain of 16-0-1, reproducibility, high sensitive and reliable.
In order to achieve the above object, in first aspect, the present invention system provides a kind of isolating nucleic acid molecule with transgenosis pawpaw strain 16-0-1 of the anti-pawpaw ring spot virus of popularity characteristic, it has: one is positioned at the right margin side areas of 5 ' end, one is positioned at the left margin side areas of 3 ' end, and a transgenic sequence that contains pawpaw ring spot virus coat protein gene (PRSV CP gene) between right margin side areas and left margin side areas, wherein the right margin side areas comprises one and has the sequence of 90% homology with sequence shown in the SEQ ID NO:29; Left boundary flanking sequence comprises one and has the sequence of 90% homology with sequence shown in the SEQ ID NO:31; And this transgenic sequence system comprises a pawpaw ring spot virus coat protein gene and an exercisable promotor that is linked to pawpaw ring spot virus coat protein gene upstream.
In second aspect, the present invention system provides a kind of primer of the transgenosis Semen Chaenomelis acid sequence that is used to increase, and it is to be selected from the group that is made of following: one has the nucleic acid fragment of at least 10 continuous sequences among shown in SEQ ID NO:29 sequence or its complementary sequence; And the nucleic acid fragment with at least 10 continuous sequences among shown in SEQ ID NO:31 sequence or its complementary sequence.
In the third aspect, the present invention system provides a kind of method that is used to detect transgenosis pawpaw strain 16-0-1, and it comprises the following steps:
Provide a pawpaw nucleic acid samples and a primer right, wherein this primer is to comprising a forward primer and a reverse primer;
Make this pawpaw nucleic acid samples and this primer to forming a pcr reaction mixture, carry out amplified reaction, obtain an amplified reaction product; And
Detect the amplified reaction product, wherein the amplified fragments if any a pre-sizing exists, and then represents this pawpaw nucleic acid samples to contain the genomic dna that stems from transgenosis pawpaw strain 16-0-1; Wherein
This forward primer system is selected from the group that is made of following person: one has the nucleic acid fragment of at least 10 continuous sequences among the sequence shown in SEQ ID NO:29; One has the nucleic acid fragment of at least 10 continuous sequences in the sequence shown in SEQ ID NO:33; And their complementary sequence; And
This reverse primer system is selected from the group that is made of following person: one has the nucleic acid fragment of at least 10 continuous sequences among the complementary sequence of sequence shown in SEQ ID NO:31; One has the nucleic acid fragment of at least 10 continuous sequences in the complementary sequence of sequence shown in SEQ ID NO:33; And their complementary sequence.
Detailed description of the invention
In the past over 10 years, in Hawaii and the Taiwan successfully cultivate commercial obtainable transgenosis pawpaw strain, it is because of having pawpaw ring spot virus (papaya ringspot virus, PRSV) coat protein (coatprotein, CP) gene resists the popularity virus resistance of the PRSV virus in source, territory variously and can have.Yet filtering out transgenosis pawpaw strain that a pair of pawpaw ring spot virus has highly an extensive stable resistance must just can obtain via long screening, field test with the method for prior art.
In view of this, the site that the T-DNA that contains the pawpaw ring spot virus coat protein that the present invention utilizes molecular biotechnology successfully to parse transgenosis pawpaw strain 16-0-1 inserts, and analysis obtains its right side, the genome sequence of left margin side, thereby isolate one and have applicable to manufacturing and have highly the extensively nucleic acid molecule of the transgenosis pawpaw of stable resistance, it has: one is positioned at the right margin side areas of 5 ' end, one is positioned at the left margin side areas of 3 ' end, and a transgenic sequence that contains pawpaw ring spot virus coat protein gene (PRSV CP gene) between right margin side areas and left margin side areas, wherein the right margin side areas comprises one and has the sequence of homology with sequence shown in the SEQID NO:29; Left boundary flanking sequence comprises one and has the sequence of homology with sequence shown in the SEQ ID NO:31; And this transgenic sequence system comprises a pawpaw ring spot virus coat protein gene and an exercisable promotor that is linked to pawpaw ring spot virus coat protein gene upstream.
On the other hand, based on the requirement of the rules of European Union and part Asian countries for the tracking of genetic modification biology, press for a kind of event-specific detection method at aforementioned transgenosis pawpaw strain (event-specific detection) in the art, this event-specific detection method has high-level efficiency and highly sensitive characteristics.
In view of this, the present invention is based on polymerase chain reaction, develops foregoing detection method, primer and test kit at the characteristic of the transgenosis pawpaw strain of PRSV CP gene.Transgenosis specificity product can be by using at the promotor in the transgenosis, terminator, selection markers and genetically modified various primer being amplified.In addition, described joint-graft polymerization enzyme chain reaction (adaptor ligation-PCR, AL-PCR) selected growing (cloned) and the sequencing in the present invention of the connecting zone (junction) between plant genome DNA that is amplified and the T-DNA inset of utilizing.
In addition, the invention provides a kind of incident-method for detecting specificity at transgenosis and flanking sequence thereof, it is to can be used for identifying specific transgenosis pawpaw strain 16-0-1.According to the designed primer that goes out of the flanking sequence on genetically modified left and right border, in the time of in being used in amplified reaction, the aspect cording of the PCR product that it amplified has reproducibility and specificity among the present invention.The present invention also confirms that PRSV CP gene is to be inserted in the genomic different positions of transgenosis pawpaw, and the number of copies of the T-DNA that inserts is made by instant PCR is tested.Therefore, incident-specificity method provided by the present invention, primer and test kit need be followed the trail of the country of genetic modification biology for laws and regulations requirement and be useful and important for protecting the intellectual property.In addition, method provided by the present invention, primer and test kit help to filter out rapidly homozygote transgenosis filial generation (homozygote-transgenic progeny) in breeding plan (breeding program).
According to the present invention, term " homology (homology " means the degree of associated between two sequences, its can sequence between identical and/or conservative (conservative) than calibration.Because the variation of nucleic acid or aminoacid sequence must not influence the characteristic that it constitutes, therefore as long as having homology to a certain degree, nucleic acid or its aminoacid sequence of encoding out do not influence characteristic or this activity of proteins of nucleotide sequence, be the present invention and contain, above-mentioned homology to a certain degree preferably at least 90%.
In the preferred embodiment of the nucleic acid molecule of isolating transgenosis pawpaw strain 16-0-1 with the anti-pawpaw ring spot virus of popularity characteristic of the present invention, wherein this promotor is a Cauliflower edge camphane virus 35S promoter (Cauliflower Mosaic Virus 35S promoter, CaMV 35S promoter).
In the preferred embodiment of the nucleic acid molecule of isolating transgenosis pawpaw strain 16-0-1 with the anti-pawpaw ring spot virus of popularity characteristic of the present invention, wherein this transgenic sequence system comprises one and has the sequence of 90% homology with sequence shown in the SEQ ID NO:34.
In the preferred embodiment of the nucleic acid molecule of isolating transgenosis pawpaw strain 16-0-1 with the anti-pawpaw ring spot virus of popularity characteristic of the present invention, foregoing nucleic acid molecule, it is made of following person: one is positioned at the right margin side areas of 5 ' end, one is positioned at the left margin side areas of 3 ' end, and a transgenic sequence that contains the pawpaw ring spot virus coat protein gene between right margin side areas and left margin side areas, wherein the right margin side areas has sequence shown in SEQ ID NO:29; Left boundary flanking sequence has sequence shown in SEQID NO:31; And this transgenic sequence has sequence shown in SEQ ID NO:33.
According to method of the present invention, the primer system of the described transgenosis Semen Chaenomelis acid sequence that is used to increase is selected from the group that is made of following: one has the nucleic acid fragment of at least 10 continuous sequences among shown in SEQ ID NO:29 sequence or its complementary sequence; And the nucleic acid fragment with at least 10 continuous sequences among shown in SEQ ID NO:31 sequence or its complementary sequence.
Preferably, aforementioned primer is to be selected from the group that is made of following: one has the nucleic acid fragment of at least 15 continuous sequences among shown in SEQ ID NO:29 sequence or its complementary sequence; And the nucleic acid fragment with at least 15 continuous sequences among shown in SEQID NO:31 sequence or its complementary sequence.
According to method of the present invention, the primer of the aforementioned transgenosis Semen Chaenomelis acid sequence that is used to increase preferably is selected from the group that is made of following primer: the Papa31 primer, and it has sequence shown in SEQ ID NO:17; And the Papa57 primer, it has sequence shown in SEQ ID NO:20.
According to the present invention, any chemistry known to " primer (primer) " can utilize in this technical field or biochemical synthetic method preparation.For example, when primer was constituted by nucleic acid, it can be by nucleic acid synthetic method preparation common in the field of genetic engineering.For example, nucleic acid can be directly synthetic by using dna synthesizer.And in order to make the nucleic acid that obtains can be comparatively stable in cell, can be further partly carry out chemical at base, candy base and the phosphoric acid of nucleic acid to modify, such as alkylation (alkylation), acylations (acylation) or other similar effects.
Term " probe " but mean and can produce the identification particular sequence and can produce material for the signal that detects, the probe of usefulness comprises the nucleic acid fragment through mark as shown here, and wherein this mark includes but not limited to radioactive substance, fluorescence dye and is subjected to matter specificity ferment etc.
According to the method that is used to detect transgenosis pawpaw strain 16-0-1 of the present invention, it comprises: provide a pawpaw nucleic acid samples and a primer right, wherein this primer is to comprising a forward primer and a reverse primer; Make this pawpaw nucleic acid samples and this primer to forming a pcr reaction mixture, carry out amplified reaction, obtain an amplified reaction product; And detect the amplified reaction product, wherein the amplified fragments if any a pre-sizing exists, and then represents this pawpaw nucleic acid samples to contain the genomic dna that stems from transgenosis pawpaw strain 16-0-1; Wherein this forward primer system is selected from the group that is made of following person: one has the nucleic acid fragment of at least 10 continuous sequences among the sequence shown in SEQ ID NO:29; Preferably, the nucleic acid fragment of at least 15 continuous sequences; An and nucleic acid fragment that has as at least 10 continuous sequences of SEQ ID NO:33 among the sequence of the 4468th to 5468 nucleotide site; Preferably, the nucleic acid fragment of at least 15 continuous sequences; And this reverse primer system is selected from the group that is made of following person: one has the nucleic acid fragment of at least 10 continuous sequences among the complementary sequence of sequence shown in SEQ ID NO:31; Preferably, the nucleic acid fragment of at least 15 continuous sequences; An and nucleic acid fragment that has as at least 10 continuous sequences of SEQ ID NO:33 among the complementary sequence of the sequence of the 1st to 1000 nucleotide site; Preferably, the nucleic acid fragment of at least 15 continuous sequences.
In a preferred embodiment of the present invention, aforesaid forward primer is to be selected from the group that is made of following primer: the Papa31 primer, and it has sequence shown in SEQ ID NO:17; The P1 primer, it has sequence shown in SEQ ID NO:13; The Papa34 primer, it has sequence shown in SEQ ID NO:15; The Papa52 primer, it has sequence shown in SEQ ID NO:14; The Papa56 primer, it has sequence shown in SEQ ID NO:19; The Papa58 primer, it has sequence shown in SEQ ID NO:21; And the N1 primer, it has sequence shown in SEQ ID NO:16; And reverse primer system is selected from the group that is made of following primer: the S18 primer, and it has sequence shown in the SEQ ID NO:11; The Papa27 primer, it has the IDNO:12 as SEQ; And the Papa57 primer, it has the NO:20 as SEQ ID.
According to the present invention, described amplified fragments (amplified fragment) means and utilizes aforementioned primer is that template is carried out the specific fragment that polymerase chain reaction amplified with the nucleic acid samples, for example use Papa31 primer (SEQ IDNO:17) to be reverse primer as forward primer and Papa27 primer (SEQ ID NO:12), genomic dna with transgenosis pawpaw strain 16-0-1 is a template, under suitable polymerase chain reaction condition, then can amplify the amplified fragments of one 241 base pairs.
According to the present invention, comprise primer, template, polysaccharase, deoxidation ribonucleoside triphosphote (deoxyribonucleotide triphosphate known to the described pcr reaction mixture technical field like this, dNTP) and reaction buffer, and can carry out an amplified reaction, obtain an amplified reaction product.In a preferred embodiment of the present invention, described pcr reaction mixture more comprises a dyestuff, and this dyestuff system can combine with amplified fragments, and accepts suitable fluorescence excitation and emit detectable fluorescent signal, for example, but is not limited to
Green.
According to the present invention, described pawpaw nucleic acid samples is can be obtained from each organ, position (such as root, stem, leaf, flower, fruit, the seed etc.) of pawpaw by any nucleic acid extracting process known in the art.Preferably utilize bromination hexadecyl trimethyl ammonium (cetyltrimethylammonium bromide, CTAB) purifying and getting from the leaf portion of pawpaw.
According to the present invention, detect the amplified reaction product and preferably be the modes such as fluorescent signal that comprise by electrophoretic analysis and directly detect amplified reaction and carry out, the mode that wherein directly detects the fluorescent signal of amplified reaction is to comprise specific probe or the use of detection through dye marker
The fluorescent signal that dyestuffs such as Green are launched behind suitable fluorescence excitation.
Description of drawings
Fig. 1 shows the side areas of the T-DNA in the transgenosis pawpaw strain, wherein Fig. 1 (a) district shows the T-DNA inset that is positioned at the pawpaw genome, Fig. 1 (b) district shows the amplification strategy of the genome sequence of right margin side, and Fig. 1 (c) district shows the amplification strategy of the genome sequence of left margin side.
The PCR of Fig. 2 use pattern 1,2,3 detects and identifies transgenosis pawpaw strain; Wherein
Fig. 2 (a) district shows the result at the Class1 detection of 35S promoter, and it utilizes the 35S promoter Auele Specific Primer 35S-F/35S-R amplification to be got the product of one 836 base pairs;
Fig. 2 (b) district shows the result at the Class1 detection of nptII, and it utilizes the nptII Auele Specific Primer npt-1/npt-2 amplification to be got the product of one 829 base pairs;
Fig. 2 (c) district shows the result at the Class1 detection of no terminator, and it utilizes no terminator Auele Specific Primer the nos-1/nos-2 amplification to be got the product of one 126 base pairs;
Fig. 2 (d) district shows the result who detects at genetically modified type 2, and it utilizes PRSV CP gene-specific primer the PRSV-F/PRSV-R amplification to be got the product of one 840 base pairs; And
Fig. 2 (e) district shows the result at CaMV 35S promoter and genetically modified type 3 detections of PRSV CP, and it utilizes primer the 35S-F/PRSV-R amplification to be got the product of one 700 base pairs.
Fig. 3 shows the sequential analysis of T-DNA right margin/pawpaw genomic dna connecting zone, wherein the T-DNA sequence with and right margin be to represent with small letter, the identification site (ATT) of SspI indicates as arrow, in addition primer locations such as Ap1, Ap2, Papa27, Papa31 and Papa32 such as figure sign; Wherein
Fig. 3 (a) district shows the genome flanking sequence (SEQ ID NO:29) of the T-DNA right margin of transgenic strain 16-0-1 and 17-0-5; And
Fig. 3 (b) district shows the genome flanking sequence (SEQ IDNO:30) of the T-DNA right margin of transgenic strain 18-2-4.
Fig. 4 shows the sequential analysis of T-DNA left margin/pawpaw genomic dna connecting zone, wherein the T-DNA sequence with and left margin be to represent with small letter, the identification site of DraI and EcoRV (TTT and GAT) indicates as arrow respectively, in addition primer locations such as Ap1, Ap2, Papa52, Papa56, Papa57, Papa58 Papa59 and N1 such as figure sign; Wherein
Fig. 4 (a) district shows the genome flanking sequence (SEQ ID NO:31) of the T-DNA left margin of transgenic strain 16-0-1 and 17-0-5; And
Fig. 4 (b) district shows the genome flanking sequence (SEQ IDNO:32) of the T-DNA left margin of transgenic strain 18-2-4.
Fig. 5 shows that the event-specific of transgenosis pawpaw strain detects, and it is to utilize genome flanking sequence and T-DNA sequence are had specific primer to producing the DNA product of crossing over T-DNA right margin and Plant Genome or the DNA product of crossing over T-DNA left margin and Plant Genome; Wherein
The demonstration of Fig. 5 (a) district utilizes primer Papa31/Papa27 to be amplified the specificity product of one 241 base pairs at 16-0-1 strain (right margin);
The demonstration of Fig. 5 (b) district utilizes primer Papa32/Papa27 to be amplified the specificity product of one 384 base pairs at 18-2-4 strain (right margin);
The demonstration of Fig. 5 (c) district utilizes primer Papa56/Papa57 to be amplified the specificity product of one 106 base pairs at 16-0-1 strain (left margin);
The demonstration of Fig. 5 (d) district utilizes primer Papa58/Papa59 to be amplified the specificity product of one 140 base pairs at 18-2-4 strain (left margin);
The demonstration of Fig. 5 (e) district utilizes primer Papa31/Papa57 to be amplified the specificity product of one 227 base pairs; And
The demonstration of Fig. 5 (f) district utilizes primer Papa32/Papa59 to be amplified the specificity product of one 345 base pairs.
Fig. 6 demonstration utilizes the Southern engram analysis to measure PRSV CP transgenosis number of copies.
Fig. 7 shows the genetically modified instant pcr amplification figure of PRSV CP, wherein Rn numerical value difference (delta Rn value) with report sub-stain from
Representing when probe is released through normalized fluorescent emission; Wherein
Fig. 7 (a) district shows 16-0-1 heterozygote T
0The typical amplification curve of plant materials;
Fig. 7 (b) district shows 16-0-1 homozygote T
1The typical amplification curve of plant materials;
Fig. 7 (c) district shows 18-2-4 heterozygote T
0The typical amplification curve of plant materials; And
Fig. 7 (d) district shows 18-2-4 homozygote T
1The typical amplification curve of plant materials.
Embodiment
The present invention will further explain by the following examples, but what should understand is that these embodiment only are the usefulness of explanation, and should not be regarded as the restriction in the enforcement of the present invention.
Embodiment
General materials and methods
1. vegetable material
Employed vegetable material is to have the CP gene of PRSV and to the transgenosis pawpaw (Cheng et al., 1996) of PRSV tool height resistance, comprising the T of 16-0-1,17-0-5 and 18-2-4 strain among the following embodiment
0The T of plant materials and 16-0-1 and 18-2-4 strain heterozygote or homozygote filial generation
1Plant materials, it all carries out micropropagation (micropropagated) (Yang et al., 1996) in the mode of tissue culture.Use not genetically modified species (varieties) as a comparison to be: (Tainung No.1 of platform farming, TN1), No. two (TainungNo.2 of platform farming, TN2), No. five (Tainung No.5 of platform farming, TN5), No. six, platform farming (Tainung No.6, TN6), day rise red (Red Ear) in (Sunrise), red prince wife (Red Lady), the fringe, the big melon of horse (Mai Tai Kua) and Thailand (Thailand).Their seedling is that (Taiwan AgriculturalResearch Institute, Fengshan torrid zone gardening test branch (Fengshan Tropical HorticulturalExperiment Branch) TARI) provides by being positioned at agricultural experiment institute of Taiwan agricultural commission of Executive Yuan.The plant of transgenic strain and non-transgenic kind is to cultivate growth in the greenhouse that TARI sets up.
2. pawpaw genomic dna (genomic DNA) purifying
The pawpaw genomic dna is purifiedly from the fresh blade of 0.5g transgenosis pawpaw strain and non-transgenic species to go out, it is with the employed method of people such as Dolyle (Doyle and Doyle, 1990) utilize bromination hexadecyl trimethyl ammonium (cetyltrimethylammonium bromide, CTAB) (SIGMA-ALDRICH Inc., St.Louis, MO, USA) purifying and getting.[poly (vinylpolypyrrolidone), PVPP) (SIGMA-ALDRICH Inc.) is added to the extraction damping fluid, in order to increase the purity of the DNA that extracts with the polyvinyl pyrrolo-indole.DNA concentration by spectrograph (U-3000 Spectrophotometer, Hitachi Instruments Inc., SanJose, CA, USA) assess the measurement OD
260, and the DNA quality is to utilize 1% agarose coagulation to analyze.
3. utilize polymerase chain reaction (polymerase chain reaction, transgenosis specific detection PCR)
Evaluation as Class1, primer is to 35S-F/35S-R, npt-1/npt-2 and nos-1/nos-2, as shown in table 1, be used for detecting Cauliflower edge camphane virus 35S promoter (Cauliflower mosaic virus 35Spromoter respectively, CaMV 35S promoter), Kang Na mycin selection markers gene npt II (kanamycin selectionmarker gene nptII) and Nuo Palin synthetic enzyme terminator (nopaline synthase terminator, nosterminator) (Hoist-Jensen et al., 2003).Identify for type 2, be to use as shown in table 1 the PRSVCP gene order to be had specific primer to PRSV-F/PRSV-R (Bau et al., 2003).Identify for type 3, be to use to 35S promoter (35S promoter) have specific primer to and PRSV CP gene had specific primer to 35S-F/PRSV-R.Pcr amplification reaction mixture (final volume 30 μ L) contain 128ng template DNA (template DNA) and 1.2unit FastStart Polymerase (RocheDiagnotics GmbH, Penzberg, Germany), 2.5mM MgCl
2, 200 μ M dNTP and 0.2 μ M primer, they are to be assigned in PCR damping fluid (50mM KCl; 10mM Tris-HCl, pH9.0; 0.1%TritonX-100).PCR is 30 recycles, one thermal cycling machine (the thermocycler) (Gene to comprise the following steps
PCR System 9700, Applied Biosystem, Foster City, CA USA) carries out: last 1 minute under 94 ℃, in order to cracking (melting), under different bonding temperature, last 1 minute, in order to binding (annealing), and last 2 minutes in 72 ℃, in order to synthetic (synthesis).The PCR product passes through 2% agarose coagulation in TAE damping fluid (Tris acetate, pH8.0; 1mM EDTA) analyzes with electrophoresis in.
The PCR product directly uses 310 Genetic Analyzer (ABI PRISM
TM, Applied Biosystems) and give sequencing.The sequencer procedure of PCR prolongation effect (PCR elongation) is to be carried out to go through 25 circulations in the aforementioned hot circulator.After the last prolongation effect, the product of this sequencing connects and precipitated, extraction and redissolve (redissolved) in sequencing electrophoretic buffer (sequencing running buffer) (Hi-Di
TMFormamide, Applied Biosystems).Sample connects and utilizes 61 centimeters kapillaries to be loaded to ABI 310, to carry out sequencing.Lasergen Software (DNASTAR, 2001, DNASTAR Inc., Madison, WI USA) is used for arranging sequence of relatively (align) amplification and specific known array.
The sequence of employed primer and bonding temperature thereof among table 1 the present invention
| Primer/probe | Target (Target) | Sequence a | Ta b (℃) | SEQ ID NO： |
| 35S-F | 35S promoter | +24755’-CAGCTATGACCATGATTACGC-3’ +2495 | 55 | 1 |
| 35S-R | 35S promoter | +32935’-TCTTGCGAAGGATAGTGG-3’ +3310 | 55 | 2 |
| nos-1 | The no terminator | +16165’-TGCCGGTCTTGCGATGAT-3’ +1633 | 55 | 3 |
| nos-2 | The no terminator | +17205’-ATGTATAATTGCGGGACTCTAA-3’ +1741 | 55 | 4 |
| npt-1 | nptII | +3555’-ATAATCTGCACCGGATCTGG-3’ +374 | 55 | 5 |
| npt-2 | nptII | +11645’-CCGCTCAGAAGAACTCGTCA-3’ +1183 | 55 | 6 |
| PRSV- F | Transgenosis | +34005’-TCCAAGAATGAAGCTGTGGA-3’ +3419 | 55 | 7 |
| PRSV- R | Transgenosis | +42205’-GTGCATGTCTCTGTTGACAT-3’ +4239 | 55 | 8 |
| Ap1 | Joint | 5’-GTAATACGACTCACTATAGGGC-3’ | 56 | 9 |
| Ap2 | Joint | 5’-ACTATAGGGCACGCGTGGT-3’ | 56 | 10 |
| S18 | The no promotor | +2135’-ACGCGCAATAATGGTTTCTGACG-3’ +235 | 56 | 11 |
| Papa27 | The no promotor | +965’-GCGTCATCGGCGGGGGTCATAA-3’ +117 | 55 | 12 |
| P1 | Transgenosis | +37045’-CAAACACTCGCGCCACTCAA-3’ +3723 | 55 | 13 |
| Papa52 | The no terminator | +45845’-TGTTGCCGGTCTTGCGATGATTAT-3’ +4607 | 55 | 14 |
| Papa34 | The no terminator | +48175’-CAACGTCGTGACTGGGAAAAC-3’ +4837 | 55 | 15 |
| N1 | The no terminator | +49525’-GCCCGCTCCTTTCGCTTTCT-3’ +4971 | 56 | 16 |
| Papa31 | The RB side | 5’-TTGTTCTAATAAGGTTGCTAC-3’ | 55 | 17 |
| Papa32 | The RB side | 5’-AATATCAAATGGACGTGTTAGTG-3’ | 55 | 18 |
| Papa56 | Left margin (LB) | +54085’-GTTATTAAGTTGTCTAAGCGTCAA-3’ +5431 | 55 | 19 |
| Papa57 | The LB side | 5’-AGACATATATCATCAAGACCATAGTAG-3’ | 55 | 20 |
| Papa58 | Left margin | +45925’-GTCTTGCGATGATTATCAT-3’ +4610 | 55 | 21 |
| Papa59 | The LB side | 5’-TGGTTATCAATATAGCAATTATGTAG-3’ | 55 | 22 |
| S9-2 | PRSV CP gene (for being used for quantitative PCR) | 5’-AGTAACGCGGCAGAGGCATA-3’ | 60 | 23 |
| S10-2 | PRSV CP gene (for being used for quantitative PCR) | 5’-GAGCCCTATCAGGTGTTTTCGA-3’ | 60 | 24 |
| S5 | Papain gene (for being used for quantitative PCR) | 5’-TGGGTTTGTCATTTGGTGATTTT-3’ | 60 | 25 |
| S6 | Papain gene (for being used for quantitative PCR) | 5’-GTCTTTCAGTGGATGTCAAGTCATTT-3’ | 60 | 26 |
| Fam c | PRSV CP gene probe | 5’-TTAGTCTCGCTAGATATGCTT-3’ | 60 | 27 |
| Vic d | The papain gene probe | 5’-CTATTGTGGGTTATTCTC-3’ | 60 | 28 |
A. the numerical value in the other sequence is represented the position in T-DNA insertion.
B.Ta is for binding temperature (annealing temperature).
C. this probe is to be marked on 5 ' end (excitation wavelength is 488nm, and emission wavelength is 518nm) with FAM.
D. this spy really is marked on 5 ' end (excitation wavelength is 488nm, and emission wavelength is 552nm) with VIC.
4. the preparation of genomic DNA fragment and connector engages reaction (adaptor ligation)
The genomic DNA fragment grown of choosing is to utilize method (Sibbert et al., the 1995) preparation that the described person of people such as Sibbert suitably modifies in addition and get.Before also got with the preceding method preparation through transgenosis pawpaw strain 16-0-1, the 17-0-5 of specificity analysis and the genomic dna of 18-2-4.DraI (New England Biolabs, Inc., Ipswich, MA, USA), SspI (Promega, Madison, WI, USA) and EcoRV different restriction enzymes such as (Promega) be used in digestion (digest) discrete genomic dna (respectively organizing quantity is 10 μ g) respectively, use producing blunt end fragment (blunt-end fragment).The joint that contains a long-chain and a complementary short chain is to prepare by binding following two complementary oligonucleotide: long-chain (48 Nucleotide): 5 '-GTAATACGACTCACTATAGGGCACGCGTGGTCGACGGCCCGGGCAGGT-3 '; And short chain (8 Nucleotide): 5 '-PO
4-ACCTGCCC-NH
2-3 '.Joint is that final volume with one 15 μ L reacts with the reaction that engages through the genomic dna of restriction enzyme digestion, and wherein contains 1.5 μ L10XFast-Link
TMGenomic dna storehouse (restricted genomic DNA libraries) and 1 μ L Fast-Link DNA Ligase (Fast-Link that Ligation Buffer, 0.75 μ L 10mM ATP, 0.5 μ L, 100 μ M upper end and lower end chain joint, 2 μ L digest through restriction enzyme
TMDNA Ligation Kits, Epicentre, Madison, WI, USA).After 15 ℃ reaction was spent the night down, reaction soln was placed in and lasts 10 minutes under 70 ℃, uses stopped reaction.Afterwards, utilize High Pure PCR Product Purification Kit (Roche Diagnostics GmbH) purification reaction product.
5. nido polymerase chain reaction (nested polymerase chain reaction)
The flanking sequence that is positioned at the T-DNA inset of pawpaw genome is to utilize the genomic DNA fragment through digestion that engages with joint to be measured in PCR step (PCR walking) mode of moving.Because long-chain comprises sequence (Zheng et al., 2001a with Ap1 and Ap2 primer tool homology (homologous); Zheng et al. 2001b), therefore is used as joint in following embodiment.The T-DNA Auele Specific Primer be designed to near right margin (right border, RB) or left margin (left border, LB) Qu Yu sequence match (match) (distinguishing) referring to Fig. 1 (a) and (b) and (c).For transgenic strain, near the primer S18 in RB zone and Papa27 be designed to start at first Nucleotide of RB+235 to+213 and+117 to+96 content array match respectively (referring to table 1 and Fig. 1 (b) district).For transgenic strain 18-2-4, near the primer P1 in LB zone and Papa52 be designed to start at first Nucleotide of RB+3704 to+3723 and+4584 to+4607 content array match respectively (referring to table 1 and Fig. 1 (c) district).For transgenic strain 16-0-1 and 17-0-5, near the primer Papa34 and the N1 in LB zone be designed to start at first Nucleotide of RB+4817 to+4837 and+4952 to+4971 (referring to table 1 and Fig. 1 (c) districts).
For the RB zone, a PCR (primary PCR) is that the reaction mixture with one 30 μ L carries out, and this reaction mixture comprises 128ng and dna fragmentation, 1.2 unitFastStart Polymerase (Roche Diagnostics GmbH), 2.5mM MgCl through restriction enzyme digestion connector engages
2, 200 μ M dNTP and 0.2 μ M primer S18 and Ap1, they are to be assigned in PCR damping fluid (50mM KCl; 10mM Tris-HCl, pH 9.0; 0.1% Triton X-100).PCR is after hot initial (the hot start) that last 1 minute under 94 ℃, continues and carries out carrying out with a fixedly denaturing step that lasts 30 seconds under 94 ℃ (denaturing step).First six circulation (six cycles) is 61 ℃ with initial bonding temperature (annealing temperature) to last 30 seconds, continues and carries out with the decreasing ratio of 1 ℃ of every circulation.And prolong step (extension step) is to carry out in 72 ℃ of modes of lasting 3 minutes in each circulation.30 extra circulations are to last 3 minutes and to carry out in the prolongation that each circulation that continues increases by 10 seconds ratio again in the sex change that lasts 30 seconds under 94 ℃, the bonding that lasts 30 seconds under 56 ℃ and in 72 ℃.PCR be in final prolongation with the reaction that under 72 ℃, lasts 5 minutes as end.Secondary PCR (Secondary PCR) is with after 1000 times of the PCR product dilutions, utilizes primer Ap2 and RB-Auele Specific Primer Papa27 (referring to table 1) to carry out.Other compositions of PCR reagent mixture (PCR reagent mixture) are identical with an aforementioned PCR.
Use has specific primer to carrying out a similar amplified reaction to the LB zone.For a PCR, use primer Papa34 and Ap1 to carry out the amplification in the LB zone of 16-0-1 and 17-0-5 strain; Use primer P1 and Ap1 to carry out the amplification (referring to table 1 and Fig. 1 (c) district) in the LB zone of 18-2-4 strain.For secondary PCR, be to use primer N1 and Ap2 to carry out at 16-0-1 and 17-0-5 strain; Be to use primer Papa52 and Ap-2 at the 18-2-4 strain.The PCR product is analyzed with electrophoresis by 2% agarose coagulation.
6. the mensuration of the genome sequence of transgenosis side
Guide according to manufacturers, to be selected from the product that nest-type PRC gets through the secondary amplification and be grown to TOPO TA carrier (TOPO TA vector) (Invitrogen, Carlsbad, CA, USA), (Qiagen GmbH, Hilden Germany) are purified into and the plastid DNA through screening utilizes Spin Miniprep Kit.Purified plastid is by 310Genetic Analyzer (Applied Biosystems) sequencing.Genome flanking sequence (genomic flankingsequences) and T-DNA border sequence are by Lasergene Software (DNASTAR Inc.) and CpGDB (pawpaw Plant Genome database, network address is http://www.plantgdb.org/CpGDB/) (Ming etal., 2008) made up and analyzed.
7. the event-specific of transgenosis pawpaw strain is identified (event-specific identification)
Detection as type 4 event-specifics, the Auele Specific Primer that will match with T-DNA sequence near RB and LB zone, comprise the Papa27,57 and 59 (as shown in table 1) that are used in the nest-type PRC, give coupling (coupled) to according to through the designed Auele Specific Primer that goes out of the genome flanking sequence of resolving, cross the zone on each T-DNA border in order to amplification (amplify); Wherein primer Papa31 and Papa56 (as shown in table 1) go out according to the RB of strain 16-0-1 strain and LB zone are designed, and primer Papa32 and Papa58 (as shown in table 1) go out according to the RB of strain 18-2-4 and LB zone are designed.PCR is that the reaction mixture with one 30 μ L carries out, and this reaction mixture comprises 1.2 unit FastStart Polymerase (Roche Diagnostics GmbH), 2.5mM MgCl
2, 200 μ M dNTP and the aforementioned suitable primer of 0.2 μ M to and 16-0-1, the 17-0-5 of 128ng and the genomic dna of 18-2-4 strain, it is to be assigned in PCR damping fluid (50mM KCl; 10mM Tris-HCl, pH 9.0; 0.1%Triton X-100).PCR carries out comprising the following steps: last 2 minutes under 94 ℃; Connect and under 94 ℃, last 1 minute,, under 61 ℃, last 1 minute, in order to binding, and last 2 minutes in 72 ℃ in order to sex change, in order to prolong, go through 30 circulations after, finally with the reaction that under 72 ℃, lasts 5 minutes as end.The PCR product is then analyzed with electrophoresis by 2% agarose coagulation.
8. be inserted with the analysis of the endogenous sequence of T-DNA
Primer to Papa31/Papa57 (at 16-0-1 and 17-0-5 the two) and Papa32/Papa52 (at 18-2-4), its its be designed to transgenic strain in the side genome sequence of T-DNA match, and be used to analyze the endogenous sequence that T-DNA inserts the non-transgenic pawpaw at place.16-0-1,17-0-5,18-2-4, TN2,16-0-1 homozygote and the homozygous sample gene group of 18-2-4 DNA are analyzed.PCR carries out in the reaction mixture of one 30 μ L, this reaction mixture comprise the aforementioned suitable primer of 0.75unit FastStartPolymerase (Roche Diagnostics GmbH), 200 μ M dNTP and 0.2 μ M to and the genomic dna of each sample of 128ng, its be assigned in the PCR damping fluid (50mM Tris-HCl, pH 9.0; 10mM KCl; 5mM (NH
4)
2SO
42mM MgCl
2, PH8.3/25 ℃).PCR carries out comprising the following steps: last 2 minutes under 94 ℃; Connect and last 1 minute, bind down in 55 ℃ and last 1 minute, and last 2 minutes, go through 30 circulations in 72 ℃ of prolongations in 94 ℃ of following sex change, finally with the prolongation reaction that under 72 ℃, lasts 10 minutes as end.The PCR product then by aforesaid 2% agarose coagulation, choosing is grown and sequencing is measured.Dna sequence analysis is to utilize BLAST formula and ORF Finder (network address: http://www.ncbi.nlm.nih.gov/) carry out.
9.Southern trace (southern blotting technique) analytical method
Analyze to 16-0-1,18-2-4, TN2,16-0-1 homozygote, 18-2-4 homozygote and with (outcorssed) 16-0-1 sample DNA of the outcross of a unidentified PRSVCP transgenic strain.Get 50 μ g by aforementioned transgenosis, non-transgenic pawpaw purifying and genomic dna react under 37 ℃ with 5 unit EcoRI (Promega) and spend the night.On one 0.8% agarose coagulation, carry out centrifugation, DNA be transferred to nylon film (nylon membrane) (AMERESCO Inc., Solon, Ohio, USA).Be to use DIGLuminescent Detection Kit (Roche Diagnositcs GmbH) prepared corresponding to the come into leaves probe (digoxigenin-labeled probe) of foxglove aglycon mark of one of PRSV CP gene by PCR.After being that 60 ℃ of following hybridizations (hybridization) spend the night, make film in lavation buffer solution (0.5XSSC, 0.1%SDS), washing under 65 ℃ with critical conditions.Probe-positivity band (probe-positive band) is that [(the 4-methoxyl group is spirally connected { 1 to 3-by making dephosphorylized chemoluminescence be subjected to matter C SPD (dephosphorylated chemiluminescence substrateCSPD), 2-diepoxy propane-3,2 '-(5 '-chlorine) ginseng ring [3.3.1.13,7] decane }-the 4-yl) phosphenylic acid) disodium (Disodium 3-(4-methoxyspiro{1,2-dioxetane-3,2 '-(5 '-chloro) tricyclo[3.3.1.13,7] phenyl phosphate decan}-4-yl))] the emission light of (Roche Diagnostics GmbH) is exposed to X-ray egative film (BioMax Light Film, Kodak, Paris France) shows.
10. utilize instant polymerase chain reaction (real time polymerase chain reaction, real time PCR) to detect the number of copies of the T-DNA that inserts
The T of 16-0-1 and 18-2-4 strain
0In the genetically modified number of copies of insertion and the homozygous filial generation of their correspondences pass through ABI
7000 Sequence Detection systems (Applied Biosystems) further analyze with the quantitatively instant polymerase chain reaction (relative quantitativereal-time PCR) of the relativity of transgenosis (PRSV CP gene), wherein use endogenous papain (papain) gene to organize (control) in contrast.The transgenosis Auele Specific Primer to S9-2/S10-2 and papain gene-specific primer to S5/S6; And the transgenosis specific probe (TaqManfluorescent dye-labeled transgene-specific probes for real-time PCR) that is used for TaqMan fluorescein stain-mark of instant PCR, Fam (transgenosis is special) and Vic (papain is special) are shown in the table 1.Following reagent is used in the amplified reaction, and utilizes sterilized water to adjust final volume to 25 μ L: each primer of 5 μ l DNA (50ng for each sample.), 0.6 μ M is to, 12.5 μ L 2X Master Mix (Applied Biosystems) and 250nM papain or transgenosis specific probes.Thermal cycle conditions is to originate in to last under 2 minutes and 95 ℃ under 50 ℃ to last 10 minutes, succeeded by 40 in lasting the circulation that lasts 1 minute under 15 seconds and 60 ℃ under 95 ℃.PCR carries out on the transparent light reaction dish in one 96 holes.Each sample repeats to test with three and utilizes SDS Software 1.1 (Applied Biosystems) and Mocrosoft Excel to analyze.
In the process of logarithm-linear stage (log-linear phase), amplified reaction is with N=N
0(1+E)
nDescribe, wherein N is the number of the molecule of amplification, and N
0Be the initial number of molecule, E is amplification efficiency (amplificationefficiency), and n is the round-robin number.If if amplification efficiency is similar in the two reaction, then the starting point concentration of sample is based on above-mentioned formula, utilize comparative Ct difference approach (comparative delta Ctmethod) (Livak and Schmittgen, 2001) to calculate, and the gene copy number is to utilize formula 2
-(Δ Δ Ct)And get Δ Δ Ct=(Ct wherein
PRSVSample-Ct
PapainSample)-(Ct PRSV
Calibrator-Ct papain
Calibrator), it is to be defined as fluorescence intensity to be promoted to this point on the baseline (baseline).
Present embodiment system utilizes the method described in " general materials and methods " to carry out the detection of Class1, type 2 and type 3.
For the detection of Class1, the CaMV 35S promoter of insertion (shown in Fig. 2 (a) district), nptII (shown in Fig. 2 (b) district) and no terminator are measured in order to the existence of incorporating at possible T-DNA.Use primer that 35S-F/35S-R is amplified one 836 base pairs (base pair, product bp) (seeing also shown in Fig. 2 (a) district) with rotation gene strain.DNA cloning (DNA amplicon) is not found in any unconverted strain.Similarly situation uses primer npt-1/npt-2 to be amplified the product (seeing also shown in Fig. 2 (b) district) of one 829 base pairs with rotation gene strain.Use no promotor Auele Specific Primer nos-1/nos-2 to be amplified the product (seeing also shown in Fig. 2 (c) district) of one 126 base pairs with rotation gene strain.
For the detection of type 2, PRSV-F/PRSV-R is used the product (seeing also shown in Fig. 2 (d) district) that amplifies one 840 base pairs with rotation gene strain according to the primer of the CP gene design of PSRV.
For the detection of type 3, according to the designed primer of CaMV 35S promoter and PSRV CP gene coded sequence to being used the product that amplifies one 700 base pairs with rotation gene strain.
Class1,2 and 3 the specificity product that detection amplified are carried out sequencing to verify its accuracy with respect to the sequence of target area.For the susceptibility (sensitivity) of analyzing these detections, in varing proportions source array from unconverted and transgenosis pawpaw the DNA extract.The result shows, when each transgenic strain was higher than 1% standard (level) when being 99: 1 (that is unconverted and transgenosis person's ratio), specific fragment can be detected.
The zone and the parsing T-DNA flanking sequence of the flanking sequence of T-DNA contained in embodiment 2 amplifications
Present embodiment system utilizes the method amplification described in " general materials and methods " to contain the zone of the flanking sequence of T-DNA, and further the amplified production of gained and the flanking sequence of sequencing analysis T-DNA are wherein grown in choosing.
The genome sequence of the side of the T-DNA inset of transgenosis pawpaw strain is amplified by AL-PCR.In nest-type PRC, use is for T-DNA and specific primer Papa27 of joint tool and AP2, the DNA cloning through SspI-digestion from 16-0-1 or 17-0-5 strain goes out the product of one 464 base pairs respectively, and amplifies the product of one 543 base pairs from the 18-2-4 strain.In nest-type PRC, use for T-DNA and specific primer N1 of joint tool and AP2, go out the product of one 771 base pairs respectively from the DNA cloning through DraI-digestion of 16-0-1 or 17-0-5 strain.In nest-type PRC, use primer Papa52 and AP2, go out the product of one 724 base pairs from the DNA cloning through EcoRV-digestion of 18-2-4 strain.
The product of the amplification of aforementioned nest-type PRC is grown and sequencing through choosing, found that, the DNA of all analyses all contain the T-DNA primer in one end and joint in its other end, this shows that the nest-type PRC product contained the genome sequence of T-DNA inset side.
Sequence with regard to the RB side of T-DNA, the DNA of the amplification of strain 16-0-1 and 17-0-5 has the T-DNA sequence (+26～+ 117) of one 92 base pairs, and shortage RB sequence (+1～+ 25), yet the sequence (shown in Fig. 3 (a) district) with Nuo Palin synthase promoter of part, and pawpaw genome sequence with SspI cleavage site (AAT) of 337 base pairs (SEQ ID NO:29) and one 48 base pair joint sequences (shown in Fig. 3 (a) district); And the RB of rotation gene strain 18-2-4DNA zone amplification and product constituted by following sequence: the genome sequence of one 422 base pairs extends the joint sequence of a SspI identification site (AAT) (SEQ ID NO:30) and one 48 base pairs; And the Nuo Palin synthase promoter sequence (+31～+ 117) of the part of one 86 base pairs, wherein do not comprise whole RB sequence (+1～+ 30) (shown in Fig. 3 (b) district).
With regard to the sequence of the LB side of T-DNA, the DNA of the amplification of strain 16-0-1 and 17-0-5 is made of following sequence: the T-DNA sequence (+4952～+ 5449) that has an incomplete T-DNA left margin sequence (16 base pair) of one 498 base pairs; And the genome sequence with DraI identification site (TTT) (SEQ IDNO:31) and 48 base pair joint sequences of one 237 base pairs (shown in Fig. 4 (a) district); And the LB of rotation gene strain 18-2-4DNA zone amplification and product constituted by following sequence: the genome sequence of one 599 base pairs has the joint sequence of an EcoRV identification site (GAT) (SEQ ID NO:32) and one 48 base pairs; And one 90 base pair and lack the T-DNA sequence (+4584～+ 4673) (shown in Fig. 3 (b) district) of LB sequence.
Present embodiment system utilizes the method described in " general materials and methods " to use Papa31 (shown in Fig. 3 (a) district) that the T-DNARB flanking sequence designing institute according to 16-0-1 and 18-2-4 gets and Papa32 (shown in Fig. 3 (b) district) primer to identify 16-0-1 and 18-2-4 transgenosis pawpaw strain.Result such as Fig. 5 (a) distinguish demonstration, utilize primer can obtain to comply with the specificity product of 241 base pairs from transgenic strain 16-0-1 and 17-0-5 to Papa31/Papa27, and can't obtain spawn from strain 18-2-4.For strain 18-2-4, can utilize primer that Papa32/Papa27 is obtained the specificity product of one 384 base pairs, yet can't obtain any other product (shown in Fig. 5 (b) district) from 16-0-1 and 17-0-5 amplification.
With regard to the LB flanking sequence, primer is designed to detect transgenosis pawpaw strain to Papa56/Papa57 (shown in Fig. 4 (a) district) and Papa58/Papa59 (shown in Fig. 4 (b) district).The result utilizes Papa56/Papa57 can amplify the specificity product of one 106 base pairs from 16-0-1 and 17-0-5 strain shown in Fig. 5 (c), and the 18-2-4 strain then denys.Relative, utilize primer can amplify the specificity product of one 140 base pairs from the 18-2-4 strain to Papa58/Papa59,16-0-1 and 17-0-5 strain then deny.
When primer is used to the PCR detection to Papa31/Papa57, can amplify the fragment (shown in Fig. 5 (e) district) of 227 base pairs of an identical sequence from strain 16-0-1,17-0-5,18-2-4, the filial generation of 18-2-4 homozygote and unconverted TN2.When primer is used in the PCR detection to Papa32/Papa59, can amplify the fragment of 345 base pairs of an identical sequence from strain 16-0-1,17-0-5,18-2-4, the filial generation of 16-0-1 homozygote and unconverted TN2, wherein the filial generation of 18-2-4 homozygote can't detect (shown in Fig. 5 (f) district).This two PCR product and then by sequencing and use the NCBI database to analyze, however find no remarkable homology (homology).Use primer that Papa31/Papa57 is increased and the PCR product with ORF Finder search do not comprise any open reading frame (open reading frame, ORF).The PCR product that uses primer that Papa32/Papa59 is increased and get is found has one 48 amino acid whose reading frames, and its BLASTP analyzes and shows in protein database without any similar sequences.
Because limiting enzyme EcoRI only produces a cutting in T-DNA, the result as shown in Figure 6, the T of 16-0-1 and 18-2-4 strain
0(kilo base pair, kb) and the single signal of 4kb, this is presented at only has the T-DNA of duplicate inset to exist in these strains to produce 10,000 base pairs respectively.The homozygote filial generation of transgenosis pawpaw and the heterozygote T of 16-0-1 and 18-2-4 strain
0The trace signal of strain (being labeled as 16-0-1homo, 18-2-4homo, 16-0-1 and 18-2-4 respectively) lays respectively at same position, and has stronger intensity.In addition, referring to shown in Figure 6, in the filial generation (being labeled as 16-0-1 outcross) that stems from 16-0-1 and the unidentified transgenic strain breeding gained that has an identical PRSV CP construct (construct), two on position have been found, wherein a fragment has the molecular size identical with the 16-0-1 strain, and another 6kb fragment obviously is derived from other transgenosis pawpaw strains.
Embodiment 5 utilizes instant polymerase chain reaction to detect the number of copies of resolving the T-DNA that inserts
The number of copies of the T-DNA that the strain that method described in the present embodiment system utilization " general materials and methods " is planted pawpaw by instant polymerase chain reaction detection commentaries on classics is contained, wherein by using the 16-0-1 strain as correction reference, utilize instant polymerase chain reaction relatively in the homozygous filial generation of the homozygote 18-2-4 of transgenosis pawpaw strain 18-2-4,16-0-1 the number of copies of T-DNA inset to analyze the connectivity (zygosity) of transgenosis pawpaw.Heterozygote T
0The multiple of plant materials changes (fold change) and is predicted to be 1, and homozygote T
1The multiple variation of plant materials is predicted to be 2.Amplification figure as shown in Figure 7.For 16-0-1 homozygote T
1Plant materials (Fig. 7 (b) district) and 18-2-4 homozygote T
1Plant materials (Fig. 7 (d) district) is similar with papain Gene Handling group.When for 16-0-1 heterozygote T
0Plant materials (Fig. 7 (a) district) and and 18-2-4T
0When plant materials is compared with PRSV CP amplification figure, find the phenomenon that one day, left avertence was moved, this shows that the number of copies of transgenosis in each group increases.The numerical value of the number of copies of the representative T-DNA inset of instant PCR-calculation is organized in table 2.Use 16-0-1 as correction reference, the relative number of copies of transgenic strain 18-2-4 is through being evaluated as 0.85, and the filial generation of 16-0-1 homozygote is 2.04, and the filial generation of 18-2-4 homozygote then is 2.12.
The number of copies analysis of table 2 transgenosis pawpaw strain
| Transgenosis pawpaw strain | ΔCt(Ct PRSV-Ct papain)±SD a | With 16-0-1 is the number of copies (2 of calibration standard -ΔΔCt) |
| 16-0-1 | 2.13±0.03 | 1.00 |
| 18-2-4 | 2.37±0.09 | 0.85 |
| The 16-0-1 homozygote | 1.11±0.04 | 2.04 |
| The 18-2-4 homozygote | 1.05±0.06 | 2.12 |
a: the mean value of three observed values
In the previous embodiment, the applicant uses PCR-to have the transgenosis pawpaw strain of extensive resistance as the technology for detection on basis for the PRSV virus strain of different geographic origin, comprising the detection method that is classified as follows according to its specific degree: the Class1 that is used to screen, be used for type 2 that gene specific detects, be used for the type 3 of construct specific detection and be used for the type 4 that event-specific detects.Because the detection method of these four kinds of degree is identical for the PCR detected result of 16-0-1 and 17-0-5, so this two strain is considered to be actually by same strain and is produced.Relative, 16-0-1 and 18-2-4 strain then be by the transformation event of two uniquenesses (transformation events) derived and independently transgenic strain, although they are derived by the species that identical T-DNA construct is transformed, and the similar degree of extensive resistance (Bau et al., 2003 have to(for) different PRSV virus strain; Bau et al., 2004).
Constituted the terminator and a selection markers (selection marker) of the termination signal that shows as regulatory gene comprising at least one target gene, a promotor as the transcription initiation signal, for being used for the plant transformed carrier by multiple unit (element).Because the transgenosis that major part is brought in the Plant Genome comprises CaMV 35S promoter, no terminator and npt II selection markers gene, these compositions can utilize Auele Specific Primer to detect in the preliminary screening analysis of transgenic plant.However, there are the promotor of more and more kinds and selection markers to be used in the middle of the Plant Transformation.According to the present invention, (Holst-Jensen et al. 2003m) is used in and detects transgenosis pawpaw strain in order to the Auele Specific Primer that detects CaMV 35S promoter, no terminator and npt II mark.Found that it is reliable that the use PCR product that these primers amplified is used to detect possible transgenosis pawpaw plant materials.Yet the preliminary screening method can not be used individually to identify specific GM crop, because these screening targets must not show that the foreign DNA inset exists, because the no terminator that stems from the 35S promoter of CaMV or stem from Ti-plastid (Ti-plasmid) is (Wolf et al., 2000) that are present in the middle of the nature.Moreover, it is generally acknowledged that the soil bacteria of the npt II selection markers that comprises one or more transposon (transposon) that belongs to the nature existence is present in (Lo et al., 2007) in the soil.
About the detection of type 2, what target gene can be for natural origin, produce but have a little modification usually, for example block or encode and use variation (codon usage alternation) (Hemmer, 1997).Moreover, the selectivity of available gene can more available promotor or the selectivity of terminator have more specificity.Therefore, have more specificity at the PCR method of target gene than the detection of Class1.The result of type 2 is used for generally confirming that specific transgenic strain comprises specific transgenosis, so the breeder can use this class detection method usually for specific function.
About the detection of type 3, positivity signal (positive signal) only has only when GM-deutero-material exists and can occur, and than type 2, can identify the GM source of DNA more specifically.Among the present invention, the applicant uses the primer based on PRSV CP gene to have the PCR product (840 base pair) that amplifies a uniqueness in the identical genetically modified transgenosis pawpaw of the PRSV CP strain certainly.Use primer to amplify another unique PCR product (700 base pair) from transgenosis pawpaw strain based on zone (35S promoter+PRSV CP) again.It is any derived from the transgenosis pawpaw strain that transforms by PRSV CP construct that molecule marker in the type 2 and 3 can be used in evaluation.Simultaneously, these marks can be as the useful tool of identifying genetically modified existence in the specific filial generation in molecular breeding (molecular bleeding) process.
About the detection of type 4, type 4 be between the DNA of pawpaw genome and insertion and the connecting zone (junction) of implantation site (integration locus) detect.This is the unique feature place of transformation event (Zheng et al, 2001; Holst-Jensen et al., 2003).Together with sequence data as can be known, AL-PCR result produces evidence to show that the T-DNA construct incorporates into really to the pawpaw genome.Secondly, it can measure the number and the integrity thereof of T-DNA inset.Also may measure simultaneously the nucleotide sequence that T-DNA inserts the genome position at place.In order to produce the various fragments pond (pools of fragements) that can engage, therefore select for use in order to produce the different restriction enzymes of blunt end cutting with joint.Therefore, can avoid missing the sequence of insertion by the various prepared products that different restriction enzyme produced.Moreover the T-DNA Auele Specific Primer is designed to as close as possible border sequence, to obtain suitable flanking sequence.On the other hand, joint be designed to have one with the staggered end of genomic dna complementary (staggered end) through restriction enzyme digestion.This can allow joint and through single-minded the engaging of genomic dna of restriction enzyme digestion, and can not engage with the dna fragmentation that ruptures in the DNA prepared product.Because the short chain joint not through phosphorylation, can avoid it to be engaged to any restricted fragment (restriction fragment), and guarantee in the heating steps first of PCR, can therefore not lose.
The event-specific PCR method be to use from the flanking sequence of T-DNA sequence designed and primer to analyze transgenosis pawpaw strain, it can be used the characterization of molecules analysis as intelligence power protection of indivedual transgenic strains.Because the whole filial generations derived from transgenic strain 16-0-1 and 18-2-4 comprise the specific T-DNA inset with identical flanking sequence, the method steps that type 4 detects can be used for producing and be used for identifying the pollen contributor of pollen flow experiment (pollen flow experiments) or be used for the molecule marker that breeding process is identified the transgenosis connectivity of indivedual filial generations.Simultaneously, these molecule markers derived from specific flanking sequence can be used to monitor specific gene transformation biology in production or sales process.The U.S. and Canada use product relevant method (product-related approach) and spontaneous mark (voluntarylabeling) for their commercialization GM crop, take manufacturing processed (technology) manufacture method methods involving (process-related approach) and legal mark (mandatory labeling) (Carter and Gruere, 2006) yet European Union member countries are more careful.Therefore, for example Taiwan and Japan have been necessary to these event-specific marks for European Union member countries and some East Asian countries.
If exist with the heterozygote state as if transgenosis, for example T
0In the example of plant materials, utilization by both sides lateral order row designed and the genome sequence of primer amplification transgenosis pawpaw should amplify two kinds of different fragments, one bigger product, it comprises the whole T-DNA that has both sides lateral order row part, and a less product, it only comprises the endogenous sequence.Employed transgenosis pawpaw product lie between the lateral order row of both sides and have a 5.4kb T-DNA construct (Cheng et al, 1996) in the previous embodiment.Obvious, the fragment of the T-DNA that is comprised whole big to can't for
The Taq polymeric enzymatic amplification goes out.Yet the position that does not have the correspondence that T-DNA inserts still can produce an amplified production that is similar to the non-transgenic pawpaw.In the homozygote filial generation, bigger amplified production can't be amplified out, because T-DNA is inserted into the chromosomal same position of antithesis, and does not therefore have the less fragment that produces from the endogenous sequence gained that does not insert.According to strain 16-0-1 and 18-2-4 designed and event-specific primer (Papa31/Papa57 and Papa32/Papa59) can distinguish self-corresponding T
0The heterozygote plant materials amplifies the specific product (referring to Fig. 5) of 227 base pairs and 345 base pairs, but can't amplify specific product from their homozygote filial generation.Genetically modified existence can add their confirmation by the detection of Class1 to 3 in the test plants.The event-specific primer then is suitable for planning to identify rapidly homozygote filial generation derived from 16-0-1 or 18-2-4 in breeding.In addition, except the transgenosis of inserting, these marks can be used for any intelligence power protection derived from the species of the tool PRSV-resistance of 16-0-1 and 18-2-4.Because a breeding such as the fruit tree of pawpaw need spend the long time, the application of these molecule markers can be shortened the breeding that the PRSVCP transgenosis is fixed to a homozygote parental generation and plans, and wherein the homozygote parental generation can be used to produce a specific hybrid species to PRSV tool resistance.
The method of modal mensuration duplicate is the Southern engram analysis, wherein through the trace of the plant genome DNA of digestion can with one corresponding to genetically modified dna probe heterozygosis mutually, to produce a useful heterozygosis pattern.The applicant can judge that having two T-DNA in the unknown transgenosis pawpaw inserts the site according to the Southern engram analysis, but the number of the band of homozygote strain does not have corresponding with number of copies (referring to Fig. 6).In actual detection, homozygote should have than the strong signal of heterozygote twice.Because the heterozygosis signal of homozygote pawpaw filial generation far is better than the heterozygote individuality (referring to Fig. 6) that stems from the filial generation of homozygote pawpaw, so the result of Southern engram analysis can be used as the important references of connectivity.
From heterozygote transgenic plant (T
0) in distinguish out s-generation homozygote strain (T
1) be to cultivate the plant lines of inheritance stability and the important step that produces the transgene performance of optimum extent.Tradition T
1Plant materials is to utilize time-consuming T
2The compartment analysis of filial generation (segregation analysis) filters out at connectivity, and it must make T
1Plant lines growth and maturity and the seed of collecting them are in the enterprising row filter of selective medium.Use the quantitatively instant PCR can relative quantification gene copy number, therefore can be used in and measure connectivity (Bubner and Baldwin, 2004; Ji et al., 2005; Prior et al., 2006; Shitara et al., 2004; Tesson et al., 2002).According to the present invention, homozygote and heterozygote transgenosis pawpaw strain can be distinguished its connectivity by instant PCR method.Therefore, for the instant PCR of the single-minded primer of transgenosis and use, can be used as a kind of quick and reliable method in order to the connectivity of identifying transgenic plant according to being used in combination of the PCR detection method of the designed event-specific primer that goes out of flanking sequence.Combine speech, its two as the rapid screening instrument in the breeding process,, be useful especially for for the long-term fruit crop of pawpaw.
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Sequence table
＜110〉the intelligent Bao Huijun model of Ye Xidong Zheng Ying ancestor great mansion Gong Yi Rong states Su Tiancai
＜120〉nucleic acid molecule of transgenosis pawpaw strain 16-0-1 and detection method and application
<130>
<160>34
<170>PatentIn?version?3.4
<210>1
<211>21
<212>DNA
＜213〉artificial sequence
<220>
＜223〉35S-F is at the primer of 35S promoter
<400>1
cagctatgac?catgattacg?c 21
<210>2
<211>18
<212>DNA
＜213〉artificial sequence
<220>
＜223〉35S-R is at the primer of 35S promoter
<400>2
tcttgcgaag?gatagtgg 18
<210>3
<211>18
<212>DNA
＜213〉artificial sequence
<220>
＜223〉nos-1 is at the primer of no terminator
<400>3
tgccggtctt?gcgatgat 18
<210>4
<211>22
<212>DNA
＜213〉artificial sequence
<220>
＜223〉nos-2 is at the primer of no terminator
<400>4
atgtataatt?gcgggactct?aa 22
<210>5
<211>20
<212>DNA
＜213〉artificial sequence
<220>
＜223〉npt-1 is at the primer of nptII
<400>5
ataatctgca?ccggatctgg 20
<210>6
<211>20
<212>DNA
＜213〉artificial sequence
<220>
＜223〉npt-2 is at the primer of nptII
<400>6
ccgctcagaa?gaactcgtca 20
<210>7
<211>20
<212>DNA
＜213〉artificial sequence
<220>
＜223〉PRSV-F is at genetically modified primer
<400>7
tccaagaatg?aagctgtgga 20
<210>8
<211>20
<212>DNA
＜213〉artificial sequence
<220>
＜223〉PRSV-R is at genetically modified primer
<400>8
gtgcatgtct?ctgttgacat 20
<210>9
<211>22
<212>DNA
＜213〉artificial sequence
<220>
＜223〉Ap1 is at the primer of joint
<400>9
gtaatacgac?tcactatagg?gc 22
<210>10
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉Ap2 is at the primer of joint
<400>10
actatagggc?acgcgtggt 19
<210>11
<211>23
<212>DNA
＜213〉artificial sequence
<220>
＜223〉S18 is at the primer of no promotor
<400>11
acgcgcaata?atggtttctg?acg 23
<210>12
<211>22
<212>DNA
＜213〉artificial sequence
<220>
＜223〉Papa27 is at the primer of no promotor
<400>12
gcgtcatcgg?cgggggtcat?aa 22
<210>13
<211>20
<212>DNA
＜213〉artificial sequence
<220>
＜223〉P1 is at genetically modified primer
<400>13
caaacactcg?cgccactcaa 20
<210>14
<211>24
<212>DNA
＜213〉artificial sequence
<220>
＜223〉Papa52 is at the primer of no terminator
<400>14
tgttgccggt?cttgcgatga?ttat 24
<210>15
<211>21
<212>DNA
＜213〉artificial sequence
<220>
＜223〉Papa34 is at the primer of no terminator
<400>15
caacgtcgtg?actgggaaaa?c 21
<210>16
<211>20
<212>DNA
＜213〉artificial sequence
<220>
＜223〉N1 is at the primer of no terminator
<400>16
gcccgctcct?ttcgctttct 20
<210>17
<211>21
<212>DNA
＜213〉artificial sequence
<220>
＜223〉Papa31 is at the primer of RB side
<400>17
ttgttctaat?aaggttgcta?c 21
<210>18
<211>23
<212>DNA
＜213〉artificial sequence
<220>
＜223〉Papa32 is at the primer of RB side
<400>18
aatatcaaat?ggacgtgtta?gtg 23
<210>19
<211>24
<212>DNA
＜213〉artificial sequence
<220>
＜223〉Papa56 is at the primer of left margin (Left Border)
<400>19
gttattaagt?tgtctaagcg?tcaa 24
<210>20
<211>27
<212>DNA
＜213〉artificial sequence
<220>
＜223〉Papa57 is at the primer of LB side
<400>20
agacatatat?catcaagacc?atagtag 27
<210>21
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉Papa58 is at the primer of left margin (Left Border)
<400>21
gtcttgcgat?gattatcat 19
<210>22
<211>26
<212>DNA
＜213〉artificial sequence
<220>
＜223〉Papa59 is at the primer of LB side
<400>22
tggttatcaa?tatagcaatt?atgtag 26
<210>23
<211>20
<212>DNA
＜213〉artificial sequence
<220>
＜223〉S9-2 is at the primer of PRSV CP gene, for being used for quantitative PCR
<400>23
agtaacgcgg?cagaggcata 20
<210>24
<211>22
<212>DNA
＜213〉artificial sequence
<220>
＜223〉S10-2 is at the primer of PRSV CP gene, for being used for quantitative PCR
<400>24
gagccctatc?aggtgttttc?ga 22
<210>25
<211>23
<212>DNA
＜213〉artificial sequence
<220>
＜223〉S5 is at the primer of papain gene, for being used for quantitative PCR
<400>25
tgggtttgtc?atttggtgat?ttt 23
<210>26
<211>26
<212>DNA
＜213〉artificial sequence
<220>
＜223〉S6 is at the primer of papain gene, for being used for quantitative PCR
<400>26
gtctttcagt?ggatgtcaag?tcattt 26
<210>27
<211>21
<212>DNA
＜213〉artificial sequence
<220>
＜223〉Fam, PRSV CP gene probe
<400>27
ttagtctcgc?tagatatgct?t 21
<210>28
<211>18
<212>DNA
＜213〉artificial sequence
<220>
＜223〉Vic, the papain gene probe
<400>28
ctattgtggg?ttattctc 18
<210>29
<211>337
<212>DNA
＜213〉pawpaw (carica papaya L.)
<400>29
tatttttaac?ataatttaat?aactgtaatc?ttattatata?ctagtaattt?aaattctcaa 60
ctccattaaa?ttcaacatta?ttcaaactta?aataaaatat?aaaattttaa?ctattagatt 120
ataatgttat?taattaccat?taaagagatt?agtagatatt?cttctatttt?ttgttatttg 180
attatcaatt?gttctaataa?ggttgctaca?cttagagaac?atctggtggt?atcaccccag 240
tcttagtttg?atagtttatt?attctgattt?tgtttgttaa?aatttctcat?tttctttgga 300
tatctgccta?ttttgaaatt?tggatactat?ctcattc 337
<210>30
<211>422
<212>DNA
＜213〉pawpaw (Carica papaya L.)
<400>30
tattgataaa?attagagtaa?tactaataaa?tgctacaaaa?ataaaagtga?caataatagt 60
aataataatg?ataaaaataa?taatataatt?aacatgacag?aataaataat?agtaacaata 120
attaataata?ataagaataa?cagtaagtat?aagttacaat?aatagagtga?gacgatagta 180
gaatggttat?aacattaata?atatcaaatg?gacgtgttag?tgatggtagc?attacaatgg 240
tattaacgtc?attattgttg?acaacaataa?tatagtaatt?atggaagtga?tgagatagag 300
gtcataagaa?ttgtaaaata?ggagacgacg?acaaaaatag?tagtagtgat?gaagaaaata 360
acaaaataac?aattatgaga?acaaagtgat?gataacatca?ataacaaatt?aataaataat 420
aa 422
<210>31
<211>237
<212>DNA
＜213〉pawpaw (Carica papaya L.)
<400>31
gctctctaaa?ttaatttatt?ttaatatttt?gtttttctac?tatggtcttg?atgatatatg 60
tctttctgca?tttaataatt?taagtagttt?ttaactaaac?agtttgggat?aaaaacgata 120
tagtttttat?atatatatat?atatatatat?atatataata?tgccatagaa?ttgctgttaa 180
tataatatca?tctattatgt?ttaattaatt?atgttctaaa?attaataata?aatttta 237
<210>32
<211>599
<212>DNA
＜213〉pawpaw (Carica papaya L.)
<400>32
aatatttttt?aaaaaaatta?ttagaatgac?aactacataa?ttgctatatt?gataaccact 60
tatttaaatt?gatgatattt?aatacagtcg?ttaatgatat?tagtatttat?taacatatat 120
aaaaaaaatt?taaatatttt?ttgaaagtaa?aaggataaat?ttgataaaat?gtcaaaataa 180
gagttaatta?aattaaaaat?aattagtata?ggagttaaaa?atggattttt?agtcttttaa 240
aaaataaaaa?aagaaaacaa?atattctttt?accgccaatg?ctaaaataat?taaataaaat 300
ccaagggtat?aagttgcggg?aagaaagcga?cctaggacta?tattttgcgg?aactacagtt 360
ggcacctacc?aattgaacaa?gtaatcgtga?aaaatgatag?aaacagaaca?ggcttgcagc 420
agcctggcga?tgccggcgcc?gactttatcg?ttactggcgg?aggattgcat?atcccttgtg 480
ctttcgctca?catcgcctca?ggacgcatgt?cgggtgtctt?tggtttagtc?ccgagtttcg 540
atccgctgcg?aaatcagacg?tggtctggaa?tcgatttttg?ccgtccgaat?acgaggata 599
<210>33
<211>5449
<212>DNA
<213>T-DNA
<400>33
aacactgata?gtttaaactg?aaggcgggaa?acgacaatct?gatcatgagc?ggagaattaa 60
gggagtcacg?ttatgacccc?cgccgatgac?gcgggacaag?ccgttttacg?tttggaactg 120
acagaaccgc?aacgttgaag?gagccactca?gccgcgggtt?tctggagttt?aatgagctaa 180
gcacatacgt?cagaaaccat?tattgcgcgt?tcaaaagtcg?cctaaggtca?ctatcagcta 240
gcaaatattt?cttgtcaaaa?atgctccact?gacgttccat?aaattcccct?cggtatccaa 300
ttagagtctc?atattcactc?tcaatccaaa?taatctgcac?cggatctgga?tcgtttcgca 360
tgattgaaca?agatggattg?cacgcaggtt?ctccggccgc?ttgggtggag?aggctattcg 420
gctatgactg?ggcacaacag?acaatcggct?gctctgatgc?cgccgtgttc?cggctgtcag 480
cgcaggggcg?cccggttctt?tttgtcaaga?ccgacctgtc?cggtgccctg?aatgaactgc 540
aggacgaggc?agcgcggcta?tcgtggctgg?ccacgacggg?cgttccttgc?gcagctgtgc 600
tcgacgttgt?cactgaagcg?ggaagggact?ggctgctatt?gggcgaagtg?ccggggcagg 660
atctcctgtc?atctcacctt?gctcctgccg?agaaagtatc?catcatggct?gatgcaatgc 720
ggcggctgca?tacgcttgat?ccggctacct?gcccattcga?ccaccaagcg?aaacatcgca 780
tcgagcgagc?acgtactcgg?atggaagccg?gtcttgtcga?tcaggatgat?ctggacgaag 840
agcatcaggg?gctcgcgcca?gccgaactgt?tcgccaggct?caaggcgcgc?atgcccgacg 900
gcgatgatct?cgtcgtgacc?catggcgatg?cctgcttgcc?gaatatcatg?gtggaaaatg 960
gccgcttttc?tggattcatc?gactgtggcc?ggctgggtgt?ggcggaccgc?tatcaggaca 1020
tagcgttggc?tacccgtgat?attgctgaag?agcttggcgg?cgaatgggct?gaccgcttcc 1080
tcgtgcttta?cggtatcgcc?gctcccgatt?cgcagcgcat?cgccttctat?cgccttcttg 1140
acgagttctt?ctgagcggga?ctctggggtt?cgaaatgacc?gaccaagcga?cgcccaacct 1200
gccatcacga?gatttcgatt?ccaccgccgc?cttctatgaa?aggttgggct?tcggaatcgt 1260
tttccgggac?gccggctgga?tgatcctcca?gcgcggggat?ctcatgctgg?agttcttcgc 1320
ccacgggatc?tctgcggaac?aggcggtcga?aggtgccgat?atcattacga?cagcaacggc 1380
cgacaagcac?aacgccacga?tcctgagcga?caatatgatc?gggcccggcg?tccacatcaa 1440
cggcgtcggc?ggcgactgcc?caggcaagac?cgagatgcac?cgcgatatct?tgctgcgttc 1500
ggatattttc?gtggagttcc?cgccacagac?ccggatgatc?cccgatcgtt?caaacatttg 1560
gcaataaagt?ttcttaagat?tgaatcctgt?tgccggtctt?gcgatgatta?tcatataatt 1620
tctgttgaat?tacgttaagc?atgtaataat?taacatgtaa?tgcatgacgt?tatttatgag 1680
atgggttttt?atgattagag?tcccgcaatt?atacatttaa?tacgcgatag?aaaacaaaat 1740
atagcgcgca?aactaggata?aattatcgcg?cgcggtgtca?tctatgttac?tagatcgggc 1800
ctcctgtcaa?tgctggcggc?ggctctggtg?gtggttctgg?tggcggctct?gagggtggtg 1860
gctctgaggg?tggcggttct?gagggtggcg?gctctgaggg?aggcggttcc?ggtggtggct 1920
ctggttccgg?tgattttgat?tatgaaaaga?tggcaaacgc?taataagggg?gctatgaccg 1980
aaaatgccga?tgaaaacgcg?ctacagtctg?acgctaaagg?caaacttgat?tctgtcgcta 2040
ctgattacgg?tgctgctatc?gatggtttca?ttggtgacgt?ttccggcctt?gctaatggta 2100
atggtgctac?tggtgatttt?gctggctcta?attcccaaat?ggctcaagtc?ggtgacggtg 2160
ataattcacc?tttaatgaat?aatttccgtc?aatatttacc?ttccctccct?caatcggttg 2220
aatgtcgccc?ttttgtcttt?ggcccaatac?gcaaaccgcc?tctccccgcg?cgttggccga 2280
ttcattaatg?cagctggcac?gacaggtttc?ccgactggaa?agcgggcagt?gagcgcaacg 2340
caattaatgt?gagttagctc?actcattagg?caccccaggc?tttacacttt?atgcttccgg 2400
ctcgtatgtt?gtgtggaatt?gtgagcggat?aacaatttca?cacaggaaac?agctatgacc 2460
atgattacgc?caagcttgca?tgcctgcagg?tccccagatt?agccttttca?atttcagaaa 2520
gaatgctaac?ccacagatgg?ttagagaggc?ttacgcagca?ggtctcatca?agacgatcta 2580
cccgagcaat?aatctccagg?aaatcaaata?ccttcccaag?aaggttaaag?atgcagtcaa 2640
aagattcagg?actaactgca?tcaagaacac?agagaaagat?atatttctca?agatcagaag 2700
tactattcca?gtatggacga?ttcaaggctt?gcttcacaaa?ccaaggcaag?taatagagat 2760
tggagtctct?aaaaaggtag?ttcccactga?atcaaaggcc?atggagtcaa?agattcaaat 2820
agaggaccta?acagaactcg?ccgtaaagac?tggcgaacag?ttcatacaga?gtctcttacg 2880
actcaatgac?aagaagaaaa?tcttcgtcaa?catggtggag?cacgacacac?ttgtctactc 2940
caaaaatatc?aaagatacag?tctcagaaga?ccaaagggca?attgagactt?ttcaacaaag 3000
ggtaatatcc?ggaaacctcc?tcggattcca?ttgcccagct?atctgtcact?ttattgtgaa 3060
gatagtggaa?aaggaaggtg?gctcctacaa?atgccatcat?tgcgataaag?gaaaggccat 3120
cgttgaagat?gcctctgccg?acagtggtcc?caaagatgga?cccccaccca?cgaggagcat 3180
cgtggaaaaa?gaagacgttc?caaccacgtc?ttcaaagcaa?gtggattgat?gtgatatctc 3240
cactgacgta?agggatgacg?cacaatccca?ctatccttcg?caagaccctt?cctctatata 3300
aggaagttca?tttcatttgg?agagaacacg?ggggactcta?gaggatcccc?gggtggtcag 3360
tcccttccat?ggcgtctaaa?aatgaagctg?tggataccgg?tctgaatgag?aagctcaaag 3420
aaaaagaaaa?gcagaaagaa?aaagaaaaag?ataaacaaca?agataaagac?aatgatggag 3480
ctagtgacgg?aaacgatgtg?tcaactagca?caaaaactgg?agagagagat?agggatgtca 3540
atgccggaac?tagtggaacc?ttcactgttc?cgaggataaa?gtcatttact?gataagatga 3600
tcttaccaag?aattaaggga?aaaactgtcc?ttaatttaaa?tcatcttctt?cagtataatc 3660
cgaaacaagt?tgacatctca?aacactcgcg?ccactcaatc?tcaatttgag?aagtggtatg 3720
agggagtgag?aaatgattat?ggccttaatg?ataacgaaat?gcaagtaatg?ttaaatggtt 3780
tgatggtttg?gtgtatcgaa?aatggtacat?ctccagatat?atctggtgtc?tgggttatga 3840
tggatgggga?aacccaagtc?gattatccca?ttaaaccttt?gattgaacac?gcaactcctt 3900
catttaggca?aatcatggct?cacttcagta?acgcggcaga?ggcatacatc?gcgaagagga 3960
atgcaactga?gaagtacatg?ccgcggtatg?gaatcaagag?aaatttgact?gacattagtc 4020
tcgctagata?tgctttcgat?ttctatgagg?tgaattcgaa?aacacctgat?agggctcgtg 4080
aagctcatat?gcagatgaag?gctgcagcgc?tacgcaatac?taatcgcaaa?atgtttggaa 4140
tggacggcag?tgtcagtaac?aaggaagaaa?acacggagag?acacacagtg?gaagatgtca 4200
acagagacat?gcactctctc?ctgggtatgc?gcaattgaat?actcgcgcta?gtgtgtttgt 4260
cgggcctggc?tcgaccctgt?ttcaccttat?aatactatgt?aagcattaga?atatagtgtg 4320
gctgcgccac?cgcttctatt?ttacagtgag?ggtagccctc?cgtgctttta?gtgttattcg 4380
agttctctga?gtctccatac?agtgtgggtg?gcccacgtgc?tattcgagcc?tcttggaatg 4440
agagaaaaaa?aaaaaaaaaa?aaaaaaaaaa?aactcgagga?attcggtacc?ccgggttcga 4500
aatcgataag?cttggatccg?gagagctcga?atttccccga?tcgttcaaac?atttggcaat 4560
aaagtttctt?aagattgaat?cctgttgccg?gtcttgcgat?gattatcata?taatttctgt 4620
tgaattacgt?taagcatgta?ataattaaca?tgtaatgcat?gacgttattt?atgagatggg 4680
tttttatgat?tagagtcccg?caattataca?tttaatacgc?gatagaaaac?aaaatatagc 4740
gcgcaaacta?ggataaatta?tcgcgcgcgg?tgtcatctat?gttactagat?cgggaattca 4800
ctggccgtcg?ttttacaacg?tcgtgactgg?gaaaaccctg?gcgttaccca?acttaatcgc 4860
cttgcagcac?atcccccttt?cgccagctgg?cgtaatagcg?aagaggcccg?caccgatcgc 4920
ccttcccaac?agttgcgcag?cctgaatggc?gcccgctcct?ttcgctttct?tcccttcctt 4980
tctcgccacg?ttcgccggct?ttccccgtca?agctctaaat?cgggggctcc?ctttagggtt 5040
ccgatttagt?gctttacggc?acctcgaccc?caaaaaactt?gatttgggtg?atggttcacg 5100
tagtgggcca?tcgccctgat?agacggtttt?tcgccctttg?acgttggagt?ccacgttctt 5160
taatagtgga?ctcttgttcc?aaactggaac?aacactcaac?cctatctcgg?gctattcttt 5220
tgatttataa?gggattttgc?cgatttcgga?accaccatca?aacaggattt?ttgcctgctg 5280
gggcaaacca?gcgtggaccg?cttgctgcaa?ctctctcagg?gccaggcggt?gaagggcaat 5340
cagctgttgc?ccgtctcact?ggtgaaaaga?aaaaccaccc?cagtacatta?aaaacgtccg 5400
caatgtgtta?ttaagttgtc?taagcgtcaa?tttgtttaca?ccacaatat 5449
<210>34
<211>4666
<212>DNA
<213>T-DNA
<400>34
gatagtttaa?actgaaggcg?ggaaacgaca?atctgatcat?gagcggagaa?ttaagggagt 60
cacgttatga?cccccgccga?tgacgcggga?caagccgttt?tacgtttgga?actgacagaa 120
ccgcaacgtt?gaaggagcca?ctcagccgcg?ggtttctgga?gtttaatgag?ctaagcacat 180
acgtcagaaa?ccattattgc?gcgttcaaaa?gtcgcctaag?gtcactatca?gctagcaaat 240
atttcttgtc?aaaaatgctc?cactgacgtt?ccataaattc?ccctcggtat?ccaattagag 300
tctcatattc?actctcaatc?caaataatct?gcaccggatc?tggatcgttt?cgcatgattg 360
aacaagatgg?attgcacgca?ggttctccgg?ccgcttgggt?ggagaggcta?ttcggctatg 420
actgggcaca?acagacaatc?ggctgctctg?atgccgccgt?gttccggctg?tcagcgcagg 480
ggcgcccggt?tctttttgtc?aagaccgacc?tgtccggtgc?cctgaatgaa?ctgcaggacg 540
aggcagcgcg?gctatcgtgg?ctggccacga?cgggcgttcc?ttgcgcagct?gtgctcgacg 600
ttgtcactga?agcgggaagg?gactggctgc?tattgggcga?agtgccgggg?caggatctcc 660
tgtcatctca?ccttgctcct?gccgagaaag?tatccatcat?ggctgatgca?atgcggcggc 720
tgcatacgct?tgatccggct?acctgcccat?tcgaccacca?agcgaaacat?cgcatcgagc 780
gagcacgtac?tcggatggaa?gccggtcttg?tcgatcagga?tgatctggac?gaagagcatc 840
aggggctcgc?gccagccgaa?ctgttcgcca?ggctcaaggc?gcgcatgccc?gacggcgatg 900
atctcgtcgt?gacccatggc?gatgcctgct?tgccgaatat?catggtggaa?aatggccgct 960
tttctggatt?catcgactgt?ggccggctgg?gtgtggcgga?ccgctatcag?gacatagcgt 1020
tggctacccg?tgatattgct?gaagagcttg?gcggcgaatg?ggctgaccgc?ttcctcgtgc 1080
tttacggtat?cgccgctccc?gattcgcagc?gcatcgcctt?ctatcgcctt?cttgacgagt 1140
tcttctgagc?gggactctgg?ggttcgaaat?gaccgaccaa?gcgacgccca?acctgccatc 1200
acgagatttc?gattccaccg?ccgccttcta?tgaaaggttg?ggcttcggaa?tcgttttccg 1260
ggacgccggc?tggatgatcc?tccagcgcgg?ggatctcatg?ctggagttct?tcgcccacgg 1320
gatctctgcg?gaacaggcgg?tcgaaggtgc?cgatatcatt?acgacagcaa?cggccgacaa 1380
gcacaacgcc?acgatcctga?gcgacaatat?gatcgggccc?ggcgtccaca?tcaacggcgt 1440
cggcggcgac?tgcccaggca?agaccgagat?gcaccgcgat?atcttgctgc?gttcggatat 1500
tttcgtggag?ttcccgccac?agacccggat?gatccccgat?cgttcaaaca?tttggcaata 1560
aagtttctta?agattgaatc?ctgttgccgg?tcttgcgatg?attatcatat?aatttctgtt 1620
gaattacgtt?aagcatgtaa?taattaacat?gtaatgcatg?acgttattta?tgagatgggt 1680
ttttatgatt?agagtcccgc?aattatacat?ttaatacgcg?atagaaaaca?aaatatagcg 1740
cgcaaactag?gataaattat?cgcgcgcggt?gtcatctatg?ttactagatc?gggcctcctg 1800
tcaatgctgg?cggcggctct?ggtggtggtt?ctggtggcgg?ctctgagggt?ggtggctctg 1860
agggtggcgg?ttctgagggt?ggcggctctg?agggaggcgg?ttccggtggt?ggctctggtt 1920
ccggtgattt?tgattatgaa?aagatggcaa?acgctaataa?gggggctatg?accgaaaatg 1980
ccgatgaaaa?cgcgctacag?tctgacgcta?aaggcaaact?tgattctgtc?gctactgatt 2040
acggtgctgc?tatcgatggt?ttcattggtg?acgtttccgg?ccttgctaat?ggtaatggtg 2100
ctactggtga?ttttgctggc?tctaattccc?aaatggctca?agtcggtgac?ggtgataatt 2160
cacctttaat?gaataatttc?cgtcaatatt?taccttccct?ccctcaatcg?gttgaatgtc 2220
gcccttttgt?ctttggccca?atacgcaaac?cgcctctccc?cgcgcgttgg?ccgattcatt 2280
aatgcagctg?gcacgacagg?tttcccgact?ggaaagcggg?cagtgagcgc?aacgcaatta 2340
atgtgagtta?gctcactcat?taggcacccc?aggctttaca?ctttatgctt?ccggctcgta 2400
tgttgtgtgg?aattgtgagc?ggataacaat?ttcacacagg?aaacagctat?gaccatgatt 2460
acgccaagct?tgcatgcctg?caggtcccca?gattagcctt?ttcaatttca?gaaagaatgc 2520
taacccacag?atggttagag?aggcttacgc?agcaggtctc?atcaagacga?tctacccgag 2580
caataatctc?caggaaatca?aataccttcc?caagaaggtt?aaagatgcag?tcaaaagatt 2640
caggactaac?tgcatcaaga?acacagagaa?agatatattt?ctcaagatca?gaagtactat 2700
tccagtatgg?acgattcaag?gcttgcttca?caaaccaagg?caagtaatag?agattggagt 2760
ctctaaaaag?gtagttccca?ctgaatcaaa?ggccatggag?tcaaagattc?aaatagagga 2820
cctaacagaa?ctcgccgtaa?agactggcga?acagttcata?cagagtctct?tacgactcaa 2880
tgacaagaag?aaaatcttcg?tcaacatggt?ggagcacgac?acacttgtct?actccaaaaa 2940
tatcaaagat?acagtctcag?aagaccaaag?ggcaattgag?acttttcaac?aaagggtaat 3000
atccggaaac?ctcctcggat?tccattgccc?agctatctgt?cactttattg?tgaagatagt 3060
ggaaaaggaa?ggtggctcct?acaaatgcca?tcattgcgat?aaaggaaagg?ccatcgttga 3120
agatgcctct?gccgacagtg?gtcccaaaga?tggaccccca?cccacgagga?gcatcgtgga 3180
aaaagaagac?gttccaacca?cgtcttcaaa?gcaagtggat?tgatgtgata?tctccactga 3240
cgtaagggat?gacgcacaat?cccactatcc?ttcgcaagac?ccttcctcta?tataaggaag 3300
ttcatttcat?ttggagagaa?cacgggggac?tctagaggat?ccccgggtgg?tcagtccctt 3360
ccatggcgtc?taaaaatgaa?gctgtggata?ccggtctgaa?tgagaagctc?aaagaaaaag 3420
aaaagcagaa?agaaaaagaa?aaagataaac?aacaagataa?agacaatgat?ggagctagtg 3480
acggaaacga?tgtgtcaact?agcacaaaaa?ctggagagag?agatagggat?gtcaatgccg 3540
gaactagtgg?aaccttcact?gttccgagga?taaagtcatt?tactgataag?atgatcttac 3600
caagaattaa?gggaaaaact?gtccttaatt?taaatcatct?tcttcagtat?aatccgaaac 3660
aagttgacat?ctcaaacact?cgcgccactc?aatctcaatt?tgagaagtgg?tatgagggag 3720
tgagaaatga?ttatggcctt?aatgataacg?aaatgcaagt?aatgttaaat?ggtttgatgg 3780
tttggtgtat?cgaaaatggt?acatctccag?atatatctgg?tgtctgggtt?atgatggatg 3840
gggaaaccca?agtcgattat?cccattaaac?ctttgattga?acacgcaact?ccttcattta 3900
ggcaaatcat?ggctcacttc?agtaacgcgg?cagaggcata?catcgcgaag?aggaatgcaa 3960
ctgagaagta?catgccgcgg?tatggaatca?agagaaattt?gactgacatt?agtctcgcta 4020
gatatgcttt?cgatttctat?gaggtgaatt?cgaaaacacc?tgatagggct?cgtgaagctc 4080
atatgcagat?gaaggctgca?gcgctacgca?atactaatcg?caaaatgttt?ggaatggacg 4140
gcagtgtcag?taacaaggaa?gaaaacacgg?agagacacac?agtggaagat?gtcaacagag 4200
acatgcactc?tctcctgggt?atgcgcaatt?gaatactcgc?gctagtgtgt?ttgtcgggcc 4260
tggctcgacc?ctgtttcacc?ttataatact?atgtaagcat?tagaatatag?tgtggctgcg 4320
ccaccgcttc?tattttacag?tgagggtagc?cctccgtgct?tttagtgtta?ttcgagttct 4380
ctgagtctcc?atacagtgtg?ggtggcccac?gtgctattcg?agcctcttgg?aatgagagaa 4440
aaaaaaaaaa?aaaaaaaaaa?aaaaaactcg?aggaattcgg?taccccgggt?tcgaaatcga 4500
taagcttgga?tccggagagc?tcgaatttcc?ccgatcgttc?aaacatttgg?caataaagtt 4560
tcttaagatt?gaatcctgtt?gccggtcttg?cgatgattat?catataattt?ctgttgaatt 4620
acgttaagca?tgtaataatt?aacatgtaat?gcatgacgtt?atttat 4666
Claims (13)
1. isolating nucleic acid molecule with transgenosis pawpaw strain 16-0-1 of the anti-pawpaw ring spot virus of popularity characteristic, it has: one is positioned at the right margin side areas of 5 ' end, one is positioned at the left margin side areas of 3 ' end, and a transgenic sequence that contains the pawpaw ring spot virus coat protein gene between right margin side areas and left margin side areas, wherein the right margin side areas comprises one and has the sequence of 90% homology with sequence shown in the SEQ ID NO:29; Left boundary flanking sequence comprises one and has the sequence of 90% homology with sequence shown in the SEQ ID NO:31; And this transgenic sequence system comprises a pawpaw ring spot virus coat protein gene and an exercisable promotor that is linked to pawpaw ring spot virus coat protein gene upstream.
2. nucleic acid molecule according to claim 1, wherein this promotor is a Cauliflower edge down virus 35S promoter.
3. nucleic acid molecule according to claim 1, wherein this transgenic sequence further comprises an antibiotics resistance gene and a terminator.
4. nucleic acid molecule according to claim 1, wherein this transgenic sequence system comprises one and has the sequence of 90% homology with sequence shown in the SEQ ID NO:33.
5. nucleic acid molecule according to claim 1, it is made of following person: one is positioned at the right margin side areas of 5 ' end, one is positioned at the left margin side areas of 3 ' end, and a transgenic sequence that contains the pawpaw ring spot virus coat protein gene between right margin side areas and left margin side areas, wherein the right margin side areas has sequence shown in SEQ ID NO:29; Left boundary flanking sequence has sequence shown in SEQ ID NO:31; And this transgenic sequence has sequence shown in SEQ ID NO:33.
6. the primer of the transgenosis Semen Chaenomelis acid sequence that is used to increase, it is to be selected from the group that is made of following:
One has the nucleic acid fragment of at least 10 continuous sequences among shown in SEQ ID NO:29 sequence or its complementary sequence; And
One has the nucleic acid fragment of at least 10 continuous sequences among shown in SEQ ID NO:31 sequence or its complementary sequence.
7. primer according to claim 1, it is to be selected from the group that is made of following primer: the Papa31 primer, it has sequence shown in SEQ ID NO:17; And the Papa57 primer, it has sequence shown in SEQ ID NO:20.
8. method that is used to detect transgenosis pawpaw strain 16-0-1, it comprises:
Provide a pawpaw nucleic acid samples and a primer right, wherein this primer is to comprising a forward primer and a reverse primer;
Make this pawpaw nucleic acid samples and this primer to forming a pcr reaction mixture, carry out amplified reaction, obtain an amplified reaction product; And
Detect the amplified reaction product, wherein the amplified fragments if any a pre-sizing exists, and then represents this pawpaw nucleic acid samples to contain the genomic dna that stems from transgenosis pawpaw strain 16-0-1; Wherein
This forward primer system is selected from the group that is made of following person: one has the nucleic acid fragment of at least 10 continuous sequences among the sequence shown in SEQ ID NO:29; One has the nucleic acid fragment of at least 10 continuous sequences in the sequence shown in SEQ ID NO:33; And their complementary sequence; And
This reverse primer system is selected from the group that is made of following person: one has the nucleic acid fragment of at least 10 continuous sequences among the complementary sequence of sequence shown in SEQ ID NO:31; One has the nucleic acid fragment of at least 10 continuous sequences in the complementary sequence of sequence shown in SEQ ID NO:33; And their complementary sequence.
9. method according to claim 8, wherein this forward primer system is selected from the group that is made of following person: one has the nucleic acid fragment of at least 10 continuous sequences among the sequence shown in SEQ ID NO:29; One has the nucleic acid fragment as at least 10 continuous sequences of SEQ ID NO:33 among the sequence of the 4468th to 5468 nucleotide site; And their complementary sequence; And
This reverse primer system is selected from the group that is made of following person: one has the nucleic acid fragment of at least 10 continuous sequences among the complementary sequence of sequence shown in SEQ ID NO:31; One has the nucleic acid fragment as at least 10 continuous sequences of SEQ ID NO:33 among the complementary sequence of the sequence of the 1st to 1000 nucleotide site; And their complementary sequence.
10. method according to claim 8, wherein
This forward primer system is selected from the group that is made of following primer: the Papa31 primer, and it has sequence shown in SEQ IDNO:17; The P1 primer, it has sequence shown in SEQ ID NO:13; The Papa34 primer, it has sequence shown in SEQ ID NO:15; The Papa52 primer, it has sequence shown in SEQ ID NO:14; The Papa56 primer, it has sequence shown in SEQ ID NO:19; The Papa58 primer, it has sequence shown in SEQ ID NO:21; And the N1 primer, it has sequence shown in SEQ ID NO:16; And
This reverse primer system is selected from the group that is made of following primer: the S18 primer, and it has sequence shown in the SEQ ID NO:11; The Papa27 primer, it has the NO:12 as SEQ ID; And the Papa57 primer, it has the NO:20 as SEQ ID.
11. method according to claim 8, wherein this forward primer is the Papa31 primer, and it has sequence shown in SEQ ID NO:17; Reverse primer is the Papa27 primer, and it has sequence shown in SEQ ID NO:12, and the pre-sizing of this amplified fragments is 241 base pairs.
12. method according to claim 8, wherein this forward primer is the Papa56 primer, and it has sequence shown in SEQ ID NO:19, and reverse primer is the Papa57 primer, it has sequence shown in SEQ ID NO:20, and the pre-sizing of this amplified fragments is 106 base pairs.
13. method according to claim 8, wherein this pawpaw nucleic acid sample source is single pawpaw plant materials; This forward primer is the Papa31 primer, and it has sequence shown in SEQ ID NO:17; And reverse primer is the Papa57 primer, and it has sequence shown in SEQ ID NO:20; And this amplified reaction system comprises and uses a polysaccharase to carry out under the polymerase chain reaction condition of 55 to 65 ℃ bonding temperature in having one; And detection amplified reaction product further comprises: the amplified fragments that detects one 227 base pairs exists, and then representing this pawpaw plant materials non-is homozygote transgenosis pawpaw strain.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102311954A (en) * | 2011-10-18 | 2012-01-11 | 福建农林大学 | Transgenic papaya 55-1 transformation event specific PCR detection method |
| CN109055592A (en) * | 2018-08-17 | 2018-12-21 | 海南医学院 | A kind of anti-ring spot virus papaya YK16-0-1 of transgenosis and its efficient qualitative, quantitative identification method of spin-off |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102311954A (en) * | 2011-10-18 | 2012-01-11 | 福建农林大学 | Transgenic papaya 55-1 transformation event specific PCR detection method |
| CN102311954B (en) * | 2011-10-18 | 2013-02-27 | 福建农林大学 | Transgenic papaya 55-1 transformation event specific PCR detection method |
| CN109055592A (en) * | 2018-08-17 | 2018-12-21 | 海南医学院 | A kind of anti-ring spot virus papaya YK16-0-1 of transgenosis and its efficient qualitative, quantitative identification method of spin-off |
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