CN110863062A - Detection method of anti-sheath blight rice line with OsPGIP1 and GAFP2 transgenic bivalent genes - Google Patents
Detection method of anti-sheath blight rice line with OsPGIP1 and GAFP2 transgenic bivalent genes Download PDFInfo
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Abstract
The invention discloses a method for detecting an OsPGIP1 and GAFP2 transgenic rice line with the resistance to banded sclerotial blight, belonging to the technical field of biology. The invention relates to a specific PCR amplification identification technology which is established by utilizing a T-DNA expression frame containing exogenous genes OsPGIP1 and GAFP2 in a transformant WYJ24-PG-1 to insert a fragment sequence and an RB-end flanking specific sequence SEQ ID NO. 1 of the insertion fragment on a rice chromosome and based on the SEQ ID NO. 1 sequence. The forward vector primer PG-P1 for PCR was designed based on the sequence 1-900 of SEQ ID NO. 1, the reverse genome primer PG-P2 was designed based on the sequence 901-1500 of SEQ ID NO. 1, and the forward genome primer PG-P3 was designed based on the position 546bp upstream of the insertion position of T-DNA. And judging whether the strain contains exogenous OsPGIP1 and GAFP2 bivalent genes according to whether the target fragment of the PG-P1 PG-P2 primer combination A, PG-P3 PG-P2 primer combination B is obtained by amplification, wherein the PCR method can be used as an effective means for identifying the transgenic rice strain WYJ24-PG-1 and the derivative line of the strain.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a detection method for identifying an OsPGIP1 and GAFP2 bivalent gene sheath blight resistant rice strain WYJ24-PG-1 and derivative strains thereof and a transformant specific sequence depended on the detection method.
Background
Rice is one of the major food crops worldwide and is widely cultivated in tropical and subtropical regions. The annual rice yield of China accounts for about half of the total grain yield of China. The rice diseases cause the yield reduction of the rice and seriously affect the quality and the commodity value of the rice. The rice sheath blight disease is an important disease which damages rice and is widely distributed in all rice production areas in the world. With the popularization of dwarf varieties and the large use of fertilizers, the occurrence of sheath blight is increasingly serious. Sheath and leaf are mainly damaged by sheath blight, so that the maturing rate of rice is reduced, the yield of rice is reduced by 15-20% generally, and even 60-70% in serious cases.
At present, the prevention and control of the sheath blight disease mainly depend on chemical pesticides, but the actual operation in the field shows that the rice sheath blight disease generally has serious harm before and after the tillering stage and the heading stage, rice groups are dense in the period, and the agent is difficult to spray to the middle and lower leaf sheaths when in use, so that the prevention and control effect is poor. Researches show that in recent years, the rice sheath blight has risen to the first disease of rice in parts of rice planting areas in southern China, and great influence is brought to the yield and the quality of rice. And the germplasm resources with high resistance to rice sheath blight which can be utilized in rice varieties and wild rice are very rare. The molecular breeding design technology taking the transgenic technology as the core can introduce resistance genes carried by rice and resistance genes of other species into the rice and perform over-expression in the rice, so that the existing variety can obtain high disease resistance. Currently, a number of transgenic rice lines have been approved for environmental release or production testing. Effective supervision of transgenic crops is a precondition and guarantee for safe utilization of the transgenic crops. The flanking sequence of the exogenous sequence insert of the transgenic crop is the most important molecular identity card of the transgenic plant line. Therefore, the flanking sequence based on the exogenous inserted fragment is an important technical data for establishing a transgenic crop line specificity detection method.
WYJ24-PG-1 is a transgenic rice line which is developed by Yangzhou university of Jiangsu province and has wide application prospect and can resist rice sheath blight. The specific identification of transgenic variety (strain) by using insertion site side sequence is the most reliable and specific detection method for identifying transgenic crop at present, and is a method for accurately identifying and distinguishing the same transformant and derived strain thereof.
The transgenic lines are identified by using flanking sequences beside the insertion sites, and the method is established in the identification of the lines of Ke-Ming-dao, Bt Shanyou 63, Ke Feng No. 6 and Ke Feng No. 8. WYJ24-PG-1 is a newly developed banded sclerotial blight resistant transgenic rice line, and no flanking sequence article report and patent of any related transgenic rice WYJ24-PG-1 exogenous gene insert fragment exist.
Disclosure of Invention
The invention aims to provide a method for detecting an OsPGIP1 and GAFP2 bivalent gene sheath blight resistant rice line, so as to improve the identification accuracy for distinguishing a same transformant and derived lines thereof.
In order to realize the technical purpose, the method for specifically identifying the transgenic variety (strain) by utilizing the flanking sequence of the insertion site is the most reliable and specific detection method for identifying the transgenic crop at present, and the adopted specific technical scheme is as follows.
A method for detecting an OsPGIP1 and GAFP2 transgenic rice line with sheath blight resistance is characterized by comprising the following steps: inserting a fragment sequence and an RB-end flanking sequence SEQ ID NO. 1 of the inserted fragment on a rice chromosome by using a T-DNA expression frame containing exogenous genes OsPGIP1 and GAFP2 in a transformant WYJ24-PG-1, and establishing a specific PCR amplification primer based on the sequence SEQ ID NO. 1; and detecting by using a specific PCR amplification primer.
The specific PCR amplification primer comprises:
forward vector primer PG-P1: 5'-TCAGATTGTCGTTTCCCGCCTTCAGTTTA-3';
reverse genome primer PG-P2: 5'-CACTACACCCAGTTCTGTTCATA-3';
a forward genome primer PG-P3:5'-TATTGTCTGCTTGCGTTGCT-3',
the rice product is WYJ 24-PG-1.
The forward carrier primer PG-P1 is a forward carrier primer designed according to a sequence from 1 to 900 bits in SEQ ID NO. 1;
the reverse genome primer PG-P2 is a reverse genome primer designed according to the sequence of 901-1500 in SEQ ID NO. 1;
the forward genome primer PG-P3 is a forward genome primer designed according to the position 546bp upstream of the insertion position of the T-DNA;
the forward carrier primer PG-P1 and the reverse genome primer PG-P2 are combined to obtain a primer combination A, and the amplified band size is 362bp band of 362 bp; combining a forward genome primer PG-P3 and a reverse genome primer PG-P2 to obtain a primer combination B, wherein the amplified band size is 854bp band of 854 bp;
respectively amplifying the rice genome DNA by using a primer combination A and a primer combination B: if only the 362bp band is amplified, the plant is indicated to be homozygote containing exogenous OsPGIP1 and GAFP2 bivalent genes; if the 362bp band and the 854bp band can be amplified simultaneously, the plant is indicated to be a heterozygote containing exogenous OsPGIP1 and GAFP2 bivalent genes; if only the 854bp band is amplified, the plant does not contain exogenous OsPGIP1 and GAFP2 bivalent gene.
The invention has the beneficial effect. The invention provides a right border flanking sequence of a transgenic rice WYJ24-PG-1 exogenous gene insertion site and a transformation event specific detection method based on the sequence, and provides a molecular characteristic and specific detection means for a transgenic rice WYJ24-PG-1 strain and a derivative line thereof.
Drawings
FIG. 1: the T-DNA region of the transformation vector pMF-PcyFBPase-GAFP2-OCS-PrbcS-OsPGIP1-35SpolyA is schematically shown in structure.
FIG. 2: the result of the rice WYJ24-PG-1RB boundary inverse PCR amplification under different restriction enzyme conditions is shown. PG-1: WYJ 24-PG-1; and M is DL2000 Marker.
FIG. 3 is a schematic diagram of the design of specific PCR detection primers for the flanking sequence of the right border of the insertion site of the exogenous gene WYJ24-PG-1 of rice. PG-P1, PG-P2 and PG-P3 represent primers for genotyping; RB and LB represent the right and left borders of T-DNA, respectively.
FIG. 4 shows a rice WYJ24-PG-1 exogenous gene insert flanking sequence specificity qualitative PCR detection electrophoretogram, a forward carrier primer PG-P1 and a reverse genome primer PG-P2 are combined to obtain a primer combination A, and a 362bp band is obtained by amplification; combining a forward genome primer PG-P3 and a reverse genome primer PG-P2 to obtain a primer combination B, amplifying to obtain a 854bp band, and respectively adopting the primer combination A and the primer combination B to carry out amplification on WYJ24-PG-1, a non-transgenic control WYJ24 and WYJ 24-PG-1/Wuyujing No. 24F2PCR identification of the generation separation population, 362bp band is amplified by WYJ24-PG-1, 854bp band is amplified by non-transgenic control WYJ24, and F of WYJ 24-PG-1/Wuyujing No. 242The segregation population has homozygote containing exogenous OsPGIP1 and GAFP2 and only amplifying 362bp bands, heterozygote containing exogenous bivalent genes and only amplifying 854bp bands and single strain containing no exogenous bivalent genes and only amplifying 854bp bands. Lane 1: WYJ 24-PG-1; lane 2: wuyujing No. 24; lanes 1-15: f from WYJ 24-PG-1/Wuyujing No. 242Randomly drawn 15F in the generation segregating population2Carrying out single plant cultivation; and M is DL1000 Marker.
Detailed Description
The technical solution of the present invention is further described in detail with reference to the accompanying drawings and specific embodiments.
The vector used by the transgenic rice WYJ24-PG-1 is shown in figure 1, a bivalent gene and a selectable marker gene HPT for transforming target genes OsPGIP1 and GAFP2 are respectively positioned in two different independent T-DNA regions, wherein one T-DNA region contains a 35S promoter and the selectable marker gene HPT; the other T-DNA region contains two green tissue expression promoters PrbcS and PcyFBPase and target genes OsPGIP1 and GAFP2, and the structure schematic diagram of the T-DNA region is shown in FIG. 1.
The invention adopts a conventional method to extract the WYJ24-PG-1DNA of the transgenic rice, utilizes an inverse PCR method to amplify to obtain a RB flanking sequence of 1500bp, the nucleotide sequence of which is shown as SEQ ID NO. 1, comprises a partial sequence of a transformation vector pMF-PcyFBPase-GAFP2-OCS-PrbcS-OsPGIP1-35SpolyA, the nucleotide sequence of which is completely identical with the nucleotide sequence of 1-900 bits in the SEQ ID NO. 1, and also comprises a rice genome sequence which is positioned at 21734-10622333 bits of the No. 1 chromosome (GeneBank ID: AP014957.1), and the nucleotide sequence of which is completely identical with the nucleotide sequence of 901-1500 bits in the SEQ ID NO. 1.
EXAMPLE 1 cloning of flanking sequence of foreign Gene insertion site of transgenic Rice WYJ24-PG-1
The reverse PCR method is an effective method for cloning flanking sequences of a T-DNA insert, and the reverse PCR method is adopted to clone the flanking sequences of the exogenous gene insertion site of the transgenic rice WYJ 24-PG-1. The method comprises the following specific steps:
extraction of rice DNA
The method for extracting the total DNA of the rice leaves by adopting a CTAB method comprises the following specific steps:
1. taking 1-2g of fresh rice leaves, shearing the fresh rice leaves into a 2mL centrifugal tube, adding steel balls, putting the centrifugal tube into liquid nitrogen for freezing, then beating the leaves into powder by using a vibration crusher, and quickly pouring out the steel balls.
2. Adding 600 μ l of 1.5% CTAB preheated at 65 deg.C, shaking once every 5min in water bath at 65 deg.C, taking out after 30min, cooling, and adding 600 μ l of chloroform solution.
3. Shaking for 30min, and centrifuging at 12000rpm for 8 min.
4. About 500. mu.l of the supernatant was added to the same volume of pre-cooled isopropanol and left at-20 ℃ for 1-3h (shaking before placing).
5.12000rpm for 8 min.
6. The supernatant was discarded, and 75% 500. mu.l ethanol was added thereto for washing.
7.12000rmp for 4min, discard the supernatant.
8. After air drying, 50. mu.l of ddH2O was added and dissolved sufficiently, and the mixture was left at 4 ℃ for further use.
Secondly, digesting the genome DNA by restriction endonuclease
The right border sequence of the T-DNA was used to select the appropriate cleavage sites EcoRI, BamHI and PstI, and the genomic DNA was digested with these endonucleases in the following order: DNA sample 1. mu.g, 1-fold Buffer 2. mu.l, restriction enzyme 1. mu.l, and sterile ddH2O to 20. mu.l; the reaction was terminated at 37 ℃ for 4 hours and then at 80 ℃ for 10 minutes, and the cleavage effect was checked by electrophoresis.
Thirdly, recovering DNA after enzyme digestion
The digestion products were recovered from the agarose gel using agarose gel DNA recovery kit (TIANGEN), the recovery procedure being described in the kit instructions.
Four, connect
Connecting system and reaction conditions: 2. mu.l of DNA, 1. mu.l of T4 DNA ligase, 4. mu.l of 10-fold T4 ligase buffer, and additionally sterilizing ddH2O to 20. mu.l; water bath at 16 ℃ for 9-12 hours, and water bath at 65 ℃ for 10min to inactivate the activity of the T4 ligase.
Amplification of flanking sequences
The forward primer PG-P1 was designed at the right border of T-DNA, and the reverse primers PG-Z1, PG-Z2 and PG-Z3 were designed near the downstream of EcoRI, BamHI and PstI cleavage sites, respectively, the sequences of the primers are shown in Table 1, the ligated fragments of 3 above were amplified by pairing PG-P1 with PG-Z1, PG-Z2 and PG-Z3, respectively, and the PCR reaction system is shown in Table 2.
The PCR amplification conditions were: pre-denaturation at 94 ℃ for 3 min; denaturation at 94 ℃ for 45S, annealing at 58 ℃ for 45S, extension at 72 ℃ for 1min, and 38 cycles; extension at 72 ℃ for 5 min.
TABLE 1 primers for reverse PCR and specific identification of WYJ24-PG-1
TABLE 2 PCR reaction system (20. mu.l)
Sixthly, electrophoresis and recovery of PCR products
The PCR amplification products were recovered from the agarose gel using the agarose gel DNA recovery kit (TIANGEN) for sequencing, and the electrophoresis pattern is shown in FIG. 2.
Seventh, PCR product sequencing and analysis
The PCR amplification product recovered in the above 6 was subjected to sequencing by Biotech Ltd of the department of Ongjingkins, and the results of the sequencing were subjected to b1ast homology comparison at NCBI (http:// www.ncbi.n1m.nih.gov /). Comparison between flanking sequences and vector backbone sequences used MegAlign in Dnastar software. The results showed that the insertion site of T-DNA was 10621734-10622333 of chromosome 1 (GeneBank ID: AP014957.1) in the rice genome.
Example 2 detection of flanking sequence specificity PCR based on insertion of exogenous gene into transgenic Rice strain WYJ24-PG-1
In order to verify the detection effect of the primers PG-P1, PG-P2 and PG-P3 in the transgenic rice WYJ24-PG-1 line and the derivative line thereof, the primers WYJ 24-PG-1/Wuyujing No. 24F 215 individuals randomly selected from the population material are separated, and PCR amplification and electrophoresis detection are carried out by adopting PG-P1 PG-P2 primer combination A, PG-P3 and PG-P2 primer combination B. The method comprises the following specific steps:
obtaining of isolated population
The transgenic rice WYJ24-PG-1 strain, the japonica rice variety Wuyujing No. 24 of Jiangsu province are hybridized to obtain F2Population from F 215 strains were randomly selected from the population and subjected to the following procedures using WYJ24-PG-1 and Wuyujing No. 24 as controls.
Second, plant genome DNA extraction was performed as in "rice DNA extraction" in example 1.
PCR detection based on insertion of flanking sequence of exogenous gene of transgenic rice strain WYJ24-PG-1
Primers PG-P1, PG-P2 and PG-P3 were designed in the transformation vector portion and the rice genomic sequence portion, respectively, based on the RB-terminal flanking sequence and the genomic sequence upstream of the T-DNA insertion position determined in example 1, the schematic design of the primers is shown in FIG. 3, and the names and specific sequences of the primers are detailed in Table 1.
Fourthly, amplifying genome DNA by adopting a conventional PCR method
Combining PG-P1 and PG-P2 to obtain a primer combination A, wherein the amplified band size is 362bp band of 362 bp; the PG-P3 and PG-P2 are combined to obtain a primer combination B, and the amplified band size is a 854bp band of 854 bp; the PCR reaction system is shown in Table 2, and the PCR amplification conditions are as follows: pre-denaturation at 94 ℃ for 3 min; denaturation at 94 ℃ for 45S, annealing at 58 ℃ for 45S, extension at 72 ℃ for 1min, and 38 cycles; extension at 72 ℃ for 5 min. And (5) carrying out electrophoretic detection on the amplification product.
Fifth, amplification and identification results
Respectively adopting a primer combination A and a primer combination B to carry out the treatment on WYJ24-PG-1, non-transgenic control WYJ24 and WYJ 24-PG-1/Wuyujing No. 24F2PCR identification of the generation separation population, 362bp band is amplified by WYJ24-PG-1, 854bp band is amplified by non-transgenic control WYJ24, and F of WYJ 24-PG-1/Wuyujing No. 242The segregation population has homozygote containing exogenous OsPGIP1 and GAFP2 and only amplifying 362bp bands, heterozygote containing exogenous bivalent genes and only amplifying 854bp bands and single strain containing no exogenous bivalent genes and only amplifying 854bp bands. The electrophoretogram is shown in figure 4, which shows that the identification method of the present invention is reliable.
The above description is only a preferred embodiment of the present invention, and all equivalent changes and modifications made in accordance with the claims of the present invention should be covered by the present invention.
Sequence listing
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Claims (3)
1. A method for detecting an OsPGIP1 and GAFP2 transgenic rice line with sheath blight resistance is characterized by comprising the following steps: inserting a fragment sequence and an RB-end flanking sequence SEQ ID NO. 1 of the inserted fragment on a rice chromosome by using a T-DNA expression frame containing exogenous genes OsPGIP1 and GAFP2 in a transformant WYJ24-PG-1, and establishing a specific PCR amplification primer based on the sequence SEQ ID NO. 1; and detecting by using a specific PCR amplification primer.
2. The method for detecting the rice sheath blight-resistant rice line with OsPGIP1 and GAFP2 transgenic bivalent genes according to claim 1, wherein the method comprises the following steps: the specific PCR amplification primer comprises:
forward vector primer PG-P1: 5'-TCAGATTGTCGTTTCCCGCCTTCAGTTTA-3';
reverse genome primer PG-P2: 5'-CACTACACCCAGTTCTGTTCATA-3';
forward genomic primer PG-P3: 5'-TATTGTCTGCTTGCGTTGCT-3';
the rice product is WYJ 24-PG-1.
3. The method for detecting the rice sheath blight-resistant rice line with OsPGIP1 and GAFP2 transgenic bivalent genes, according to claim 2, wherein the method comprises the following steps:
the forward carrier primer PG-P1 is a forward carrier primer designed according to a sequence from 1 to 900 bits in SEQ ID NO. 1;
the reverse genome primer PG-P2 is a reverse genome primer designed according to the sequence of 901-1500 in SEQ ID NO. 1;
the forward genome primer PG-P3 is a forward genome primer designed according to the position 546bp upstream of the insertion position of the T-DNA;
the forward carrier primer PG-P1 and the reverse genome primer PG-P2 are combined to obtain a primer combination A, and the amplified band size is 362bp band of 362 bp; combining a forward genome primer PG-P3 and a reverse genome primer PG-P2 to obtain a primer combination B, wherein the amplified band size is 854bp band of 854 bp;
respectively amplifying the rice genome DNA by using a primer combination A and a primer combination B: if only the 362bp band is amplified, the plant is indicated to be homozygote containing exogenous OsPGIP1 and GAFP2 bivalent genes; if the 362bp band and the 854bp band can be amplified simultaneously, the plant is indicated to be a heterozygote containing exogenous OsPGIP1 and GAFP2 bivalent genes; if only the 854bp band is amplified, the plant does not contain exogenous OsPGIP1 and GAFP2 bivalent gene.
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