CN110872635A - Detection method of transgenic bivalent sheath blight-resistant rice strain WYJ24-PG-10-1 - Google Patents

Abstract

The invention discloses a detection method of an OsPGIP1 and GAFP2 transgenic rice sheath blight resistant rice strain WYJ24-PG-10-1, 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-10-1 to insert a fragment sequence and a specific sequence SEQ ID NO 1 beside an RB end of the insertion fragment on a rice chromosome and based on the sequence SEQ ID NO 1. The forward vector primer PG-S1 for PCR was designed based on the sequence 1-1301 of SEQ ID NO. 1, the reverse genomic primer PG-S2 was designed based on the sequence 1302-1900 of SEQ ID NO. 1, and the forward genomic primer PG-S3 was designed based on the position 699bp 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-S1 PG-S2 primer combination M, PG-S3 PG-S2 primer combination N is obtained by amplification, wherein the PCR method can be used as an effective means for identifying the transgenic rice strain WYJ24-PG-10-1 and a derivative line of the strain.

Description

Detection method of bivalent sheath blight resistant rice line WYJ24-PG-10-1

technical field

The invention belongs to the field of biotechnology, in particular to a detection method for identifying OsPGIP1 and GAFP2 bivalent gene sheath blight resistance rice line WYJ24-PG-10-1 and its derivative lines and the transformant specific sequence it depends on.

Background technique

Rice is one of the major food crops in the world and is widely grown in tropical and subtropical regions. The annual output of rice in my country accounts for about half of the country's total grain output. Rice diseases have reduced rice yield and seriously affected the quality and commercial value of rice. Rice sheath blight is an important disease that harms rice and is widely distributed in all rice-producing regions of the world. With the promotion of dwarf varieties and the extensive use of fertilizers, the occurrence of sheath blight is becoming more and more serious. Sheath blight mainly damages leaf sheaths and leaves, reducing rice seed setting rate and increasing shriveled grains. Generally, it can cause 15% to 20% yield reduction, and even 60% to 70% yield reduction in severe cases.

At present, the prevention and control of sheath blight mainly relies on chemical pesticides. However, in the actual field operation, it is found that the rice sheath blight is generally seriously harmful before and after the tillering and heading stages. During this period, the rice population is relatively dense, and it is difficult to spray the pesticide to the middle. Lower leaf sheath, so that the control effect is not good. Studies have shown that sheath blight has risen to the number one disease of rice in some rice-growing areas in southern China in recent years, which has had a great impact on rice yield and quality. However, germplasm resources with high resistance to rice sheath blight available in rice varieties and wild rice are very rare. Molecular breeding design technology centered on transgenic technology can introduce resistance genes carried by rice itself and resistance genes of other species into rice, and overexpress in rice, so that existing varieties can acquire high disease resistance characteristics. Several transgenic rice lines have been approved for environmental release or production trials. Effective supervision of genetically modified crops is the guarantee for the safe use of genetically modified crops. The sequences flanking the inserts of exogenous sequences in transgenic crops are the most important molecular identifications of transgenic plant lines. Therefore, the flanking sequence of the exogenous insert is an important technical information for establishing a line-specific detection method of transgenic crops.

WYJ24-PG-10-1 is a transgenic rice line developed by Yangzhou University in Jiangsu Province, which is resistant to rice sheath blight and has broad application prospects [Shang Hongyan, creation of a new rice germplasm by transgenic bivalent sheath blight resistance gene (D), Yangzhou University, 2017]. Using the sequences flanking the insertion site to specifically identify transgenic varieties (lines) is the most reliable and specific detection method for identifying transgenic crops at present, and it is a method for accurately identifying and distinguishing the same transformant and its derived lines.

The transgenic lines were identified by the sequences flanking the insertion site, and this method has been established in the identification of Bt Shanyou 63, Kemodao, Kefeng 8 and Kefeng 6 lines. WYJ24-PG-10-1 is the newly developed sheath blight-resistant transgenic rice line, and there are no articles and patents on the flanking sequence of the insertion fragment of any related transgenic rice WYJ24-PG-10-1 foreign gene.

SUMMARY OF THE INVENTION

In view of the deficiencies in the prior art, the object of the present invention is to provide a detection method of the sheath blight resistance rice line WYJ24-PG-10-1 transgenic OsPGIP1 and GAFP2 bivalent genes, to improve the ability to distinguish the same transformant and its derivative lines. Identification accuracy.

In order to achieve the above technical purpose, the present invention utilizes the sequence flanking the insertion site to specifically identify transgenic varieties (lines), which is the most reliable and specific detection method for identifying transgenic crops at present. The specific technical scheme adopted is as follows.

A method for detecting sheath blight resistance rice line WYJ24-PG-10-1 transfected with bivalent genes of OsPGIP1 and GAFP2, characterized in that: using the T gene containing foreign genes OsPGIP1 and GAFP2 in the transformant WYJ24-PG-10-1 - The DNA expression cassette is inserted into the rice chromosome with the sequence of the insert fragment and the sequence flanking the RB end of the insert fragment SEQ ID NO: 1, and based on the sequence of SEQ ID NO: 1, a specific PCR amplification primer is established; then the specific PCR amplification primer is used test.

The specific PCR amplification primers include:

Forward vector primer PG-S1: 5'-CACCCCCATATGACTTATCCTACTGGATTC-3';

Reverse genome primer PG-S2:5'-CCAGCACTTACTGTTCGGTTT-3';

Forward genome primer PG-S3:5'-GGCTGGAAGTAGTTGAAGGC-3',

The rice line is WYJ24-PG-10-1.

The forward carrier primer PG-S1 is a forward carrier primer designed according to the 1-1301 position sequence in SEQ ID NO: 1;

The reverse genome primer PG-S2 is a reverse genome primer designed according to the sequence 1302-1900 in SEQ ID NO: 1;

The forward genome primer PG-S3 is a forward genome primer designed at 699bp upstream of the T-DNA insertion position;

Described forward carrier primer PG-S1 and reverse genome primer PG-S2 are combined to obtain primer combination M, and the amplified band size is a 604bp band of 604bp; forward genome primer PG-S3 and reverse genome primer PG -S2 is combined to obtain primer combination N, and the amplified band size is an 864bp band of 864bp;

Amplify rice genomic DNA with primer combination M and primer combination N respectively: if only the 604bp band is amplified, it means that the plant is a homozygous containing exogenous OsPGIP1 and GAFP2 bivalent genes; if it can be amplified at the same time The 604bp band and the 864bp band indicate that the plant is a hybrid containing exogenous OsPGIP1 and GAFP2 bivalent genes; if only the 864bp band is amplified, it indicates that the plant does not contain exogenous OsPGIP1 and GAFP2 bivalent gene.

As a preferred technical solution of the present application, the rice line WYJ24-PG-10-1 includes a parent, a derivative line or a variety.

As a preferred technical solution of the present application, the PCR reaction system is: template DNA 2.0 μL, 10×PCRBuffer 2.0 μL, dNTP 0.4 μL, forward primer 0.4 μL, reverse primer 0.4 μL, Taq enzyme 0.2 μL, ddH ₂ 0 to 20 μL.

As a preferred technical solution of the present application, PCR amplification conditions are: 94°C pre-denaturation for 3 min; 94°C denaturation for 45S, 58°C annealing for 45S, 72°C extension for 1 min, 38 cycles; 72°C extension for 5 min.

It is well known to those skilled in the art that the above-mentioned specific PCR primers can be prepared by artificial sequence synthesis, and the existing state can be in the form of powder or solution.

The present invention also protects a kit for homozygous identification of rice line WYJ24-PG-10-1, including the above-mentioned specific PCR amplification primers.

Wherein, the kit includes conventional reagents for PCR amplification, and/or conventional reagents for electrophoresis detection.

beneficial effect

The invention provides the right border flanking sequence of the transgenic rice WYJ24-PG-10-1 exogenous gene insertion site, and a specific detection method for the transformation event based on this sequence, which are the transgenic rice WYJ24-PG-10-1 line and Its derivatives provide molecular characterization and specific detection means.

Description of drawings

Figure 1: Schematic diagram of the structure of the T-DNA region of the transformation vector pMF-PcyFBPase-GAFP2-OCS-PrbcS-OsPGIP1-35SpolyA.

Figure 2: Inverse PCR amplification results of rice WYJ24-PG-10-1RB boundary under different restriction endonuclease conditions; PG-10: WYJ24-PG-10-1; M: DL2000 Marker.

Figure 3: Schematic diagram of primer design for specific PCR detection of sequences flanking the right border of the foreign gene insertion site in rice WYJ24-PG-10-1; PG-S1, PG-S2 and PG-S3 represent primers for genotyping; RB and LB denote the right and left borders of T-DNA, respectively.

Figure 4: Electropherogram of the specific qualitative PCR detection of the flanking sequence of the foreign gene insertion of rice WYJ24-PG-10-1. The combination of the forward vector primer PG-S1 and the reverse genome primer PG-S2 yields the primer combination M, which is amplified by 604bp band; forward genome primer PG-S3 and reverse genome primer PG-S2 are combined to obtain primer combination N, amplify to obtain 864bp band, using primer combination M and primer combination N to WYJ24-PG-10-1, PCR identification of the non-transgenic control WYJ24 and the F ₂ segregated population of WYJ24-PG-10-1/Wuyunjing 24 showed that only a 604bp band was amplified in WYJ24-PG-10-1, and only a 604bp band was amplified in the non-transgenic control WYJ24. An 864bp band was added, and the F ₂ segregated population of WYJ24-PG-10-1/Wuyunjing 24 appeared to have only a 604bp band that was homozygous for the bivalent genes of OsPGIP1 and GAFP2, and at the same time amplified The heterozygote containing the exogenous bivalent gene in the 604bp and 864bp bands, and the single plant without the exogenous bivalent gene in which only the 864bp band was amplified; lane 1: WYJ24-PG-10-1; lane 2: Wuyunjing 24; Lanes 1-15: 15 F2 individuals randomly selected from the F2 generation segregating population of _{WYJ24 -} PG-10-1/Wuyunjing 24; M: DL1000 Marker.

Detailed ways

The present invention will be described in further detail below in conjunction with the embodiments. If the reagents or equipment used are not marked with the manufacturer, they are regarded as conventional products that can be purchased through the market.

The schematic diagram of the T-DNA region structure of the vector used in transgenic rice WYJ24-PG-10-1 is shown in Figure 1. The bivalent genes used to transform the target genes OsPGIP1 and GAFP2 and the selectable marker gene HPT are located in two different independent T-DNA regions. One T-DNA region contains 35S promoter and selectable marker gene HPT; the other T-DNA region contains two green tissue expression promoters PrbcS and PcyFBPase, and target genes OsPGIP1 and GAFP2.

In the present invention, the conventional method is used to extract the DNA of transgenic rice WYJ24-PG-10-1, and the reverse PCR method is used to amplify the RB flanking sequence of 1900bp, the nucleotide sequence of which is shown in SEQ ID NO: 1, and the sequence includes the transformation vector The partial sequence of pMF-PcyFBPase-GAFP2-OCS-PrbcS-OsPGIP1-35SpolyA, its nucleotide sequence is exactly the same as the nucleotide sequence of positions 1-1301 in SEQ ID NO: 1, the sequence also includes the rice genome sequence, located in The nucleotide sequence of positions 4265676-4266375 of rice chromosome 3 (GeneBank ID: AF334813.1) is identical to the nucleotide sequence of positions 1302-1900 in SEQ ID NO:1.

experiment material

1. Plant material: The transgenic rice lines WYJ24-PG-10-1 and Wuyunjing 24 are conventional rice.

2. Reagents: Taq DNA Ploymerase (TIANGEN), restriction endonuclease (Takara), 10X Loading Buffer (TaKaRa), PCR amplification primers (Nanjing Qingke Biotechnology Co., Ltd.), Spanish agar tank (BIOWEST).

3. Experimental equipment: PCR instrument (Thermo Fisher Scientific 2720), high-speed centrifuge (Eppendorf 5424R), pipette (Eppendorf), electrophoresis instrument (Beijing Liuyi Instrument Factory DYY-10C), gel imaging system (Tanon2500), Horizontal electrophoresis tank (Bio-Rad).

Example 1 Cloning of sequences flanking the insertion site of foreign genes in transgenic rice WYJ24-PG-10-1

Inverse PCR method is a very effective method for cloning the flanking sequences of T-DNA inserts. We cloned the flanking sequences of foreign gene insertion sites of transgenic rice WYJ24-PG-10-1 by inverse PCR method.

It includes the following specific steps:

1. Extraction of rice DNA

The total DNA of rice leaves was extracted by CTAB method. The specific steps are as follows:

1. Take 1-2g of fresh rice leaves, cut them into a 2mL centrifuge tube, add steel balls, put the centrifuge tube in liquid nitrogen to freeze, then use a vibrating pulverizer to grind the leaves into powder, and quickly pour out the steel balls.

2. Add 600 μL of 1.5% CTAB preheated at 65°C, in a 65°C water bath, shake once every 5 minutes, take out and cool after 30 minutes, and add 600 μL of chloroform solution.

3. Shake for 30 minutes, and centrifuge at 11900 rpm for 8 minutes.

4. Take about 500 μL of supernatant, add the same volume of pre-cooled isopropanol, and place it at -20°C for 1-3 hours (shake before release).

5. Centrifuge at 11900rpm for 8min.

6. Discard the supernatant and add 75% 500 μL ethanol for washing.

7. Centrifuge at 11900 rmp for 4 min and discard the supernatant.

8. After drying, add 50 μL ddH ₂ O to fully dissolve, and store at 4°C for later use.

2. Restriction endonuclease digestion of genomic DNA

Select appropriate restriction sites EcoRI, BamHI and PstI according to the right border sequence of T-DNA, and then digest genomic DNA with these endonucleases respectively. Restriction system: DNA sample 1μg, 1x buffer 2μL, restriction endonucleases Dicer 1 μL, supplemented with sterile ddH ₂ O to 20 μL; digested at 37°C for 4 hours, then terminated the reaction at 80°C for 10 minutes, and detected the effect of enzyme digestion by electrophoresis.

3. Recovering the digested DNA

The enzyme cleavage product was recovered from the agarose gel using the agarose gel DNA recovery kit (TIANGEN), and the recovery steps were referred to the kit instructions.

4. Connection

Ligation system and reaction conditions: DNA 2 μL, T4 DNA ligase 1 μL, 10-fold T4 ligase buffer 4 μL, supplemented with sterile ddH ₂ O to 20 μL; 16°C water bath for 9-12 hours, 65°C water bath for 10 minutes to inactivate T4 ligase activity.

5. Flanking sequence amplification

Design forward primer PG-S1 at the right border of T-DNA, and design reverse primers PG-R1, PG-R2 and PG-R3 near the downstream of EcoRI, BamHI and PstI restriction sites, respectively. The primer sequences are shown in Table 1. , and PG-S1 was paired with PG-Z1, PG-Z2 and PG-Z3 to amplify the connected fragments in the above 3, respectively. The PCR reaction system is shown in Table 2.

PCR amplification conditions were: pre-denaturation at 94°C for 3 min; denaturation at 94°C for 45S, annealing at 58°C for 45S, extension at 72°C for 1 min, 38 cycles; extension at 72°C for 5 min.

Table 1. Primers used for inverse PCR and WYJ24-PG-10-1 specific identification

Table 2. PCR reaction system (20 μL)

6. PCR product electrophoresis and recovery

The PCR amplification product was recovered from the agarose gel using the agarose gel DNA recovery kit (TIANGEN) for sequence determination.

7. PCR product sequencing and analysis

The PCR amplification products recovered in the above 6 were sent to Nanjing Qingke Biotechnology Co., Ltd. for sequencing, and the sequencing results were performed on NCBI (http://www.ncbi.n1m.nih.gov/) to compare the sequence homology with b1ast. Comparisons between flanking sequences and between flanking sequences and vector backbone sequences were performed using MegAlign in Dnastar software. The results showed that the T-DNA insertion position was 4265676-4266375 of the third chromosome in the rice genome (GeneBank ID: AF334813.1).

Example 2 Sequence-specific PCR detection based on insertion of foreign genes in transgenic rice WYJ24-PG-10-1 line

In order to verify the detection effect of primers PG-S1, PG-S2 and PG-S3 in transgenic rice WYJ24-PG-10-1 line and its derived lines, we tested the F of WYJ24-PG-10-1/Wuyunjing 24. Fifteen individual plants randomly selected from the _2nd generation isolated population material were amplified by PCR and detected by electrophoresis using PG-S1 PG-S2 primer combination M and PG-S3, PG-S2 primer combination N. It includes the following specific steps:

The acquisition of segregated groups

The transgenic rice line WYJ24-PG-10-1, a japonica rice variety Wuyunjing 24 in Jiangsu Province, was obtained by hybridization to obtain the F2 population, and ₁₅ plants _were randomly selected from the F2 population. Japonica No. 24 was used as a control to carry out the following operations.

2. Extraction of plant genomic DNA is the same as "Rice DNA extraction" in Example 1.

3. PCR detection based on the insertion of flanking sequences of foreign genes in transgenic rice line WYJ24-PG-10-1

According to the RB end flanking sequence and the upstream genome sequence of the T-DNA insertion position determined in Example 1, primers PG-S1, PG-S2 and PG-S3 were designed in the transformation vector part and the rice genome sequence part respectively. The schematic diagram of primer design is shown in Figure 3, the primer names and specific sequences are shown in Table 1.

4. Amplify genomic DNA by conventional PCR method

Combining PG-S1 and PG-S2 to obtain primer combination M, the amplified band size is a 604bp band of 604bp; PG-S3 and PG-S2 are combined to obtain primer combination N, and the amplified band size is 864bp The PCR reaction system is shown in Table 2. The PCR amplification conditions are: pre-denaturation at 94°C for 3 min; denaturation at 94°C for 45S, annealing at 58°C for 45S, extension at 72°C for 1 min, 38 cycles; extension at 72°C for 5 min. Amplification products were detected by electrophoresis.

5. Amplification identification results

PCR identification of WYJ24-PG-10-1, non-transgenic control WYJ24 and WYJ24-PG-10-1/Wuyunjing 24 F ₂ segregated populations using primer combination M and primer combination N, respectively, WYJ24-PG -10-1 only amplified 604bp band, non-transgenic control WYJ24 only amplified 864bp band, WYJ24-PG-10-1/Wuyunjing ₂₄ F2 isolated population only amplified 604bp band The homozygote containing the exogenous OsPGIP1 and GAFP2 bivalent genes, the heterozygote containing the exogenous bivalent gene that amplified 604bp and 864bp bands at the same time, and the exogenous bivalent gene that only amplified the 864bp band. of a single plant. The electropherogram is shown in Fig. 4, which shows that the identification method of the present invention is reliable.

The schematic diagram of the T-DNA region structure of the vector used in transgenic rice WYJ24-PG-10-1 is shown in Figure 1. The bivalent genes used to transform the target genes OsPGIP1 and GAFP2 and the selectable marker gene HPT are located in two different independent T-DNA regions. One T-DNA region contains 35S promoter and selectable marker gene HPT; the other T-DNA region contains two green tissue expression promoters PrbcS and PcyFBPase, and target genes OsPGIP1 and GAFP2.

In the present invention, the conventional method is used to extract the DNA of transgenic rice WYJ24-PG-10-1, and the reverse PCR method is used to amplify the RB flanking sequence of 1900bp, the nucleotide sequence of which is shown in SEQ ID NO: 1, and the sequence includes the transformation vector The partial sequence of pMF-PcyFBPase-GAFP2-OCS-PrbcS-OsPGIP1-35SpolyA, its nucleotide sequence is exactly the same as the nucleotide sequence of positions 1-1301 in SEQ ID NO: 1, the sequence also includes the rice genome sequence, located in The nucleotide sequence of positions 4265676-4266375 of rice chromosome 3 (GeneBank ID: AF334813.1) is identical to the nucleotide sequence of positions 1302-1900 in SEQ ID NO:1.

Example 3

The examples in this group provide a kit for homozygous identification of transgenic rice line WYJ24-PG-10-1. The kit includes the PG-S1, PG-S2 and PG-S3 primers described in Example 1.

The kit also includes conventional reagents for PCR amplification, and/or conventional reagents for electrophoretic detection. According to the records of this application, for the purpose of "identification of homozygous heterozygosity of transgenic rice line WYJ24-PG-10-1", those skilled in the art can reasonably select the conventional reagents for PCR amplification and/or electrophoresis reagents in the field .

The protection content of the present invention is not limited to the above embodiments. Variations and advantages that can occur to those skilled in the art without departing from the spirit and scope of the inventive concept are included in the present invention, and the appended claims are the scope of protection.

sequence listing

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Claims (6)

1. A detection method of an OsPGIP1 and GAFP2 bivalent gene banded sclerotial blight resistant rice strain WYJ24-PG-10-1 is characterized in that a T-DNA expression frame containing exogenous genes OsPGIP1 and GAFP2 in a transformant WYJ24-PG-10-1 is utilized to insert a fragment sequence and an RB end flanking sequence SEQ ID NO 1 of the insert fragment on a rice chromosome, and a specific PCR amplification primer is established based on the SEQ ID NO 1 sequence; and detecting by using a specific PCR amplification primer.

2. The method for detecting the rice sheath blight-resistant rice strain WYJ24-PG-10-1 by transferring OsPGIP1 and GAFP2 bivalent genes as claimed in claim 1, wherein the specific PCR amplification primers comprise:

forward vector primer PG-S1: 5'-CACCCCCATATGACTTATCCTACTGGATTC-3';

reverse genomic primer PG-S2: 5'-CCAGCACTTACTGTTCGGTTT-3';

forward genomic primer PG-S3: 5'-GGCTGGAAGTAGTTGAAGGC-3'.

3. The method for detecting the rice sheath blight-resistant rice line WYJ24-PG-10-1 with OsPGIP1 and GAFP2 as transgenic bivalent genes according to claim 2,

the forward carrier primer PG-S1 is designed according to a sequence 1-1301 in SEQ ID NO. 1;

the reverse genome primer PG-S2 is a reverse genome primer designed according to the sequence 1302-1900 in SEQ ID NO. 1;

the forward genome primer PG-S3 is a forward genome primer designed according to the position 699bp upstream of the insertion position of the T-DNA;

the forward carrier primer PG-S1 and the reverse genome primer PG-S2 are combined to obtain a primer combination M, and the amplified band size is 604bp band of 604 bp; combining a forward genome primer PG-S3 and a reverse genome primer PG-S2 to obtain a primer combination N, wherein the amplified band size is 864bp bands of 864 bp;

respectively amplifying the rice genome DNA by using a primer combination M and a primer combination N: if only the 604bp band is amplified, the plant is a homozygote containing exogenous OsPGIP1 and GAFP2 bivalent genes; if the 604bp band and the 864bp band can be amplified simultaneously, the plant is a hybrid containing exogenous OsPGIP1 and GAFP2 bivalent genes; if only the 864bp band is amplified, the plant does not contain exogenous OsPGIP1 and GAFP2 bivalent genes.

4. The method for detecting the rice sheath blight-resistant rice strain WYJ24-PG-10-1 with OsPGIP1 and GAFP2 bivalent genes as claimed in claim 2, wherein the PCR reaction system is as follows: template DNA 2.0. mu.L, 10 XPCR Buffer 2.0. mu. L, dNTP 0.4.4. mu.L, forward primer 0.4. mu.L, reverse primer 0.4. mu. L, Taq enzyme 0.2. mu.L, plus ddH₂O to 20. mu.L.

5. The detection method of the OsPGIP1 and GAFP2 transgenic sheath blight rice strain WYJ24-PG-10-1 as claimed in claim 2, wherein 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.

6. A kit for pure heterozygous identification of rice line WYJ24-PG-10-1, comprising the specific PCR amplification primers of claim 2.

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