CN113046457A - Flanking sequence of insertion site of GhSOCL1 transgenic cotton and specificity identification method thereof - Google Patents
Flanking sequence of insertion site of GhSOCL1 transgenic cotton and specificity identification method thereof Download PDFInfo
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Abstract
The invention discloses a flanking sequence of a cotton insertion site of a GhSOCL1 transgenic gene and a specificity identification method thereof, belonging to the technical field of biology. The flanking sequences comprise left flanking sequences and/or right flanking sequences; the 1 st to 912 th site sequences of the left flank sequence are derived from the genome sequence of the Zhongmian 24, and the 913 th to 1348 th site sequences of the left flank sequence are derived from the exogenous insert sequence of GhSOCL1 transgenic cotton; the 1 st to 466 th site sequences of the right flank sequence are derived from the exogenous insertion fragment sequence of cotton transformed with GhSOCL1 gene, and the 467 th to 1269 th site sequences of the right flank sequence are derived from the genome sequence of Zhongmiao 24. The invention designs the specific primer through the flanking sequence, and can judge whether the sample to be detected contains GhSOCL1 transgenic cotton component or not through whether the designed primer and the PCR product of the sample to be detected contain the specific amplification band or not.
Description
Technical Field
The invention relates to the technical field of biology, in particular to a flanking sequence of a cotton insertion site of a GhSOCL1 transgenic gene and a specificity identification method thereof.
Background
Cotton is one of the leading economic crops in the world, and is a raw material and an important strategic material for industries such as textile, fine chemical engineering and the like. Short season cotton (Short sea cotton) is used as an important cotton variety resource in China, can well solve the conflict between grain and cotton in land competition, and plays an important role in optimizing the agricultural industrial structure. The earliness is an important target character in short-season cotton breeding, is a quantitative character with complex relationship, and is influenced by various factors such as a growth period, a plant growth and development state and the like. Flowering time directly affects crop maturity, while flower bud differentiation affects flowering time. Therefore, the flowering related gene is cloned, and expression analysis and transgenic function verification are carried out on the flowering related gene, so that high-quality gene resources can be provided for short-season cotton breeding.
With the development of biotechnology, transgenic technology is a new way for improving important traits of crops, and transgenic breeding has the advantages of realizing cross-species gene discovery, widening the utilization range of genetic resources, realizing the directional efficient transfer of known functional genes, enabling organisms to obtain specific traits required by human beings, and providing a new technical way for breeding new varieties of high-yield, high-quality and high-resistance agricultural organisms. The GhSOCL1 gene is cloned from cotton by the Chinese agricultural academy of sciences cotton institute, and the cDNA sequence of the GhSOCL1 gene is introduced into the cotton institute 24 (national approval GS 08001-1997) of the cotton variety by an agrobacterium-mediated method to obtain a transgenic cotton plant FJ 334; compared with a non-transgenic cotton variety, the GhSOCL1 transgenic cotton FJ334 plant has the characteristics of obviously reducing the number of proud buds, increasing the plant height, increasing the number of single plant bolls, increasing the weight of the single bolls and the like.
The specific detection of the transgenic event and the derived strain or variety thereof is an important technical means for realizing the effective supervision and management of the transgenic plant and ensuring the healthy development of the transgenic industry. The flanking sequence of the exogenous insertion fragment and the detection method established based on the flanking sequence are important basis for effective supervision of the transgenic plant and the product thereof. At present, although related patents and literatures report that the flanking sequences are inserted into transgenic plants exogenously, no article or patent report related to the exogenous insertion of the flanking sequences into GhSOCL1 transgenic cotton is found.
Disclosure of Invention
The embodiment of the invention aims to provide a flanking sequence of a cotton insertion site of a transgenic GhSOCL1 gene to solve the problems in the background technology.
In order to achieve the above purpose, the embodiments of the present invention provide the following technical solutions:
the flanking sequence of the cotton insertion site of GhSOCL1 transgenic gene includes left flanking sequence and/or right flanking sequence; the nucleotide sequence of the left flanking sequence is shown as a sequence table SEQ ID NO. 2, and the nucleotide sequence of the right flanking sequence is shown as a sequence table SEQ ID NO. 3; the 1 st to 912 th site sequences of the left flank sequence are derived from the genome sequence of the Zhongmian 24, and the 913 th to 1348 th site sequences of the left flank sequence are derived from the exogenous insert sequence of GhSOCL1 transgenic cotton; the 1 st to 466 th site sequences of the right flank sequence are derived from the exogenous insertion fragment sequence of GhSOCL1 gene-transferred cotton, and the 467 th to 1269 th site sequences of the right flank sequence are derived from the genome sequence of the Zhongmian cotton plant 24.
Another objective of the embodiments of the present invention is to provide a primer for identifying the specificity of the flanking sequence of the cotton insertion site of the transgenic GhSOCL1 gene, wherein the primer is designed according to the flanking sequence.
As a preferable embodiment of the present invention, the primer includes a first primer and/or a second primer; the first primer comprises a first forward primer designed according to site 1-912 sequences of the left flanking sequence and a first reverse primer designed according to site 913-1348 sequences of the left flanking sequence; the second primer comprises a second forward primer designed according to the 1 st to 466 th site sequences of the right flank sequence and a second reverse primer designed according to the 467 th to 1269 th site sequences of the right flank sequence.
As another preferable scheme of the embodiment of the invention, the nucleotide sequence of the first forward primer is shown as SEQ ID NO. 4 of the sequence table; the nucleotide sequence of the first reverse primer is shown as a sequence table SEQ ID NO. 5.
As another preferable scheme of the embodiment of the invention, the nucleotide sequence of the second forward primer is shown as SEQ ID NO. 6 of the sequence table; the nucleotide sequence of the second reverse primer is shown in a sequence table SEQ ID NO. 7.
Another objective of the embodiments of the present invention is to provide a kit for identifying specificity of a flanking sequence of a cotton insertion site of a transgenic GhSOCL1 gene, which includes a Polymerase Chain Reaction (PCR) premix and the above primers.
Another objective of the embodiments of the present invention is to provide a method for specifically identifying a flanking sequence of a cotton insertion site of a transgenic GhSOCL1 gene, which comprises the following steps:
obtaining genome DNA of a sample to be detected as a DNA template;
mixing the DNA template, the PCR premix solution and the primers to obtain a PCR reaction solution;
carrying out PCR amplification reaction on the PCR reaction solution to obtain a PCR reaction product;
carrying out gel electrophoresis detection on the PCR reaction product to judge whether a specific band exists in the PCR reaction product; if a specific band exists in the PCR reaction product, the sample to be detected contains the component of GhSOCL1 transgenic cotton.
In another preferable embodiment of the present invention, in the step, the gel electrophoresis detection is agarose gel electrophoresis detection.
Compared with the prior art, the embodiment of the invention has the beneficial effects that:
according to the embodiment of the invention, the flanking sequence of the exogenous insertion fragment of GhSOCL1 transgenic cotton is firstly determined, and the specific primer is designed according to the flanking sequence, so that whether the sample to be detected contains GhSOCL1 transgenic cotton components can be judged according to whether the primer and the PCR product of the sample to be detected contain specific amplification bands, and the method can simply, conveniently and accurately detect the GhSOCL1 transgenic cotton, thereby facilitating the effective supervision and management of the GhSOCL1 transgenic cotton.
Drawings
FIG. 1 is a structural map of a GhSOCL1 gene-transgenic cotton transformation vector pBI121-GhSOCL1 provided by the embodiment of the invention.
FIG. 2 is a diagram showing gel electrophoresis obtained in example 7 of the present invention.
FIG. 3 is a diagram showing gel electrophoresis obtained in example 8 of the present invention.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention. In addition, the apparatus and reagents used in the following examples are commercially available ones unless otherwise specified.
Example 1
This example provides flanking sequences of a cotton insertion site for transforming the GhSOCL1 gene, which include a left flanking sequence; the nucleotide sequence of the left flanking sequence is shown as a sequence table SEQ ID NO. 2, and the length is 1348 bp; the 1 st to 912 th site sequences of the left flank sequence are derived from the genome sequence of the Zhongmian 24, and the 913 th to 1348 th site sequences of the left flank sequence are derived from the exogenous insert sequence of GhSOCL1 transgenic cotton. Wherein, the left flanking sequence refers to the left border flanking sequence of the exogenous insertion fragment of GhSOCL1 gene-transferred cotton.
Example 2
This example provides flanking sequences of a cotton insertion site for transforming the GhSOCL1 gene, which include a right flanking sequence; the nucleotide sequence of the right flanking sequence is shown as a sequence table SEQ ID NO. 3, and the length of the right flanking sequence is 1269 bp; the 1 st to 466 th site sequences of the right flank sequence are derived from the exogenous insertion fragment sequence of GhSOCL1 gene-transferred cotton, and the 467 th to 1269 th site sequences of the right flank sequence are derived from the genome sequence of the Zhongmian cotton plant 24. Wherein, the right flank sequence refers to the right border flanking sequence of the exogenous insertion fragment of GhSOCL1 gene-transferred cotton.
In addition, the method for obtaining the left flank sequence and the right flank sequence provided in the above embodiments 1 to 2 is as follows:
(1) extracting genome DNA of a GhSOCL1 transgenic cotton plant: placing young leaves of a GhSOCL 1-transgenic cotton plant into a centrifuge tube, then placing a sample into liquid nitrogen, and carrying out oscillation and smashing on a proofing machine to obtain the sample; then, adding 800 muL of cetyl trimethyl ammonium bromide cracking liquid preheated at 65 ℃ into the sample, simultaneously adding beta-mercaptoethanol to prevent oxidation, uniformly mixing until no obvious layering occurs, placing the mixture in a water bath at 65 ℃ for 40 minutes, and slowly reversing and uniformly mixing once every 10 min; after water bath, adding 800 muL of chloroform/isoamyl alcohol mixed solution with the volume ratio of 24:1, and repeatedly reversing until no layering occurs; then, the mixture was centrifuged at 12000rpm for 10 minutes at 4 ℃; sucking the supernatant by using a tip-free gun head, wherein the supernatant is about 600 mu L, and transferring the supernatant to another 2mL centrifuge tube; adding ice-cooled isopropanol with the volume of 0.8 time, slightly inverting for several times until flocculent DNA is generated, centrifuging at 12000rpm for 1 minute, and pouring out the ice-cooled isopropanol; adding 75% ethanol, washing for 3 times, and washing with anhydrous ethanol for 1 time; pouring off the absolute ethyl alcohol, and adding 200 muL ddH after drying2And dissolving the O to obtain the genome DNA of the GhSOCL1 transgenic cotton plant, and storing at 4 ℃ for later use. Wherein, the GhSOCL1 transgenic cotton is a transgenic plant FJ334 of the cotton research institute of Chinese academy of agricultural sciences, which is obtained by introducing the cDNA sequence of the GhSOCL1 gene into a cotton institute 24 (national approval GS 08001-1997) in a cotton variety by utilizing an agrobacterium-mediated method, and the structural map of a transformation vector pBI121-GhSOCL1 is shown in figure 1.
(2) Entrust Beijing and kang biotechnology limited company to carry on resequencing analysis to the above-mentioned genomic DNA which changes GhSOCL1 gene cotton plant, specifically, after changing GhSOCL1 gene cotton plant's genomic DNA to detect qualified, use the method of mechanical breaking (supersonic wave) to segment DNA, then carry on segment purification, end repair, 3' end add A, connect sequencing joint to fragmented DNA, reuse agarose gel electrophoresis to carry on the size selection of fragment, carry on PCR amplification and form the sequencing library, the library of library built carries on the quality control of the library first, the qualified library of quality control carries on the sequencing with Illumina HiSeq platform; the off-line Data was filtered to obtain Clean Data, and then reads were aligned to Gossypium hirsutum (AD 1), NAU association database and Mapped Data obtained on the insert sequence in the designated reference genomic cotton genome database (http:// www.cottonfgd.org /), and used for exogenous insert search. The specific searching method comprises the following steps: firstly, finding junction reads which contain a reference genome and an insertion sequence in the read; then, determining an insertion position and a direction according to the comparison information of the connection reads; finally, these reads are clustered to determine the insertion location and length, and whether there are pairs of junction clusters. Finding out the following two types of Paired _ end reads according to the comparison result: the first type is that a reference genome sequence is aligned on a reads at one end, and an insertion sequence is aligned on a reads at the other end; the second type is that a part of reads at either end is aligned with the reference genome sequence, and the other part is aligned with the insert sequence. And (3) comparing the reference genome by adopting BWA, selecting all reads capable of comparing the exogenous insertion sequences, and carrying out local assembly. And respectively comparing the exogenous insertion sequence and the reference genome result by using Blastn according to the assembled contig, selecting regions of the contig sequence compared to the chromosome, and carrying out IGV screenshot verification on bwa comparison results of the regions to obtain the insertion position information of the exogenous insertion fragment. The insertion position of the exogenous insertion fragment of the GhSOCL1 transgenic cotton is verified to be D07 chromosome 30270907 of Zhongmian cotton plant 24, and sequences with insertion sites of 1901bp in upstream and downstream lengths in a reference cotton genome are obtained, wherein the nucleotide sequence of 30270907-30270998 sites of the Gomian cotton plant 24 is shown as SEQ ID NO: 1.
(3) And designing PCR detection primers according to the sequence of the exogenous insertion fragment of the cotton with the GhSOCL1 transgenic gene and the upstream and downstream sequences of the insertion site in a reference genome of the cotton. Wherein, the nucleotide sequences of the GhSOCL1 transgenic cotton left boundary flanking sequence amplification primer pair are shown as SEQ ID NO. 4-5, and are respectively: 5'-TGCTCTCGATATCGCACACT-3' and 5'-AGATTGTCGTTTCCCGCCTT-3';the nucleotide sequences of the GhSOCL1 transgenic cotton right border flanking sequence amplification primer pair are shown as SEQ ID NO. 6-7, and respectively are as follows: 5'-TGATGGTTCACGTAGTGGGC-3', and 5'-ATGTTCGTGCCCGTGTAACT-3'. Then, taking the genomic DNA of the GhSOCL1 transgenic cotton plant as a DNA template, and respectively carrying out PCR amplification reaction by using the two groups of primers to obtain two groups of PCR amplification products, wherein a PCR reaction system (10 uL) is as follows: 5 muL of PCR premixed solution; 0.8 muL of primer pairs (0.4 muL of each of 2 primers); 2 muL of DNA template; ddH2O2.2 muL. The PCR reaction conditions are as follows: pre-denaturation at 94 deg.C for 2min, denaturation at 94 deg.C for 30S, annealing at 55 deg.C for 30S, extension at 72 deg.C for 30S, and 30 cycles; final extension at 72 deg.C for 2min, and storage at 4 deg.C. And respectively detecting the two groups of PCR amplification products by using 1% agarose gel electrophoresis, purifying the PCR products by using a gel recovery kit, entrusting a prokaryote biotechnology company to perform sequencing verification, and comparing a sequencing result with an exogenous insertion sequence and a reference genome sequence to finally obtain a left boundary flanking sequence (namely the left flanking sequence) and a right boundary flanking sequence (namely the right flanking sequence) of the exogenous insertion fragment of the GhSOCL1 transgenic cotton. It should be noted that, as the PCR premix, commercially available 2 × Taq MasterMix (Dye) can be used.
Example 3
This example provides a primer for identifying the specificity of the flanking sequence of the cotton insertion site of the GhSOCL1 gene, which is designed according to the left flanking sequence provided in example 1 above. Specifically, the primer comprises a first primer; the first primer comprises a first forward primer designed according to site 1-912 sequences of the left flanking sequence and a first reverse primer designed according to site 913-1348 sequences of the left flanking sequence; wherein, the nucleotide sequence of the first forward primer is shown as a sequence table SEQ ID NO. 4 and is as follows: 5'-TGCTCTCGATATCGCACACT-3', respectively; the nucleotide sequence of the first reverse primer is shown as a sequence table SEQ ID NO. 5 and comprises the following components: 5'-AGATTGTCGTTTCCCGCCTT-3' are provided.
Example 4
This example provides a primer for identifying the specificity of the flanking sequence of the cotton insertion site of the GhSOCL1 gene, which is designed according to the right flanking sequence provided in example 2 above. Specifically, the primer comprises a second primer; the second primer comprises a second forward primer designed according to the 1 st to 466 th site sequences of the right flank sequence and a second reverse primer designed according to the 467 th to 1269 th site sequences of the right flank sequence; wherein the nucleotide sequence of the second forward primer is shown as a sequence table SEQ ID NO. 6 and is: 5'-TGATGGTTCACGTAGTGGGC-3', respectively; the nucleotide sequence of the second reverse primer is shown as a sequence table SEQ ID NO. 7 and is as follows: 5'-ATGTTCGTGCCCGTGTAACT-3' are provided.
Example 5
This example provides a kit for identifying the specificity of flanking sequences of a cotton insertion site of a transgenic GhSOCL1 gene, which comprises a PCR premix and the primers provided in the above example 3. As the PCR premix, 2 × Taq MasterMix (Dye) which is commercially available is used.
Example 6
This example provides a kit for identifying the specificity of flanking sequences of a cotton insertion site of a transgenic GhSOCL1 gene, which comprises a PCR premix and the primers provided in the above example 4. As the PCR premix, 2 × Taq MasterMix (Dye) which is commercially available is used.
Example 7
The embodiment provides a method for detecting whether a sample to be detected contains GhSOCL1 transgenic cotton, which is detected by using the kit provided in the embodiment 5, and specifically comprises the following steps:
(1) extracting the genome DNA of a sample to be detected as a DNA template according to the method for extracting the genome DNA of the GhSOCL1 transgenic cotton plant.
(2) 2 muL of DNA template, 5 muL of PCR premixed solution, 0.4 muL of first forward primer, 0.4 muL of first reverse primer and 2.2 muL of ddH2And O, mixing to obtain a PCR reaction solution.
(3) Placing the PCR reaction solution in a PCR instrument for PCR amplification reaction to obtain a PCR reaction product; wherein, the PCR reaction conditions are as follows: pre-denaturation at 94 deg.C for 2min, denaturation at 94 deg.C for 30S, annealing at 55 deg.C for 30S, extension at 72 deg.C for 30S, and 30 cycles; final extension at 72 deg.C for 2min, and storage at 4 deg.C.
(4) Carrying out 1% agarose gel electrophoresis detection on the PCR reaction product, and dyeing by using nucleic acid dye to judge whether a specific band exists in the PCR reaction product; if a specific band exists in the PCR reaction product, the sample to be detected contains the component of GhSOCL1 transgenic cotton. Wherein, the GhSOCL1 gene-transferred cotton comprises the parents, plants, tissues, seeds and the like of derived lines.
The method is used for detecting samples of a leaf of GhSOCL1 gene-transferred cotton, a seed of GhSOCL1 gene-transferred cotton, a root of the seed of GhSOCL1 gene-transferred cotton, non-transgenic Zhongbaozhu 24, Zhongbaozhu 50, corn, wheat and the like, and clear water is used as a negative control group, and the gel electrophoresis pattern obtained by detection is shown in figure 2, wherein M is DNA molecular weight standard (DL 5000), 1 is the leaf of GhSOCL1 gene-transferred cotton, 2 is the seed of GhSOCL1 gene-transferred cotton, 3 is the root of GhSOCL1 gene-transferred cotton, 4 is the non-transgenic Zhongbaozhu 24, 5 is the Zhongbaozhu 50, 6 is the corn, 7 is the wheat and 8 is the clear water. As can be seen from the figure, only the sample of the leaf, seed and root of GhSOCL1 transgenic cotton generates a specific amplification band with the length of 724bp, and the other samples do not generate specific amplification bands, which shows that the detection method provided by the embodiment of the invention can accurately detect whether the sample is GhSOCL1 transgenic cotton or cotton containing GhSOCL1 transgenic cotton.
Example 8
The embodiment provides a method for detecting whether a sample to be detected contains GhSOCL1 transgenic cotton, which is detected by using the kit provided in the embodiment 6, and specifically comprises the following steps:
(1) extracting the genome DNA of a sample to be detected as a DNA template according to the method for extracting the genome DNA of the GhSOCL1 transgenic cotton plant.
(2) 2 muL of DNA template, 5 muL of PCR premixed solution, 0.4 muL of second forward primer, 0.4 muL of second reverse primer and 2.2 muL of PCR premixed solutionddH2And O, mixing to obtain a PCR reaction solution.
(3) Placing the PCR reaction solution in a PCR instrument for PCR amplification reaction to obtain a PCR reaction product; wherein, the PCR reaction conditions are as follows: pre-denaturation at 94 deg.C for 2min, denaturation at 94 deg.C for 30S, annealing at 55 deg.C for 30S, extension at 72 deg.C for 30S, and 30 cycles; final extension at 72 deg.C for 2min, and storage at 4 deg.C.
(4) Carrying out 1% agarose gel electrophoresis detection on the PCR reaction product, and dyeing by using nucleic acid dye to judge whether a specific band exists in the PCR reaction product; if a specific band exists in the PCR reaction product, the sample to be detected contains the component of GhSOCL1 transgenic cotton. Wherein, the GhSOCL1 gene-transferred cotton comprises the parents, plants, tissues, seeds and the like of derived lines.
The method is used for detecting samples of a leaf of GhSOCL1 gene-transferred cotton, a seed of GhSOCL1 gene-transferred cotton, a root of the seed of GhSOCL1 gene-transferred cotton, non-transgenic Zhongbaozhu 24, Zhongbaozhu 50, corn, wheat and the like, and clear water is used as a negative control group, and the gel electrophoresis graph obtained by detection is shown in figure 3, wherein M is DNA molecular weight standard (DL 5000), 1 is the leaf of GhSOCL1 gene-transferred cotton, 2 is the seed of GhSOCL1 gene-transferred cotton, 3 is the root of the leaf of GhSOCL1 gene-transferred cotton, 4 is the non-transgenic Zhongbaozhu 24, 5 is the Zhongbaozhu 50, 6 is the corn, 7 is the wheat and 8 is the clear water. As can be seen from the figure, only the sample of the leaf, seed and root of GhSOCL1 transgenic cotton generates a specific amplification band with the length of 502bp, and the other samples do not generate specific amplification bands, which shows that the detection method provided by the embodiment of the invention can accurately detect whether the sample is GhSOCL1 transgenic cotton or cotton containing GhSOCL1 transgenic cotton.
In light of the foregoing description of the preferred embodiment of the present invention, many modifications and variations will be apparent to those skilled in the art without departing from the spirit and scope of the invention. The technical scope of the present invention is not limited to the content of the specification, and must be determined according to the scope of the claims.
SEQUENCE LISTING
<110> Cotton research institute of Chinese academy of agricultural sciences
<120> flanking sequence of insertion site of GhSOCL1 transgenic cotton and specificity identification method thereof
<160> 7
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1901
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 1
attacaatat cacatatact taccatttgg ctgaatttag accttcatat actcatatac 60
tttcatttac ctcatataaa ttttattatc acgattgaag taactaactt accttgatat 120
catgtattat tcaaccattg catacttgac ctatcatgta ctaaataatt ggtcgagacc 180
ataataaagt tgcacaatta gtgctctttc tcatctaacc aaattgctct cgatatcgca 240
cacttagtcc ctgttattca acatcatcat tttaatttcg cacacttagt gcccattatt 300
caacatcgtc attttagttt cgcacactta gtgtcgttca tatagctgta gctatcccat 360
actcgcacac ttagtgctaa atattgataa attcattcat gtctatttct actttccgac 420
taaaacatac tcagctatat atatatttgc atactttcat ttcggcatat ataagtaata 480
tttatttaga aattatagca tctatttgat tataatctta cctcggatag tgataaaccg 540
gaacagaacg atcaatcaac gactttattt tttccctgat ccaagttcga tttctttaat 600
tcatgatcta aacacattca aatttaactc attcaaacat aatttcatcc aatttcatcc 660
aaaaacacat aattgggtaa cttaccattt tacccctgat atttaacatt tttacaattc 720
agtccaaatt gcacaaaaca aaaaatattc caaattaatc acaatgtagt ttggtcggat 780
attccctagt ctcatatagg tccttgcatg tcgtttattt cacaatttag tccctcaatt 840
taatatttct tacaatttaa ctcaaaataa tcaaattcat caaaaattca aaaacaaaca 900
ttataatcta ttacatatct tttattttct accatcaacc aacaaaaatc acaaactatc 960
atcaatggca catcacaaaa tcgtcataaa ttccaaaaat taaagcatga attttgaaga 1020
acacgaagca ataatctcag aaacataaaa attataaaaa aacgattaaa aactaacctt 1080
aatttaagct caaccaaggc cgaatataac aaagcttcaa ctatttctat ttctttcaac 1140
attcggtgat ggtggatgaa caatccacta acatttttta ttttttatta ttataatata 1200
cgagcatgtg gcttggtcgt gtgacccaac ttcaaaagtt acacgggcac gaacataggc 1260
tgggacacgg ccgtgtgtct taggtcgtgt gtccctattt tgattgttac aaggcctaag 1320
acaagggtgt gtgtctcagc cgtgtgagtc acacagccta ggcacacgac cgtgtgaccc 1380
ctgcagttca aattttctaa atttttccag aactttttga atgattccaa tttagtccct 1440
aattgtttct aaagtgtttt tagggcatcg aaggctcgat taagggacta tatgcatgta 1500
gatgaactat ctatgccatg tttatgttga ggtactaaat gtatgtttta aagttaaaat 1560
tttatgataa tgctctgtga cactattttg acgatggata cggattaggg gtgttacaat 1620
tatattatag aggactaggt caaaaagacc taggaagtac aagtgattga aattgatgtg 1680
agagaggccc aaaacccctc atagaacatg aagggggagg aaaccctagt agaaatacaa 1740
gggtgggacg ttcacccctc tcctactcta agaaggatgt tgtttttctg tttaaaataa 1800
acttctacaa ctctacaagg gttctacctt ttattcctat aaatatctgg caccagtaga 1860
gctatttaca caacttttaa gcgattatca ttctatcgaa a 1901
<210> 2
<211> 1348
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 2
attacaatat cacatatact taccatttgg ctgaatttag accttcatat actcatatac 60
tttcatttac ctcatataaa ttttattatc acgattgaag taactaactt accttgatat 120
catgtattat tcaaccattg catacttgac ctatcatgta ctaaataatt ggtcgagacc 180
ataataaagt tgcacaatta gtgctctttc tcatctaacc aaattgctct cgatatcgca 240
cacttagtcc ctgttattca acatcatcat tttaatttcg cacacttagt gcccattatt 300
caacatcgtc attttagttt cgcacactta gtgtcgttca tatagctgta gctatcccat 360
actcgcacac ttagtgctaa atattgataa attcattcat gtctatttct actttccgac 420
taaaacatac tcagctatat atatatttgc atactttcat ttcggcatat ataagtaata 480
tttatttaga aattatagca tctatttgat tataatctta cctcggatag tgataaaccg 540
gaacagaacg atcaatcaac gactttattt tttccctgat ccaagttcga tttctttaat 600
tcatgatcta aacacattca aatttaactc attcaaacat aatttcatcc aatttcatcc 660
aaaaacacat aattgggtaa cttaccattt tacccctgat atttaacatt tttacaattc 720
agtccaaatt gcacaaaaca aaaaatattc caaattaatc acaatgtagt ttggtcggat 780
attccctagt ctcatatagg tccttgcatg tcgtttattt cacaatttag tccctcaatt 840
taatatttct tacaatttaa ctcaaaataa tcaaattcat caaaaattca aaaacaaaca 900
ttataatcta ttctgatagt ttaaactgaa ggcgggaaac gacaatctga tcatgagcgg 960
agaattaagg gagtcacgtt atgacccccg ccgatgacgc gggacaagcc gttttacgtt 1020
tggaactgac agaaccgcaa cgttgaagga gccactcagc cgcgggtttc tggagtttaa 1080
tgagctaagc acatacgtca gaaaccatta ttgcgcgttc aaaagtcgcc taaggtcact 1140
atcagctagc aaatatttct tgtcaaaaat gctccactga cgttccataa attcccctcg 1200
gtatccaatt agagtctcat attcactctc aatccaaata atctgcaccg gatctggatc 1260
gtttcgcatg attgaacaag atggattgca cgcaggttct ccggccgctt gggtggagag 1320
gctattcggc tatgactggg cacaacag 1348
<210> 3
<211> 1269
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 3
ttcttccctt cctttctcgc cacgttcgcc ggctttcccc gtcaagctct aaatcggggg 60
ctccctttag ggttccgatt tagtgcttta cggcacctcg accccaaaaa acttgatttg 120
ggtgatggtt cacgtagtgg gccatcgccc tgatagacgg tttttcgccc tttgacgttg 180
gagtccacgt tctttaatag tggactcttg ttccaaactg gaacaacact caaccctatc 240
tcgggctatt cttttgattt ataagggatt ttgccgattt cggaaccacc atcaaacagg 300
attttcgcct gctggggcaa accagcgtgg accgcttgct gcaactctct cagggccagg 360
cggtgaaggg caatcagctg ttgcccgtct cactggtgaa aagaaaaacc accccagtac 420
attaaaaacg tccgcaatgt gttattaagt tgtctaagcg tcaattgccg aatataacaa 480
agcttcaact atttctattt ctttcaacat tcggtgatgg tggatgaaca atccactaac 540
attttttatt ttttattatt ataatatacg agcatgtggc ttggtcgtgt gacccaactt 600
caaaagttac acgggcacga acataggctg ggacacggcc gtgtgtctta ggtcgtgtgt 660
ccctattttg attgttacaa ggcctaagac aagggtgtgt gtctcagccg tgtgagtcac 720
acagcctagg cacacgaccg tgtgacccct gcagttcaaa ttttctaaat ttttccagaa 780
ctttttgaat gattccaatt tagtccctaa ttgtttctaa agtgttttta gggcatcgaa 840
ggctcgatta agggactata tgcatgtaga tgaactatct atgccatgtt tatgttgagg 900
tactaaatgt atgttttaaa gttaaaattt tatgataatg ctctgtgaca ctattttgac 960
gatggatacg gattaggggt gttacaatta tattatagag gactaggtca aaaagaccta 1020
ggaagtacaa gtgattgaaa ttgatgtgag agaggcccaa aacccctcat agaacatgaa 1080
gggggaggaa accctagtag aaatacaagg gtgggacgtt cacccctctc ctactctaag 1140
aaggatgttg tttttctgtt taaaataaac ttctacaact ctacaagggt tctacctttt 1200
attcctataa atatctggca ccagtagagc tatttacaca acttttaagc gattatcatt 1260
ctatcgaaa 1269
<210> 4
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 4
tgctctcgat atcgcacact 20
<210> 5
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 5
agattgtcgt ttcccgcctt 20
<210> 6
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 6
tgatggttca cgtagtgggc 20
<210> 7
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 7
atgttcgtgc ccgtgtaact 20
Sequence listing
<110> Cotton research institute of Chinese academy of agricultural sciences
<120> flanking sequence of insertion site of GhSOCL1 transgenic cotton and specificity identification method thereof
<141> 2019-12-24
<160> 7
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1901
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 1
attacaatat cacatatact taccatttgg ctgaatttag accttcatat actcatatac 60
tttcatttac ctcatataaa ttttattatc acgattgaag taactaactt accttgatat 120
catgtattat tcaaccattg catacttgac ctatcatgta ctaaataatt ggtcgagacc 180
ataataaagt tgcacaatta gtgctctttc tcatctaacc aaattgctct cgatatcgca 240
cacttagtcc ctgttattca acatcatcat tttaatttcg cacacttagt gcccattatt 300
caacatcgtc attttagttt cgcacactta gtgtcgttca tatagctgta gctatcccat 360
actcgcacac ttagtgctaa atattgataa attcattcat gtctatttct actttccgac 420
taaaacatac tcagctatat atatatttgc atactttcat ttcggcatat ataagtaata 480
tttatttaga aattatagca tctatttgat tataatctta cctcggatag tgataaaccg 540
gaacagaacg atcaatcaac gactttattt tttccctgat ccaagttcga tttctttaat 600
tcatgatcta aacacattca aatttaactc attcaaacat aatttcatcc aatttcatcc 660
aaaaacacat aattgggtaa cttaccattt tacccctgat atttaacatt tttacaattc 720
agtccaaatt gcacaaaaca aaaaatattc caaattaatc acaatgtagt ttggtcggat 780
attccctagt ctcatatagg tccttgcatg tcgtttattt cacaatttag tccctcaatt 840
taatatttct tacaatttaa ctcaaaataa tcaaattcat caaaaattca aaaacaaaca 900
ttataatcta ttacatatct tttattttct accatcaacc aacaaaaatc acaaactatc 960
atcaatggca catcacaaaa tcgtcataaa ttccaaaaat taaagcatga attttgaaga 1020
acacgaagca ataatctcag aaacataaaa attataaaaa aacgattaaa aactaacctt 1080
aatttaagct caaccaaggc cgaatataac aaagcttcaa ctatttctat ttctttcaac 1140
attcggtgat ggtggatgaa caatccacta acatttttta ttttttatta ttataatata 1200
cgagcatgtg gcttggtcgt gtgacccaac ttcaaaagtt acacgggcac gaacataggc 1260
tgggacacgg ccgtgtgtct taggtcgtgt gtccctattt tgattgttac aaggcctaag 1320
acaagggtgt gtgtctcagc cgtgtgagtc acacagccta ggcacacgac cgtgtgaccc 1380
ctgcagttca aattttctaa atttttccag aactttttga atgattccaa tttagtccct 1440
aattgtttct aaagtgtttt tagggcatcg aaggctcgat taagggacta tatgcatgta 1500
gatgaactat ctatgccatg tttatgttga ggtactaaat gtatgtttta aagttaaaat 1560
tttatgataa tgctctgtga cactattttg acgatggata cggattaggg gtgttacaat 1620
tatattatag aggactaggt caaaaagacc taggaagtac aagtgattga aattgatgtg 1680
agagaggccc aaaacccctc atagaacatg aagggggagg aaaccctagt agaaatacaa 1740
gggtgggacg ttcacccctc tcctactcta agaaggatgt tgtttttctg tttaaaataa 1800
acttctacaa ctctacaagg gttctacctt ttattcctat aaatatctgg caccagtaga 1860
gctatttaca caacttttaa gcgattatca ttctatcgaa a 1901
<210> 2
<211> 1348
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 2
attacaatat cacatatact taccatttgg ctgaatttag accttcatat actcatatac 60
tttcatttac ctcatataaa ttttattatc acgattgaag taactaactt accttgatat 120
catgtattat tcaaccattg catacttgac ctatcatgta ctaaataatt ggtcgagacc 180
ataataaagt tgcacaatta gtgctctttc tcatctaacc aaattgctct cgatatcgca 240
cacttagtcc ctgttattca acatcatcat tttaatttcg cacacttagt gcccattatt 300
caacatcgtc attttagttt cgcacactta gtgtcgttca tatagctgta gctatcccat 360
actcgcacac ttagtgctaa atattgataa attcattcat gtctatttct actttccgac 420
taaaacatac tcagctatat atatatttgc atactttcat ttcggcatat ataagtaata 480
tttatttaga aattatagca tctatttgat tataatctta cctcggatag tgataaaccg 540
gaacagaacg atcaatcaac gactttattt tttccctgat ccaagttcga tttctttaat 600
tcatgatcta aacacattca aatttaactc attcaaacat aatttcatcc aatttcatcc 660
aaaaacacat aattgggtaa cttaccattt tacccctgat atttaacatt tttacaattc 720
agtccaaatt gcacaaaaca aaaaatattc caaattaatc acaatgtagt ttggtcggat 780
attccctagt ctcatatagg tccttgcatg tcgtttattt cacaatttag tccctcaatt 840
taatatttct tacaatttaa ctcaaaataa tcaaattcat caaaaattca aaaacaaaca 900
ttataatcta ttctgatagt ttaaactgaa ggcgggaaac gacaatctga tcatgagcgg 960
agaattaagg gagtcacgtt atgacccccg ccgatgacgc gggacaagcc gttttacgtt 1020
tggaactgac agaaccgcaa cgttgaagga gccactcagc cgcgggtttc tggagtttaa 1080
tgagctaagc acatacgtca gaaaccatta ttgcgcgttc aaaagtcgcc taaggtcact 1140
atcagctagc aaatatttct tgtcaaaaat gctccactga cgttccataa attcccctcg 1200
gtatccaatt agagtctcat attcactctc aatccaaata atctgcaccg gatctggatc 1260
gtttcgcatg attgaacaag atggattgca cgcaggttct ccggccgctt gggtggagag 1320
gctattcggc tatgactggg cacaacag 1348
<210> 3
<211> 1269
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 3
ttcttccctt cctttctcgc cacgttcgcc ggctttcccc gtcaagctct aaatcggggg 60
ctccctttag ggttccgatt tagtgcttta cggcacctcg accccaaaaa acttgatttg 120
ggtgatggtt cacgtagtgg gccatcgccc tgatagacgg tttttcgccc tttgacgttg 180
gagtccacgt tctttaatag tggactcttg ttccaaactg gaacaacact caaccctatc 240
tcgggctatt cttttgattt ataagggatt ttgccgattt cggaaccacc atcaaacagg 300
attttcgcct gctggggcaa accagcgtgg accgcttgct gcaactctct cagggccagg 360
cggtgaaggg caatcagctg ttgcccgtct cactggtgaa aagaaaaacc accccagtac 420
attaaaaacg tccgcaatgt gttattaagt tgtctaagcg tcaattgccg aatataacaa 480
agcttcaact atttctattt ctttcaacat tcggtgatgg tggatgaaca atccactaac 540
attttttatt ttttattatt ataatatacg agcatgtggc ttggtcgtgt gacccaactt 600
caaaagttac acgggcacga acataggctg ggacacggcc gtgtgtctta ggtcgtgtgt 660
ccctattttg attgttacaa ggcctaagac aagggtgtgt gtctcagccg tgtgagtcac 720
acagcctagg cacacgaccg tgtgacccct gcagttcaaa ttttctaaat ttttccagaa 780
ctttttgaat gattccaatt tagtccctaa ttgtttctaa agtgttttta gggcatcgaa 840
ggctcgatta agggactata tgcatgtaga tgaactatct atgccatgtt tatgttgagg 900
tactaaatgt atgttttaaa gttaaaattt tatgataatg ctctgtgaca ctattttgac 960
gatggatacg gattaggggt gttacaatta tattatagag gactaggtca aaaagaccta 1020
ggaagtacaa gtgattgaaa ttgatgtgag agaggcccaa aacccctcat agaacatgaa 1080
gggggaggaa accctagtag aaatacaagg gtgggacgtt cacccctctc ctactctaag 1140
aaggatgttg tttttctgtt taaaataaac ttctacaact ctacaagggt tctacctttt 1200
attcctataa atatctggca ccagtagagc tatttacaca acttttaagc gattatcatt 1260
ctatcgaaa 1269
<210> 4
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 4
tgctctcgat atcgcacact 20
<210> 5
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 5
agattgtcgt ttcccgcctt 20
<210> 6
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 6
tgatggttca cgtagtgggc 20
<210> 7
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 7
atgttcgtgc ccgtgtaact 20
Claims (8)
1. The flanking sequence of the cotton insertion site of the GhSOCL1 transgenic gene is characterized by comprising a left flanking sequence and/or a right flanking sequence; the nucleotide sequence of the left flanking sequence is shown as a sequence table SEQ ID NO. 2, and the nucleotide sequence of the right flanking sequence is shown as a sequence table SEQ ID NO. 3; the 1 st to 912 th site sequences of the left flank sequence are derived from the genome sequence of the Zhongmian 24, and the 913 th to 1348 th site sequences of the left flank sequence are derived from the exogenous insert sequence of GhSOCL1 transgenic cotton; the 1 st to 466 th site sequences of the right flank sequence are derived from the exogenous insertion fragment sequence of GhSOCL1 gene-transferred cotton, and the 467 th to 1269 th site sequences of the right flank sequence are derived from the genome sequence of the Zhongmian cotton plant 24.
2. Primer for identifying the specificity of the flanking sequence of the cotton insertion site of the GhSOCL1 gene, wherein the primer is designed according to the flanking sequence of claim 1.
3. The primer for identifying the specificity of the flanking sequence of the cotton insertion site of the GhSOCL1 transgenic gene of claim 2, wherein the primer comprises a first primer and/or a second primer; the first primer comprises a first forward primer designed according to site 1-912 sequences of the left flanking sequence and a first reverse primer designed according to site 913-1348 sequences of the left flanking sequence; the second primer comprises a second forward primer designed according to the 1 st to 466 th site sequences of the right flank sequence and a second reverse primer designed according to the 467 th to 1269 th site sequences of the right flank sequence.
4. The primer for identifying the specificity of the flanking sequence of the insertion site of the cotton transgenic with the GhSOCL1 gene as claimed in claim 3, wherein the nucleotide sequence of the first forward primer is shown in SEQ ID NO. 4 of the sequence table; the nucleotide sequence of the first reverse primer is shown as a sequence table SEQ ID NO. 5.
5. The primer for identifying the specificity of the flanking sequence of the insertion site of the cotton transgenic with the GhSOCL1 gene according to claim 3, wherein the nucleotide sequence of the second forward primer is shown in a sequence table SEQ ID NO. 6; the nucleotide sequence of the second reverse primer is shown in a sequence table SEQ ID NO. 7.
6. A kit for identifying the specificity of a flanking sequence of a cotton insertion site of a GhSOCL1 transgenic gene comprises a PCR premix, and is characterized by further comprising the primer as claimed in any one of claims 2 to 5.
7. The method for specifically identifying the flanking sequence of the insertion site of the GhSOCL1 transgenic cotton is characterized by comprising the following steps of:
obtaining genome DNA of a sample to be detected as a DNA template;
mixing a DNA template, a PCR premix and the primer according to any one of claims 2-5 to obtain a PCR reaction solution;
carrying out PCR amplification reaction on the PCR reaction solution to obtain a PCR reaction product;
carrying out gel electrophoresis detection on the PCR reaction product to judge whether a specific band exists in the PCR reaction product; if a specific band exists in the PCR reaction product, the sample to be detected contains the component of GhSOCL1 transgenic cotton.
8. The method for identifying the specificity of the flanking sequences of the insertion site of the GhSOCL1 transgenic cotton according to claim 7, wherein the gel electrophoresis detection is agarose gel electrophoresis detection.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101240278A (en) * | 2007-02-09 | 2008-08-13 | 中国农业科学院植物保护研究所 | Side sequence of exogenous insert of transgene paddy strain Kemingdao 1 |
| WO2015196874A1 (en) * | 2014-06-25 | 2015-12-30 | 创世纪种业有限公司 | Cotton plant event ac20-3 and primer and method for the detection thereof |
| CN109929850A (en) * | 2017-12-15 | 2019-06-25 | 中国种子集团有限公司 | Anti- nematode Cotton Transformation event GHP10 |
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2019
- 2019-12-27 CN CN201911373956.5A patent/CN113046457A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101240278A (en) * | 2007-02-09 | 2008-08-13 | 中国农业科学院植物保护研究所 | Side sequence of exogenous insert of transgene paddy strain Kemingdao 1 |
| WO2015196874A1 (en) * | 2014-06-25 | 2015-12-30 | 创世纪种业有限公司 | Cotton plant event ac20-3 and primer and method for the detection thereof |
| CN109929850A (en) * | 2017-12-15 | 2019-06-25 | 中国种子集团有限公司 | Anti- nematode Cotton Transformation event GHP10 |
Non-Patent Citations (1)
| Title |
|---|
| XIAOHONG ZHANG ET AL.: ""Functional characterization of GhSOC1 and GhMADS42 homologs fromupland cotton (Gossypium hirsutum L.)"", 《PLANT SCIENCE》, vol. 242, pages 178 - 186 * |
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