CN108060258B - Primer group, kit and method for pure heterozygous identification of cotton MON15985 transformant - Google Patents
Primer group, kit and method for pure heterozygous identification of cotton MON15985 transformant Download PDFInfo
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Abstract
The invention provides a primer group for detecting pure heterozygosis of a cotton MON15985 transformant, belonging to the technical field of biology. The primer group comprises the following three primers: 15985-GF: 5'-GCCCAACTCACTCATTTAGA-3', 15985-TR: 5'-TTCCTGACGGCAATCGACAC-3', 15985-GR: 5'-GGGCAAAGTTTTAACGTGGC-3' are provided. The invention also provides a kit and a detection method based on the primer group. The primer group provided by the invention can detect whether the individual material of the cotton to be detected contains the MON15985 transformant or not, and identify the pure heterozygosis state of a specific individual in the variety, so that the primer group is suitable for detecting the degree of homozygosity and selecting the breeding seeds.
Description
Technical Field
The invention belongs to the technical field of molecular biology, and particularly relates to a primer group, a kit and a method for pure heterozygous identification of a cotton MON15985 transformant.
Background
The transgenic cotton is one of main transgenic crops planted globally and is also the transgenic crop with the largest planting area in China. In 2016, the transgenic cotton planting area in China is 280 ten thousand hectares, and accounts for about 95 percent of the total planting area of the cotton.
"Baoling cotton II" (Bollgard II, also known as Baoling cotton MON15985) is an insect-resistant cotton product developed by Monsanto corporation, which was first approved for commercial planting in the United states in 2002 and then approved for consumption or planting in countries such as Australia, Japan, New Zealand, the European Union. Chinese approval was obtained in 2006 to allow import for use as a processing feedstock (Nongji 'an's letter (2006) No. 052).
Monsanto inserted cry1Ac gene into the genome of cotton variety Coker 312 by Agrobacterium-mediated transformation to obtain cotton boll MON 531. And the cotton boll MON15985 is obtained by retransformation by a gene gun method and inserting cry2Ab gene into the genome of cotton boll MON 531. Cotton boll MON15985 therefore contains two different transgene insertion sites.
The transgenic trait is the same as other biological traits, and can be determined to be homozygous or heterozygous according to the segregation condition in the progeny generated by selfing or crossing. In the breeding process, obtaining homozygote is a very critical step and is also an important factor for ensuring the seed quality. The traditional method for distinguishing the pure heterozygosis of the transgenic plants is carried out by detecting whether the first filial generation generated by self-crossing is separated and calculating the separation ratio, so that the time and the labor are consumed, and the precious resources of the first filial generation are wasted. The homozygous plant can be quickly and efficiently screened, and the breeding process of the transgenic plant can be remarkably accelerated, so that the method for identifying the pure heterozygous transgenic plant has important application value.
Identification of pure heterozygosity of transgenic plants at the molecular level the pure heterozygosity state of individual plants or individual seeds can be identified by amplifying the sequence of the transgene insertion site and the genomic sequences at both ends by PCR methods. The cotton planted at present mostly belongs to upland cotton varieties, and the genome of the upland cotton is allopetraploid, namely, the uploid consists of two diploids with high similarity. Therefore, when pure hybrids of the transgenic cotton are identified, the amplification of the genome at the insertion site is interfered by the sequence on the homologous chromosome, and the identification is difficult.
Disclosure of Invention
In view of the above problems and needs in the art, the present invention compares the difference between the cotton genomic sequence at the insertion site of the MON15985 transformant and its homologous chromosomal sequence, designs and verifies specific primer combinations, establishes a method for rapidly identifying pure heterozygosity of the MON15985 transformant, detects that the cotton material to be tested is the MON15985 transformant, and identifies the pure heterozygosity of a specific individual.
The technical scheme provided by the invention is as follows:
the invention provides a primer group for detecting a cotton MON15985 transformant, which is characterized by comprising the following three primers:
15985-GF：5’-GCCCAACTCACTCATTTAGA-3’，
15985-TR：5’-TTCCTGACGGCAATCGACAC-3’，
15985-GR：5’-GGGCAAAGTTTTAACGTGGC-3’。
the 3 primers are adopted to amplify the genome DNA of the cotton material to be detected, and electrophoresis detection is carried out on the amplified primers, so that whether the cotton material to be detected contains the MON15985 transformant or not and the pure heterozygous type of the MON15985 transformant can be visually and quickly identified through electrophoresis band display; the conventional qualitative detection method for the MON15985 transformant in the prior art can only detect whether the MON15985 transformant contains, but can not further know the pure heterozygous type of the MON15985 transformant; the conventional means for knowing the pure heterozygous type of the MON15985 transformant is to judge whether the cotton is homozygous or heterozygous through the segregation condition in progeny generated by selfing or hybridizing cotton materials, and the method is obviously tedious, time-consuming and labor-consuming. By adopting the primer group, DNA can be extracted by sampling at any stage of cotton plant growth, whether the MON15985 transformant exists or not and a pure heterozygous type can be quickly and conveniently obtained by utilizing PCR and electrophoresis, time and labor are saved, and the method has remarkable progress. Moreover, the invention also verifies the accuracy of the primer group and the related PCR detection of the MON15985 transformant, and finds that the result of the PCR qualitative detection adopting the primer group is completely consistent with the pure heterozygous type of the MON15985 transformant obtained by the conventional selfing and hybridization methods, so that the primer group not only can creatively and rapidly detect the pure heterozygous type of the MON15985 transformant by a molecular biological method, but also can ensure very high accuracy.
As is well known to those skilled in the art, the above primers can be prepared by artificial sequence synthesis, and can be present in the form of a powder or a solution.
In a second aspect, the invention provides a kit for pure heterozygous identification of cotton MON15985 transformant, which is characterized by comprising the primer group. The kit based on the primer group can also solve the problem of qualitative detection of the MON15985 transformant and the pure heterozygous type thereof in the cotton material, and obtain the same effect.
In a further embodiment, the kit further comprises conventional reagents for performing PCR amplification, and/or conventional reagents for performing electrophoretic detection.
Specifically, the conventional reagents for performing PCR amplification include: dNTP, DNA polymerase, PCR buffer solution, ultrapure water, or a commercial PCR reaction mixture containing the above substances; the conventional reagents for performing electrophoretic detection include: electrophoresis buffer solution, nucleic acid dye and agarose gel;
further, the conventional reagent for performing electrophoresis detection also includes a DNA Marker; the nucleic acid dye is ethidium bromide; the commercial PCR reaction mixed solution is Go
GreenMaster Mix。
Based on the primer group and the kit, the method comprising the steps can quickly and easily know whether the MON15985 transformant exists or not and the specific pure heterozygous type of the MON15985 transformant from the cotton material.
In a third aspect of the invention, there is provided a method for pure heterozygous identification of a cotton MON15985 transformant, comprising: and carrying out PCR amplification on the DNA of the cotton material to be detected by adopting the primer group and/or the kit.
Specifically, the reaction system of the PCR amplification is as follows: go
0.5 muL/muL of GreenMaster Mix, 0.5 mumol/L of primer, 0.1 muL/muL of DNA template of the cotton material to be detected and the balance of ultrapure water;
reaction procedure for the PCR amplification: pre-denaturation at 95 deg.C for 4min, denaturation at 95 deg.C for 30s, annealing at 56 deg.C for 30s, and extension at 72 deg.C for 30s for 1 cycle, 35 cycles in total, and keeping the temperature at 72 deg.C for 10 min.
Further, the method further comprises: and carrying out electrophoresis detection on the PCR amplification result, and judging the electrophoresis detection result.
More specifically, the electrophoresis refers to: placing the PCR amplification product on agarose gel with the mass-volume ratio of 0.8%, and carrying out electrophoresis at the voltage of 120V for 20 minutes;
the interpretation electrophoresis detection result refers to: if the electrophoresis result only shows a 225bp strip, the cotton material sample to be detected contains a homozygous MON15985 transformant;
if the electrophoresis result shows that the 225bp and 153bp bands simultaneously appear, the cotton material sample to be detected contains the heterozygous MON15985 transformant;
if the electrophoresis result shows that only 153bp bands appear, the cotton material sample to be tested does not contain the MON15985 transformant.
The fourth aspect of the invention provides application of the primer group in preparation of a cotton MON15985 transformant detection reagent, which is characterized in that 3 primers shown in SEQ NO. ID 1-3 are placed in a commodity packaging box marked with the application of cotton MON15985 transformant detection.
The invention discloses a rapid and efficient pure heterozygous detection method by analyzing the sequence information of the insertion sites of the cry1Ac gene and the cry2Ab gene which are disclosed, wherein the insertion sites are respectively located on chromosome 5 and chromosome 13 of the upland cotton genome, and meanwhile, the invention researches the insertion on the chromosome 13 of the cry2Ab gene (namely the MON15985 transformant mentioned herein).
A large number of experiments prove that the pure heterozygous type of the cotton MON15985 transformant obtained by detection of the primer group/kit/method and the pure heterozygous distribution ratio of the randomly extracted selfed seeds are highly matched with the selfing separation ratio of 1: 2: 1; in addition, the results of pure heterozygous identification of the same batch of selfed seeds by simultaneously adopting the primer group/kit/method disclosed by the invention and the conventional method are compared, and the results of the two are highly consistent, so that the accuracy of the primer group/kit/method disclosed by the invention in identifying the pure heterozygous state of the MON15985 transformant is up to 100%, the method is not limited by the growth stage of the plant, and the pure heterozygous type of the MON15985 transformant can be obtained by sampling at any growth stage of the plant. Therefore, the method provides convenience for specific identification of the cotton MON15985 transformant, and has the advantages of rapidness, accuracy, low required experimental conditions and very high practical value.
Drawings
FIG. 1 is a schematic representation of the primer positions for PCR detection of the MON15985 transformant.
FIG. 2 is a schematic diagram showing the design of identifying primers 15985-GF and 15985-GR; a, picture A: 15985-GF; and B, drawing: 15985-GR; wherein, 1: MON15985 transformant genomic sequence; 2: cotton chr13 chromosome sequence; 3: cotton chr14 chromosome sequence.
FIG. 3.MON15985 transformant detection method validation profile; wherein, lanes 1-3: homozygous single plant of MON15985 transformant; 4-6: MON15985 transformant heterozygous individual; 7-9: single strain without MON15985 transformant; 10-12: blank control; m: molecular weight marker DL 2000.
FIG. 4 is a single seed identification map; wherein, lanes 1-24: 24 seeds; m: molecular weight marker DL 2000.
Detailed Description
The invention will be further illustrated with reference to the following specific examples. These examples are intended to illustrate the invention and are not intended to limit the scope of the invention. Experimental procedures without specific conditions noted in the following examples, generally followed by conventional conditions, such as molecular cloning of Sambrook et al: the conditions described in the Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 2001), or the conditions as recommended by the manufacturer of the apparatus or reagents.
Reagent
Molecular biological reagents such as dNTPs, Taq DNA polymerase, DL2000Marker and the like are purchased from Dalibao bioengineering, Inc.
Other biochemical reagents are imported split charging or domestic analytical purification. The primers were synthesized by Beijing Sanbo Biotechnology, Inc.
Laboratory apparatus
A PCR amplification instrument: model S1000(Bio-Rad Co., Ltd.)
Nucleic acid electrophoresis apparatus: DYY-III type nucleic acid electrophoresis apparatus (Beijing Liuyi instrument factory)
Gel imaging system: BiospecrumAC (UVP Co., Ltd.)
Other instruments include: centrifuge, constant temperature heating plate, electronic balance, incubator, etc.
Sources of biological material
The cotton variety Ji Cotton 169 (the producer: Jinnantianren seeds Co., Ltd., production lot: 20161105, approval number: national Cotton check 2010001) used in the experimental examples of the present invention was purchased from the Cotton seed market in Jiangsu province in the early 2017. The homozygous material and the heterozygous material of the MON15985 transformant are obtained by single plant screening identification, selfing propagation and the like after greenhouse planting in plant protection research institute of Chinese agricultural academy of sciences.
The present group of embodiments provides a primer set for pure heterozygous identification of cotton MON15985 transformant. All embodiments of this group have the following common features: the primer group comprises the following three primers:
15985-GF：5’-GCCCAACTCACTCATTTAGA-3’，
15985-TR：5’-TTCCTGACGGCAATCGACAC-3’，
15985-GR：5’-GGGCAAAGTTTTAACGTGGC-3’。
based on the above 3 specific primers and the technical purpose that the above primer set is "for pure heterozygous identification of cotton MON15985 transformant", it is well known in the art that the basic function of the "primers" is to perform PCR amplification, so that the skilled person can reasonably adopt the conventional PCR detection in the art to solve the technical problem of the present invention and obtain the expected technical effect.
The 3 primers can be prepared by artificial synthesis.
This group of embodiments provides a kit for pure heterozygous identification of cotton MON15985 transformants. All embodiments of this group have the following common features: the kit comprises a primer set as described in any of group 1 embodiments.
In a further embodiment, the kit further comprises conventional reagents for performing PCR amplification, and/or conventional reagents for performing electrophoretic detection. One skilled in the art can rationally select reagents conventional in the art for PCR amplification and/or electrophoretic reagents for the purpose of "identifying pure heterozygous type of MON15985 transformant" based on the teachings herein. For example:
in specific embodiments, the conventional reagents for performing PCR amplification include: dNTP, DNA polymerase, PCR buffer solution, ultrapure water, or a commercial PCR reaction mixture containing the above substances; the conventional reagents for performing electrophoretic detection include: electrophoresis buffer, nucleic acid dye and agarose gel. Each of the above reagents has technical meanings conventionally understood by those skilled in the art, and is commercially available. The skilled person can make routine adjustments and substitutions to the above-mentioned reagents in order to obtain better results of PCR amplification or electrophoresis detection.
In more specific embodiments, the conventional reagents for performing electrophoretic assaysAlso comprises a DNA Marker; the DL2000Marker used in the DNA Marker herein can, of course, be substituted for other DNA markers commonly used in the art. The nucleic acid dye is ethidium bromide, and can also replace other nucleic acid dyes commonly used in the field, such as Gel Green and Gel Red; the commercial PCR reaction mixed solution is Go
GreenMaster Mix. Other common PCR Mix products can also be selected by those skilled in the art.
This group of examples provides a method for pure heterozygous identification of cotton MON15985 transformants. In all embodiments of this group, the method has the following common features: the method comprises the following steps: and (2) carrying out PCR amplification on the DNA of the cotton material to be detected by adopting the primer group in any one of the group 1 embodiment and/or the kit in any one of the group 2 embodiment. Those skilled in the art can perform virtual alignment according to the primer sequences provided by the present invention, thereby predicting possible electrophoretic band results based on the virtual results of the sequences, and thus interpreting the detection meaning represented by the electrophoretic results showing different size bands.
In a specific embodiment, the reaction system for PCR amplification is: go
0.5 muL/muL of GreenMaster Mix, 0.5 mumol/L of primer, 0.1 muL/muL of DNA template of the cotton material to be detected and the balance of ultrapure water; regarding the reaction system, the skilled person can routinely adjust it for the purpose of obtaining better PCR results, for example, in the following experimental example 2, a set of most preferred reaction systems is used: go
GreenMaster Mix 10. mu.L, primers 757-GF, 757-GR and 757-TR (10. mu. mol/L) each 1. mu.L, DNA template 2. mu.L, and ultrapure water was supplemented to a total volume of 20. mu.L. Reaction procedure for the PCR amplification: pre-denaturation at 95 ℃ for 4min, denaturation at 95 ℃ for 30s, 56Annealing at 72 deg.C for 30s, extending at 72 deg.C for 30s for 1 cycle, performing 35 cycles, and maintaining at 72 deg.C for 10 min.
In a further embodiment, the method further comprises: and carrying out electrophoresis detection on the PCR amplification result, and judging the electrophoresis detection result.
In some embodiments, the electrophoresis refers to: placing the PCR amplification product on agarose gel with the mass-volume ratio of 0.8%, and carrying out electrophoresis at the voltage of 120V for 20 minutes;
the interpretation electrophoresis detection result refers to: if the electrophoresis result only shows a 225bp strip, the cotton material sample to be detected contains a homozygous MON15985 transformant; if the electrophoresis result shows that the 225bp and 153bp bands simultaneously appear, the cotton material sample to be detected contains the heterozygous MON15985 transformant; if the electrophoresis result shows that only 153bp bands appear, the cotton material sample to be tested does not contain the MON15985 transformant.
The method provided by the group of embodiments can refer to the specific operation described in experimental example 2 in the operation of each step or link.
Example of group 4, application of primers of the present invention
This group of embodiments provides use of a primer set as described in any of group 1 embodiments in the preparation of a test agent for cotton MON15985 transformant. All embodiments of this group share the following common features: the use comprises the following steps: 3 primers shown in SEQ NO. ID 1-3 were placed in a commercial package labeled for detection of cotton MON15985 transformant.
It is within the scope of the present invention to use the primer sets of the present invention to detect the behavior of the MON15985 transformant on any scale sold for commercial purposes.
Experimental example 1 primer design
The key point of the invention is the design of the forward primer and the reverse primer on the cotton genome of the MON15985 transformant.
The insertion site of the MON15985 transformant was found to be located on chromosome 13 of the cotton variety TM-1 genome (https:// www.ncbi.nlm.nih.gov/genome/.
Sequence 15985-GF was retrieved by aligning the cotton genome sequence upstream of the insertion site of the MON15985 transformant with the chr14 chromosomal sequence: 5'-GCCCAACTCACTCATTTAGA-3' differs from chr14 by 3 bases (FIG. 2A), and was therefore used as the forward genomic primer.
In the same way, the cotton genome sequence downstream of the insertion site of the MON15985 transformant was aligned with the chr14 chromosome sequence one by one, and the sequence 15985-GR: 5'-GGGCAAAGTTTTAACGTGGC-3' differs from chr14 by 2 bases (FIG. 2B) and was therefore used as the reverse genomic primer.
The invention designs primers on the genome at two sides of the insertion site of the MON15985 transformant, and designs primers on an insertion sequence, wherein the three primers are as follows:
15985-GF：5’-GCCCAACTCACTCATTTAGA-3’，
15985-TR：5’-TTCCTGACGGCAATCGACAC-3’，
15985-GR: 5'-GGGCAAAGTTTTAACGTGGC-3', as shown in fig. 1.
Wherein the 15985-GF/15985-TR amplification region is a connecting region of a 5' end transgenic sequence of the MON15985 transformant and a cotton genome, and the 15985-GF/15985-TR amplification sequence of the MON15985 transformant is 225bp, and is shown as Seq ID No. 4; 15985-GF/15985-GR amplified fragment is a cotton genome sequence, is located at 34995391-34995543 of the chr13 chromosome of the genome of the cotton variety TM-1 (Gossypium hirsutum), and has an amplified sequence of 153 bp. Whereas in the genome containing the MON15985 transformant, the fragment between the primers at the insertion site is a region including the entire insert, which is expected to exceed 10kb, such a long fragment cannot be amplified under ordinary PCR conditions; thus, only 153bp amplification products of the cotton genome sequence, as shown in Seq ID No.5, were obtained in samples without the MON15985 transformant;
the three primers are used together, and two bands can be amplified in a heterozygote.
Can be used for rapidly and accurately identifying the pure heterozygous state of the MON15985 transformant in a field cotton single plant or single cotton seed by using a conventional PCR method.
Experimental example 2 qualitative PCR detection method of Cotton MON15985 transformant
Experimental methods and procedures
1. Sample pretreatment
The cotton material planted in the greenhouse is obtained by respectively taking single plant leaves.
2. Cotton genome DNA extraction
The extraction of total DNA from cotton material was carried out according to the manual of plant DNA extraction kit (TIANGEN Biotech Co., Ltd.). Mu.l of the extracted DNA solution was subjected to electrophoresis on a 0.8% agarose gel, and the quality of the extracted DNA was preliminarily judged based on the brightness and band pattern. The concentration and purity of the extracted DNA were determined using an ultraviolet spectrophotometer.
3. Detection of greenhouse planted single plant cotton material
The established qualitative PCR detection method of the MON15985 transformant consisting of 3 primers 15985-GF (genome forward primer), 15985-GR (genome reverse primer) and 15985-TR (insert reverse primer) is adopted to select the greenhouse cotton material single strains which are homozygous, heterozygous and do not contain the MON15985 transformant for verification.
And (3) PCR reaction system: go
10 μ L of GreenMaster Mix, 1 μ L of each of primers 15985-GF, 15985-GR and 15985-TR (10 μmol/L), 2 μ L of DNA template, and a total volume of 20 μ L of ultrapure water. Reaction procedure: pre-denaturation at 95 ℃ for 4min, denaturation at 95 ℃ for 30s, annealing at 56 ℃ for 30s, extension at 72 ℃ for 30s, amplification reaction for 35 cycles, and heat preservation at 72 ℃ for 10 min.
In the MON15985 transformant heterozygous sample, three primers respectively amplify the cotton genome sequence between the insertion sites and the 5' junction region sequence of the insertion sequence, thereby obtaining two amplification products of 225 and 153 bp;
in the homozygous samples of the MON15985 transformant, only 225bp amplification products of 15985-GF (genome forward primer) and 15985-TR (insert reverse primer) amplification, which are sequences of the 5' joining region of the insert, were obtained; while the whole region of the included insert (MON15985 transformant) between the forward and reverse primers of the genome is expected to exceed 10kb, and amplification cannot be obtained under the common PCR condition;
in contrast, only 153bp amplification products of the cotton genomic sequence, namely 15985-GF (genome forward primer) and 15985-GR (genome reverse primer) amplification products, were obtained in samples not containing the MON15985 transformant.
The validation results showed that only one 225bp fragment was amplified from homozygous MON15985 transformant plants (lanes 1-3), both 225bp and 153bp bands from heterozygous plants (lanes 4-6), and only one 153bp band from plants without MON15985 transformant (lanes 7-9), which is completely consistent with the expected results, as shown in fig. 3.
Therefore, the qualitative PCR method and the primer set of the MON15985 transformant established by the invention can effectively identify the MON15985 transformant and the existence state thereof.
4. Qualitative PCR detection of MON15985 transformant in cotton seed sample
Randomly extracting 24 seeds of the heterozygous single plant Ji cotton 169 and adopting the established MON15985 transformant specificity qualitative PCR method for identification (figure 4). The results showed that the MON15985 transformant was homozygous, heterozygous and null in 24 seeds of 5, 13 and 6 seeds, respectively. The pure heterozygosity separation ratio of the randomly extracted 24 selfed seeds is 5: 13: 6, and the pure heterozygosity separation ratio basically accords with the genetic separation ratio of the selfing of the MON15985 transformant heterozygote of 1: 2: 1, so that the accuracy of the primer group/kit/method in the pure heterozygosity identification aspect can be proved to be higher.
The method and kit of the invention can accurately identify whether a single cotton material contains the MON15985 transformant, identify the pure heterozygous state of the MON15985 transformant, and can also be used for identifying the proportion of homozygous or heterozygous individuals in a population.
Experimental example 3 verification of identification accuracy of primer set/kit/method of the present invention
The experimental example verifies the accuracy of the primer group/kit in the pure heterozygous identification of the MON15985 transformant, and the method comprises the following steps: the result of identifying the pure heterozygous type of the MON15985 transformant by the primer group PCR of the invention is compared with the prior conventional means: the results of determining the pure heterozygous type of the MON15985 transformant through segregation in cotton material selfing or progeny produced by crossing are compared.
The specific operation is as follows: sowing seeds of a cotton variety Ji cotton 169, naming the seeds as T1 generation seeds, and numbering each seed; the conventional method needs to harvest self-bred cotton seeds (named as 'T2 generation seeds') after the batch of cotton plants grow up and mature, observe and record the phenotype separation condition (total number of no less than 16 single seeds) of the T2 generation seeds to judge the phenotype of the corresponding T1 seeds. If all the T2 generation seeds are MON15985 transformants, the corresponding T1 generation seeds are homozygous MON15985 transformants; corresponding T1 generation seeds are heterozygous MON15985 transformants if T2 generation seeds partially harbor MON15985 transformants and partially do not contain MON15985 transformants; if all of the T2 seeds did not contain the MON15985 transformant, the corresponding T1 seeds were negative for the MON15985 transformant.
By adopting the primer/kit/method, the pure heterozygous type of the MON15985 transformant of each seed can be identified only by obtaining the DNA extracted from the leaves of the seedling after the ' Ji cotton 169 ' heterozygous single plant selfing seed (T1 generation seed) ' is sown and the seedling is grown out, and the result obtained by identifying each numbered seed through the method is recorded.
Compared with the conventional method, the method of the invention has completely consistent pure heterozygous identification result of the MON15985 transformant, which shows that the accuracy of the primer group/kit/method for pure heterozygous identification of the MON15985 transformant is 100%.
SEQUENCE LISTING
<110> institute of plant protection of Chinese academy of agricultural sciences
<120> primer group, kit and method for pure heterozygous identification of cotton MON15985 transformant
<130> P180033/ZBS
<160> 5
<170> PatentIn version 3.5
<210> 1
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> 15985-GF
<400> 1
<210> 2
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> 15985-TR
<400> 2
<210> 3
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> 15985-GR
<400> 3
<210> 4
<211> 225
<212> DNA
<213> Artificial Sequence
<220>
<223> MON15985 transformant primer positive amplification sequence
<400> 4
gcccaactca ctcatttaga cattttcctt gctttgagcc ttagtaccaa caaccctact 60
ttgatcaagg taagtgagtt cctcttggtt gtcgagtttt tatcaatggt ggagccttgc 120
taatacggtt atcaaatgta gtttcacagt ggtgccgatt ctctttcttc tttctatagt 180
ggtctccctt tttaatctct tctttgtgtc gattgccgtc aggaa 225
<210> 5
<211> 153
<212> DNA
<213> Artificial Sequence
<220>
<223> MON15985 transformant primer negative amplification sequence
<400> 5
gcccaactca ctcatttaga cattttcctt gctttgagcc ttagtaccaa caacaaccca 60
tttcaaatga atactttccc gcccggcctg ttgactacca ctaggcccta tagtcccttt 120
tttattcccc tgggccacgt taaaactttg ccc 153
Claims (10)
1. The primer group for pure heterozygous identification of the cotton MON15985 transformant is characterized by consisting of the following three primers:
15985-GF：5’-GCCCAACTCACTCATTTAGA-3’，
15985-TR：5’-TTCCTGACGGCAATCGACAC-3’，
15985-GR：5’-GGGCAAAGTTTTAACGTGGC-3’。
2. a kit for pure heterozygous identification of cotton MON15985 transformant comprising the primer set of claim 1.
3. The kit of claim 2, further comprising conventional reagents for performing PCR amplification and/or conventional reagents for performing electrophoretic detection.
4. The kit according to claim 2 or 3, wherein the conventional reagents for performing PCR amplification comprise: dNTP, DNA polymerase, PCR buffer solution, ultrapure water, or a commercial PCR reaction mixture containing the above substances;
the conventional reagents for performing electrophoretic detection include: electrophoresis buffer, nucleic acid dye and agarose gel.
5. The reagent according to claim 4The kit is characterized in that the conventional reagent for electrophoresis detection also comprises a DNA Marker; the nucleic acid dye is ethidium bromide; the commercial PCR reaction mixed solution refers to
GreenMaster Mix。
6. A method for pure heterozygous identification of a cotton MON15985 transformant, comprising: performing PCR amplification on DNA of a cotton material to be detected by using the primer group of claim 1 and/or the kit of any one of claims 2 to 5.
7. The method of claim 6, wherein the reaction system of the PCR amplification is:
0.5 muL/muL of GreenMaster Mix, 0.5 mumol/L of primer, 0.1 muL/muL of DNA template of the cotton material to be detected and the balance of ultrapure water;
reaction procedure for the PCR amplification: pre-denaturation at 95 deg.C for 4min, denaturation at 95 deg.C for 30s, annealing at 56 deg.C for 30s, and extension at 72 deg.C for 30s for 1 cycle, 35 cycles in total, and keeping the temperature at 72 deg.C for 10 min.
8. The method of claim 6 or 7, further comprising: and carrying out electrophoresis detection on the PCR amplification result, and judging the electrophoresis detection result.
9. The method of claim 8, wherein the electrophoresis refers to: placing the PCR amplification product on agarose gel with the mass-volume ratio of 0.8%, and carrying out electrophoresis at the voltage of 120V for 20 minutes;
the interpretation electrophoresis detection result refers to: if the electrophoresis result only shows a 225bp strip, the cotton material sample to be detected contains a homozygous MON15985 transformant;
if the electrophoresis result shows that the 225bp and 153bp bands simultaneously appear, the cotton material sample to be detected contains the heterozygous MON15985 transformant;
if the electrophoresis result shows that only 153bp bands appear, the cotton material sample to be tested does not contain the MON15985 transformant.
10. The use of the primer set of claim 1 in the preparation of a test agent for cotton MON15985 transformant detection, wherein 3 primers shown in SEQ No. id 1-3 are placed in a commercial package labeled for cotton MON15985 transformant detection.
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