CN116103326B - Plasmid standard molecule for specificity detection of transgenic soybean strain and application thereof - Google Patents

Plasmid standard molecule for specificity detection of transgenic soybean strain and application thereof Download PDF

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CN116103326B
CN116103326B CN202210999568.3A CN202210999568A CN116103326B CN 116103326 B CN116103326 B CN 116103326B CN 202210999568 A CN202210999568 A CN 202210999568A CN 116103326 B CN116103326 B CN 116103326B
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transgenic soybean
strain
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plasmid
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CN116103326A (en
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曹际娟
胡冰
郑秋月
刘军山
朴永哲
李想
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Dalian Minzu University
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
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    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/686—Polymerase chain reaction [PCR]
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
    • C12Q1/6895—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for plants, fungi or algae
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    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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Abstract

The invention relates to the technical field of biology, and provides a plasmid standard molecule for detecting the specificity of a transgenic soybean strain and application thereof. The plasmid standard molecule comprises a fusion fragment of a strain-specific boundary sequence for identifying transgenic components of 17 transgenic soybeans, wherein the sequence of the fusion fragment of the strain-specific boundary sequence is shown as SEQ ID NO: 1. Experiments prove that the plasmid standard molecule provided by the invention can be simultaneously suitable for the identification of 17 transgenic soybean strains, and can be commercially popularized and applied.

Description

Plasmid standard molecule for specificity detection of transgenic soybean strain and application thereof
Technical Field
The invention relates to the technical field of biology, in particular to a plasmid standard molecule for detecting the specificity of a transgenic soybean strain and application thereof.
Background
Soybean is not only a major source of oil crops and vegetable proteins in humans, but also an important industrial raw material, and plays an important role in food safety and national economy. In the last decade, the cultivation of transgenic plants breaks through, a batch of new varieties are provided, obvious effects are exerted in improving crop production, and huge potential is also revealed. However, there is a great deal of debate about the safety of transgenic crops. Transgenic soybean lines are numerous, some lines are approved for entering the environment in China, but the entering of the environment still needs to be declared and approved for specific lines; some strains such as DAS81419-2 and DAS68416 have not been approved for import in China. The transgenic crop strain facing the unauthorized entry has important practical significance for detecting and identifying the transgenic soybean strain, and the transgenic crop strain is safe to grains, safe to seeds, ecological environment and possible risks brought by consumers.
The identification of transgenic soybean strain is an important detection item for the detection and identification of transgenic products, and relates to grain safety and seed safety in China. The method for detecting the transgenic soybean strain by using the real-time fluorescence quantitative Polymerase Chain Reaction (PCR) comprises 17 kinds of identification of the transgenic soybean strain in GB/T19495.4-2018 'method for detecting the transgenic product by using the real-time fluorescence qualitative Polymerase Chain Reaction (PCR)' method for detecting the transgenic product 'by using the GB/T19495.5-2018'. However, at present, only a few single molecular standard samples of 1-4 transgenic soybean lines exist, but the single or few transgenic soybean line molecular standard samples have low use efficiency and small line number, and the requirement of simultaneously identifying 17 transgenic soybean lines cannot be met, so that the efficiency in the practical application of identifying the transgenic soybean lines is affected.
Therefore, it is highly desirable to provide a standard molecule capable of simultaneously being applied to the specific detection of 17 transgenic soybean lines, so as to meet the requirement of simultaneously identifying 17 transgenic soybean lines based on real-time fluorescence PCR, thereby improving the identification efficiency of the transgenic soybean lines.
Disclosure of Invention
The invention aims to provide a plasmid standard molecule which can be simultaneously applied to the specificity detection of 17 transgenic soybean lines so as to meet the requirement of simultaneously identifying 17 transgenic soybean lines, thereby improving the identification efficiency of the transgenic soybean lines.
In a first aspect of the embodiment of the invention, a recombinant plasmid is provided, wherein the recombinant plasmid can be simultaneously applicable to real-time fluorescence PCR qualitative and quantitative detection for identifying 17 transgenic soybean lines, and comprises fusion fragments for identifying 17 transgenic soybean line specific boundary sequences, and the sequences of the fusion fragments are shown as SEQ ID NO: 1.
In the embodiment of the invention, the fusion fragment comprises the following 17 boundary sequence gene fragments specific to transgenic soybean lines: 305523, MON89788, CV127, MON87769, DP-356043-5, MON87701, MON87705, DAS-68416-4, DAS-44406-6, MON87751, MON87708, DAS81419-2, A2704-12, FG72, A5547-127, GTS40-3-2 and SYHT0H2, having the sequences set forth in SEQ ID NO:2 to 18.
The strain-specific border sequence refers to a sequence linked to the plant genome and transgene, and may be referred to as a target sequence.
The fusion fragment provided by the embodiment of the invention is obtained by splicing the specific boundary sequence gene fragments of the 17 transgenic soybean strains sequentially according to the following splicing sequence (from 5 'end to 3') of: 305523, MON89788, CV127, MON87769, DP-356043-5, MON87701, MON87705, DAS-68416-4, DAS-44406-6, MON87751, MON87708, DAS81419-2, A2704-12, FG72, A5547-127, GTS40-3-2, and SYHT0H2.
In a preferred embodiment, the fusion fragment of the line-specific border sequence has a sequence length of 1768bp. The structure of the recombinant plasmid provided by the embodiment of the invention, which comprises the fusion fragments of the 17 transgenic soybean strain specific border sequences, is shown in figure 1.
In a preferred embodiment, the fusion fragment of the line-specific border sequence is prepared by the steps of:
extracting 17 transgenic soybean strain specific boundary sequence gene fragments; splicing the 17 transgenic soybean strain specific boundary sequence gene fragments according to a preset fragment splicing sequence to obtain spliced sequence fragments; converting the spliced sequence fragment into escherichia coli to realize cloning conversion, so as to obtain converted escherichia coli; culturing the transformed escherichia coli to obtain transformed escherichia coli colonies; screening the colony of the transformed escherichia coli for a sequence shown as SEQ ID NO:1, and a fusion fragment of a line-specific border sequence as set forth in seq id no.
In the embodiment of the invention, the recombinant plasmid can be prepared by inserting the fusion fragment of the strain-specific border sequence into the multiple cloning site of any commercial plasmid vector.
In a preferred embodiment, the recombinant plasmid is prepared by inserting (e.g., pMD 19-T) a fusion fragment of the strain-specific border sequence into the multiple cloning site of a plasmid backbone vector.
In a second aspect of an embodiment of the present invention, there is provided a kit for detecting a transgenic soybean line, characterized in that the kit comprises a recombinant plasmid as described in the first aspect.
The recombinant plasmid provided in the first aspect or the kit provided in the second aspect of the embodiment of the invention can be simultaneously suitable for identifying 17 transgenic soybean strains, so that the identification efficiency is greatly improved.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings used in the embodiments or the description of the prior art will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.
FIG. 1 is a schematic structural diagram of a recombinant plasmid comprising fusion fragments of 17 transgenic soybean line-specific border sequences provided in example 1 of the present invention;
FIG. 2 is a schematic view of splice sequencing of fusion fragments comprising 17 transgenic soybean line-specific border sequences provided in example 1 of the present invention;
FIG. 3 is a real-time fluorescence PCR identification result of 17 transgenic soybean lines simultaneously identified by using the plasmid standard molecules provided in example 2 of the present invention;
FIG. 4 is a graph showing the results of real-time fluorescent PCR assays for providing line specificity of other transgenic crop plants according to the examples of the present invention.
Detailed Description
In order to make the technical problems, technical schemes and beneficial effects to be solved more clear, the invention is further described in detail below with reference to the embodiments. It should be understood that the specific examples described herein are for purposes of illustration only and that the embodiments of the invention are not limited thereto.
Unless defined otherwise, technical terms used in the following examples have the same meaning as commonly understood by one of ordinary skill in the art to which the present invention pertains. The experimental reagents used in the following examples are all conventional biochemical reagents unless otherwise specified; the dosage of the experimental reagent is the dosage of the reagent in the conventional experimental operation if no special description exists; the experimental methods are conventional methods unless otherwise specified. Unless otherwise indicated, all experimental equipment, materials, reagents, etc. used in the examples of the present invention are commercially available.
The real-time fluorescence PCR technology is a gold standard which is widely accepted at home and abroad and is used for detecting transgene rapidly and accurately. GB/T19495.4-2018 (real-time fluorescence qualitative Polymerase Chain Reaction (PCR) detection method for detecting transgenic products) and GB/T19495.5-2018 (real-time fluorescence quantitative Polymerase Chain Reaction (PCR) detection method for detecting transgenic products) are published in China, wherein 17 methods for identifying transgenic soybean strains are included, but target fragment DNA sequences related to strain identification specific fragments are not published, and particularly a molecular standard sample matched with the standard is lacked. Furthermore, the DNA sequences involved in the recombinant construct inserted into the soybean genome and the border sequences flanking the insertion site leading to a superior soybean line are the only targets for line identification whose DNA sequences are secret information of seed development companies and are not externally published. The above reasons make it very difficult to prepare a molecular standard sample for simultaneously identifying the specificity of 17 transgenic soybean lines, but only a few single molecular standard samples of 1-4 transgenic soybean lines are provided, so that the molecular standard sample for simultaneously identifying the specificity of 17 transgenic soybean lines is not available at home and abroad, and inconvenience is brought to the identification of 17 transgenic soybean lines in grains, seeds and agricultural products in China. The standard sample is used as a measuring tool in the detection industry, and plays an indispensable role in the fields of calibrating measuring instruments and devices, evaluating analysis methods, quality control in the detection process, evaluating the operation technical level of analysts and the like. There is currently much research on molecular standard samples of transgenic soybean single lines, but there is little or no research on molecular standard samples for simultaneous identification of 17 transgenic soybean lines.
In the subsequent research process, the inventor finds that the single or few transgenic soybean strain molecular standard samples have low use efficiency and small strain quantity, can not meet the requirement of simultaneously identifying 17 transgenic soybean strains, and influence the efficiency in the practical application of identifying the transgenic soybean strains. Therefore, research and development of a molecular standard sample for simultaneously identifying 17 transgenic soybean lines are performed to realize simultaneous identification of 17 transgenic soybean lines, so that the identification efficiency is improved, and the method has important significance for further promoting research and development of specificity identification of the transgenic soybean lines.
The invention takes the commercialized 17 transgenic soybean strains as raw materials, obtains the 17 transgenic soybean strain specific boundary sequence gene fragments through PCR analysis and gene sequencing, then splices the strain specific boundary sequence gene fragments through a molecular splicing technology to form a section of fusion fragment of strain specific boundary sequence, synthesizes plasmid artificially, extracts plasmid DNA, and performs linearization treatment to obtain plasmid standard molecules which can be simultaneously applied to 17 transgenic soybean strains of real-time fluorescence PCR detection 305523, MON89788, CV127, MON87769, DP-356043-5, MON87701, MON87705, DAS-68416-4, DAS-44406-6, MON87751, MON87708, DAS81419-2, A2704-12, FG72, A5547-127, GTS40-3-2 and SYHT0H2. The plasmid standard molecules (recombinant plasmids) included fusion fragments of line-specific border sequences of transgenic soybean 305523, MON89788, CV127, MON87769, DP-356043-5, MON87701, MON87705, DAS-68416-4, DAS-44406-6, MON87751, MON87708, DAS81419-2, A2704-12, FG72, A5547-127, GTS40-3-2, and SYHT0H2 lines, and the suitability of the plasmid standard molecules for use in the 17 transgenic soybean lines described above was verified. The verification result shows that the plasmid standard molecule provided by the invention can be suitable for real-time fluorescence PCR and simultaneously identify the 17 transgenic soybean strains, and can meet the requirement of 17 strain specificity detection for simultaneously identifying the transgenic soybean in national standards GB/T19495.4-2018 and GB/T19495.5-2018.
Example 1 preparation of fusion fragments for identification of 17 transgenic Soybean line specific border sequences
The method comprises the steps of taking commercialized 17 transgenic soybeans (30553, MON89788, CV127, MON87769, DP-356043-5, MON87701, MON87705, DAS-68416-4, DAS-44406-6, MON87751, MON87708, DAS81419-2, A2704-12, FG72, A5547-127, GTS40-3-2 and SYHT0H 2) as raw materials, screening, detecting, identifying, PCR analyzing and determining the sequence of a specific DNA fragment of the 17 transgenic soybeans, extracting the strain specific boundary sequence gene fragments of the 17 transgenic soybeans, carrying out sequence analysis and molecular splicing design on the 17 strain specific boundary sequence fragments, eliminating interference of homologous sequences and hairpin structures on multi-channel detection, and inserting cleavage site base sequence intervals to form a DNA sequence which is 1768bp in size and contains fusion fragments of 17 soybean specific boundary sequences, wherein the DNA sequence is shown as SEQ ID NO: 1. Wherein, the structure diagram of the recombinant plasmid containing the fusion fragment of 17 transgenic soybean strain specific boundary sequences is shown in figure 1. A schematic of the splice ordering of the border sequences of the fusion fragments comprising the 17 transgenic soybean line-specific border sequences is shown in fig. 2.
The sequence information of the specific border sequence gene fragments (target sequences) of the 17 transgenic soybean lines used in the examples of the present invention is shown in table 1 below.
TABLE 1 boundary target sequence information for specific identification of 17 transgenic soybean lines
Example 2 preparation of plasmid Standard molecules (recombinant plasmids) comprising fusion fragments for identification of specific border sequences of 17 transgenic Soybean lines
(1) Preparation of DNA fragment and cloning transformation: synthesizing single-stranded small fragment DNA according to the DNA sequence of the fusion fragment of the strain-specific border sequence prepared in the embodiment 1, and splicing the single-stranded small fragment DNA into a complete double-stranded DNA fragment by a PCR method; and then carrying out molecular ligation reaction, and converting the fusion product into escherichia coli to realize cloning transformation.
(2) Positive clone detection and sequencing: and (2) taking the escherichia coli colony in the step (2), carrying out PCR detection, detecting whether the length of the inserted fragment in the contained plasmid is 1768bp, extracting plasmid DNA from the plasmid with the length of the inserted fragment being 1768bp, and determining the sequence of the extracted plasmid DNA to ensure that the inserted sequence is completely consistent with the sequence with the length of 1768bp provided in the example 1 (namely, consistent with the sequence shown in SEQ ID NO: 1).
(3) Large amount of plasmid extraction and plasmid linearization: and extracting plasmids by using a commercial plasmid extraction kit, and then carrying out linearization treatment of single enzyme digestion, concentration measurement and copy number calculation to obtain a standard sample of the plasmid standard molecule. The standard sample has a mass concentration standard value of 0.953+ -0.162 ng/. Mu.L and a corresponding copy number standard value of 4.9X10 8 copies/μL。
Then, the plasmid linearized in the step (3) can be split into 500 sample bottles, 100. Mu.L/bottle, sealed and stored for a long period of time under the condition of-20 ℃. Randomly extracting samples for uniformity and stability test and evaluation, and ensuring that the uniformity and stability of the molecular standard samples meet the requirements of national standard samples.
Example 3 characterization of plasmid Standard molecules
Randomly extracting a sealed storage sample of the plasmid standard molecules prepared in the embodiment 2 of the invention, and identifying whether the plasmid standard molecules meet the requirement for simultaneously detecting the specificity of 17 transgenic soybean strains by simultaneously measuring the specificity characteristic values of 17 transgenic soybean strains by adopting a detection method in national standards GB/T19495.4-2018 and GB/T19495.5-2018.
The plasmid standard molecule provided in the embodiment 2 of the invention can be simultaneously suitable for the identification of 17 transgenic soybean lines through identification, and the real-time fluorescence PCR identification result is shown in figure 3. As can be seen from FIG. 3, 17 transgenic soybean lines determined simultaneously with the plasmid standard molecule provided in example 2 of the present invention were positive, and typical S-type positive amplification curves were obtained in the specific assays of 305523, MON89788, CV127, MON87769, DP-356043-5, MON87701, MON87705, DAS-68416-4, DAS-44406-6, MON87751, MON87708, DAS81419-2, A2704-12, FG72, A5547-127, GTS40-3-2 and SYHT0H2 lines.
The primer probe sequences and the boundary target sequence information of the specific identification of the 17 transgenic soybean strains adopted in the embodiment of the invention are shown in the following table 2.
TABLE 2 primer probe sequence information for specific identification of 17 transgenic soybean lines
In addition, the embodiment of the invention also selects some common other transgenic crop lines (such as a transgenic corn MON810 line, a transgenic corn MON863 line, a transgenic rape RT73 line and the like) for real-time fluorescence PCR amplification test, and the test result is shown in figure 4. As can be seen from FIG. 4, no obvious S-shaped curve was generated when the PCR amplification was performed in real time with other transgenic crop line specific primers. Therefore, the plasmid standard molecule provided by the invention does not have unexpected amplification when being practically applied to detecting 17 transgenic soybean lines, and has high specificity for identifying 17 transgenic soybean lines.
Example 4 evaluation of Performance test for homogeneity and stability of plasmid Standard molecule Standard sample
1. Uniformity test of Standard sample
Sampling and uniformity inspection are carried out by repeating the measuring method according to the random sequence in the general principle and statistical method of standard sample fixed value of standard sample working guide (3) of GB/T15000.3-2008. 15 bottles are randomly extracted from samples which are split into minimum packages (100 mu L/bottle) for uniformity test, each bottle is repeatedly tested for 2 times, ct values of 17 strain-specific fragments are measured by adopting a method in national standards GB/T19495.4-2018 and GB/T19495.5-2018, and uniformity of characteristic values in standard samples is evaluated by a single factor analysis of variance (F test). When F ratio is smaller than critical value F 0.05 (14, 15) no significant difference between the inter-unit variance and the intra-unit variance, indicating that the strain characteristic magnitudes in the standard samples are sufficiently uniform; f ratio of>Critical value F 0.05 (14, 15) the inter-cell variance was significantly different from the intra-cell variance, indicating that the strain characteristic values in the molecular standard samples were non-uniform.
The plasmid standard molecule standard sample prepared in the example 2 of the present invention was subjected to uniformity test, and the statistical analysis results of the test are shown in Table 1 below.
TABLE 1 uniformity variance analysis results
As shown in the above Table 1, the F ratio of the characteristic value of the standard molecular sample of the plasmid prepared in the embodiment 2 of the present invention is between 0.83 and 2.36, which is less than F critical value F0.05 (14, 15) =2.42, which indicates that there is no obvious difference between the bottles and the inside of the bottles under the 95% confidence probability, the uniformity of the characteristic value of the standard sample is good, and the expected use purpose of the uniform and consistent value transmission of the standard sample can be satisfied.
2. Stability test of Standard sample
1. Long term stability test
Sampling is carried out according to a random sequence repeated measurement method in GB/T15000.3-2008 general principle and statistical method of standard sample fixed value of standard sample working guide (3) so as to carry out long-term preservation stability investigation. The strain 305523 is used as a representative, and the Ct value is measured by adopting the methods of national standards GB/T19495.4-2018 and GB/T19495.5-2018. For long-term stability test, according to the sampling principle of the time interval before and after the time interval, the packaged samples selected in the embodiment are stored for a long time under the low temperature condition of minus 20 ℃, the samples are randomly selected for long-term stability test every 2 months in the first year and every 3 months in the second year, 3 standard samples are extracted each time, and the test is repeated for 2 times for each sample. The standard sample stability test method recommended according to GB/T15000.3 uses straight lines as an empirical model for statistical tests. If the Ct value of 305523 is equal to or less than the value of Ct of 30423 (0.95,n-2) ×s (b1) The transgenic soybean strain characteristic value of the molecular standard sample is not obviously changed in a trend in the stability test period, namely the stability is good.
The results of the statistical analysis of the long-term stability test performed on the molecular standard sample prepared in example 2 of the present invention are shown in Table 2 below.
Table 2 results of analysis of Linear empirical model of Long-term stability represented by strain 305523
Project Trend analysis Project Trend analysis
Result total average Y 13.439 Time average value X 10.9
Average slope b 1 0.00453 Mean intercept b 0 13.38987
Standard deviation s 0.19612 s 2 0.03846
s(b 1 ) 0.00787 │b 1 │ 0.00453
Critical value t% 0.95,n-2 ) 2.26 t( 0.95,n-2 )×s(b 1 ) 0.01780
As is clear from table 2, stability was measured as represented by 305523 strain. Through statistical test of linear empirical model, the Ct value of the transgenic strain Ct value of the plasmid standard molecular standard sample prepared in example 2 of the invention is 0.00453, which is smaller than t (0.95,n-2) ×s (b1) This indicatesThe plasmid standard molecular standard sample has good stability and meets the stability performance requirement of the national standard sample.
2. Short term stability test under transportation conditions
Sampling is carried out according to a random sequence repeated measurement method in GB/T15000.3-2008 general principle and statistical method of standard sample fixed value of standard sample working guide (3), and short-term stability investigation under transportation conditions is carried out. Considering that the short-term stability of the standard sample is mainly the influence of short-term transportation under the condition of temperature change on the characteristic value, the experimental study simulates the transportation extreme condition, and the sample is stored for a short time under the extreme condition between-20 ℃ and 37 ℃ for short-term stability study under the transportation condition. Specifically, short-term stability study is performed on a standard sample of plasmid molecules under transportation conditions selected to be within a range of-20 ℃ to 37 ℃ (-20 ℃,0 ℃ to 4 ℃,25 ℃ and 37 ℃). The standard plasmid molecular sample is placed at-20 deg.c for 7 days, 0-4 deg.c for 7 days, 25 deg.c for 7 days and 37 deg.c for 7 days before being tested. 3 bottles of standard samples (m=3, n=2) are selected under each temperature condition, and Ct values are measured by adopting the methods of national standards GB/T19495.4-2018 and GB/T19495.5-2018. Short term stability was analyzed using linear regression variance. After transport conditions, if statistic p > 0.05, regression was not significant (p <0.05 was significant for 95% confidence level), indicating no significant change in strain characteristic values for the plasmid molecular standard samples at-20 ℃ and 37 ℃ and good short term stability.
The results of the short-term stability test performed on the plasmid standard molecule standard sample prepared in example 2 of the present invention are shown in Table 3 below.
Table 3 results of short-term stability Linear regression analysis of variance represented by the 305523 strain
As can be seen from Table 3, the short-term stability was determined on behalf of the 305523 strain, and the plasmid standard molecule provided in example 2 of the present invention was subjected to the above simulated transportation conditions by linear regression analysis of variance under the 95% confidence probability, the significance p value of the 305523 strain characteristic value in the plasmid standard molecular standard sample is 0.144 and is more than 0.05, which shows that the transgene strain characteristic value of the plasmid standard molecular standard sample is not changed significantly after transportation under the transportation simulation conditions of-20 ℃ and 37 ℃, and the short-term stability is good.
3. Combined fixed value for simultaneously measuring 17 strain characteristic values of plasmid standard molecule standard sample
In the test, the 17 strain characteristic values of the plasmid standard molecules prepared in the embodiment 2 of the invention are jointly determined by 8 different test units. Specifically, 24 bottles of plasmid standard molecular standard samples which are qualified through uniformity and stability test and provided in the embodiment 2 of the invention are randomly extracted, 3 bottles of standard samples are randomly distributed to each test unit, the specific characteristic values of 17 strains are measured by each test unit through the national standard GB/T19495.4-2018 and GB/T19495.5-2018 methods, the measurement is repeated for 2 times (m=3 and n=2) for each bottle of standard samples, and the measurement value data of 8 test units are summarized, wherein the summarized result is shown in the table 4 below.
TABLE 4 characterization of 17 transgenic Soybean lines in Standard samples simultaneously with characterization of the fixed value results
As is clear from Table 4 above, there were no abnormal values (e.g., undetected values, etc.) among the 17 strain characteristic values of the plasmid standard molecule standard sample prepared in example 2 of the present invention by 8 test units, indicating that all the data passed the test. In addition, 8 test units are positive for the detection of the specific characteristic values of 17 transgenic soybean strains in the plasmid standard molecules prepared in the embodiment 2 of the invention, which indicates that the plasmid standard molecules provided in the embodiment of the invention can be simultaneously applicable to qualitative and quantitative detection of the transgenic components of 17 transgenic soybeans.
The plasmid standard molecular standard sample provided by the embodiment of the invention has moderate uniformity, stability and uncertainty, replication sustainability, and the uniformity and stability of the standard sample meet the performance requirements of national standard samples, can meet the detection method verification, detection kit evaluation, detection process quality control, capability verification and personnel assessment for simultaneously identifying 17 transgenic strains in soybeans, and fills the blank that no such standard sample exists at home and abroad at present.
The above embodiments are only for illustrating the technical solution of the present invention, and are not limiting; although the invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical scheme described in the foregoing embodiments can be modified or some technical features thereof can be replaced by equivalents; such modifications and substitutions do not depart from the spirit and scope of the technical solutions of the embodiments of the present invention, and are intended to be included in the scope of the present invention.
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Claims (5)

1. The recombinant plasmid is characterized by being simultaneously applicable to real-time fluorescence PCR qualitative and quantitative detection for identifying 17 transgenic soybean lines, and comprises fusion fragments for identifying 17 transgenic soybean line specific boundary sequences, wherein the sequences of the fusion fragments are shown as SEQ ID NO:1 is shown in the specification;
the fusion fragment comprises the following 17 transgenic soybean strain-specific border sequence gene fragments: 305523, MON89788, CV127, MON87769, DP-356043-5, MON87701, MON87705, DAS-68416-4, DAS-44406-6, MON87751, MON87708, DAS81419-2, A2704-12, FG72, A5547-127, GTS40-3-2 and SYHT0H2, having the sequences set forth in SEQ ID NO:2 to 18;
the fusion fragment is obtained by splicing 17 transgenic soybean strain specific boundary sequence gene fragments in sequence according to the following splicing sequence: 305523, MON89788, CV127, MON87769, DP-356043-5, MON87701, MON87705, DAS-68416-4, DAS-44406-6, MON87751, MON87708, DAS81419-2, A2704-12, FG72, A5547-127, GTS40-3-2, SYHT0H2;
the sequence length of the fusion fragment of the strain-specific boundary sequence is 1768bp;
primer probe sequence information for specific identification of 17 transgenic soybean lines is shown in the following table:
2. the recombinant plasmid according to claim 1, wherein the fusion fragment of the strain-specific border sequence is prepared by:
extracting 17 transgenic soybean strain specific boundary sequence gene fragments;
splicing the 17 transgenic soybean strain specific boundary sequence gene fragments according to a preset fragment splicing sequence to obtain spliced sequence fragments;
converting the spliced sequence fragment into escherichia coli to realize cloning conversion, so as to obtain converted escherichia coli;
culturing the transformed escherichia coli to obtain transformed escherichia coli colonies;
screening the colony of the transformed escherichia coli for a sequence shown as SEQ ID NO:1, and a fusion fragment as shown in 1.
3. The recombinant plasmid according to any one of claims 1 to 2, wherein the recombinant plasmid is prepared by inserting the fusion fragment into a multicloning site of a pMD19-T plasmid backbone vector.
4. A kit for detecting a transgenic soybean line, comprising the recombinant plasmid of any one of claims 1-3.
5. Use of a recombinant plasmid according to any one of claims 1 to 3 or a kit according to claim 4 for strain identification of transgenic soybeans.
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