Primer, kit and detection method for identifying homozygosity and heterozygosity of transgenic maize LD05 and application of primer, kit and detection method

Abstract

The invention provides a homozygosity and heterozygosity identification method for insect-resistant and herbicide-resistant transgenic maize LD05. The method can accurately distinguish LD05 homozygote from LD05 heterozygote so as to quickly obtain a pure line, effectively shorten the time for introducing excellent characters into a conventional parent selfing line and accelerate the biological breeding process.

Description

Technical Field

[0001] The present invention relates to the field of biological breeding, and specifically provides a detection method for rapidly identifying homozygotes and heterozygotes of transgenic corn LD05 and its application. Background Art

[0002] Affected by the genotype of corn, only a few inbred line materials are suitable for existing corn genetic transformation methods (Li Yu, Wang Tianyu. Current status and prospects of research and application of corn transgenic technology. Corn Science, 2018, 26(2): 1-15). Excellent target traits are mainly introduced into the parental inbred lines of corn through backcrossing. After multiple generations of backcrossing, the target traits and phenotypes of the backcross materials are close to the expected ones, and then the homozygous individuals are obtained through selfing (Zhou Hongchang, Song Wei, Wang Fengge, et al. Screening of foreground primers and background primers in molecular marker-assisted selection breeding of corn head smut [J]. Molecular Plant Breeding, 2011, 9(04): 450-456). Therefore, it is crucial to accurately and quickly introduce the target traits of transgenic corn into conventional inbred lines for breeding. It is of great significance to use molecular detection methods to effectively shorten the backcrossing process during the backcrossing process.

[0003] The pure heterozygous identification method is mainly used to determine the genetic purity of breeding materials, which is of great significance for confirming the genetic background of materials and improving breeding efficiency. Homozygotes can ensure the stability of the genetic characteristics of the variety and contribute to the stability and consistency of the traits; while heterozygotes need to be selected in the offspring to stabilize these traits. Identifying the pure heterozygous characteristics of breeding materials in advance can provide breeders with more suitable parents to achieve more efficient breeding goals. At the same time, it can minimize the waste of resources and breeding failures caused by the use of non-homozygous materials. The application range of the pure heterozygous identification method is very wide. In variety selection, especially in the development of new germplasm resources, accurate identification of homozygotes can ensure that the selected varieties have the expected genetic characteristics; when conducting germplasm resource identification and evaluating existing germplasm resources, it is often necessary to understand their purity and heterozygosity; when conducting genomic research, ensuring the consistency of the genetic background of the research materials is an important prerequisite for obtaining accurate experimental results. Traditional methods for pure and heterozygous identification include molecular marker technologies such as SSR and SNP and phenotypic analysis. In some breeding materials with rich diversity, the identification of homozygous and heterozygous may be affected by the complexity of the genetic background, which increases the difficulty of identification and makes it difficult to ensure the accuracy and stability of the identification results. With the development of genome sequencing and bioinformatics, with the help of powerful data analysis capabilities, high-throughput sequencing technology and high-precision molecular marker data analysis have been applied to the selection and breeding of homozygous parents, which can ensure the reliability and accuracy of the results, but will increase the cost and cycle of testing. For transgenic breeding materials, the exogenous genes of homozygotes can be stably inherited to ensure that the offspring have the same genetic characteristics; while heterozygotes will cause the offspring to lose exogenous genes and target traits due to factors such as gene separation and recombination. Therefore, the homozygous state of the transgenic is very important for maintaining the genetic stability of the target traits of the transgenic variety.

[0004] The insect-resistant and herbicide-resistant transgenic corn LD05 is a new transgenic corn material with insect-resistant and herbicide-resistant properties obtained by Shandong Academy of Agricultural Sciences using Agrobacterium-mediated method to introduce the new insect-resistant fusion gene m2cryAb-vip3A and the herbicide-resistant gene bar into the receptor material in series, and backcrossing with the backbone inbred line Zheng 58 as the recurrent parent. The high-generation transgenic backcross material obtained after multiple generations of backcrossing has the target traits of insect resistance and herbicide resistance, and its agronomic traits are also close to those of the backcross parent. At this time, it is very necessary to establish an accurate and specific transgenic pure heterozygous identification method and quickly obtain the homozygous line of the target gene to ensure the genetic stability of the target traits of the offspring.

[0005] The present invention designs multiple sets of different PCR primers for amplification at the left and right boundaries of the LD05 insertion sequence, which can quickly, accurately and high-throughput identify the pure heterozygosity of the exogenous insertion sequence, and establishes an efficient identification method for homozygous transgenic corn LD05, thereby effectively shortening the breeding cycle and providing useful information for the rapid industrial application of excellent transformants. Summary of the invention

[0006] The purpose of the present invention is to provide a rapid and accurate detection method for identifying homozygous transgenic corn LD05.

[0007] To achieve the above purpose, the present invention designs multiple sets of different PCR primers at the left and right borders of the LD05 insertion sequence to conduct amplification experiments, and screens out a primer combination that can be used to quickly identify the pure heterozygosity of transgenic corn LD05.

[0008] The present invention provides a primer pair for identifying homozygous transgenic corn LD05, characterized in that the primer pair comprises a primer pair A consisting of sequence primers shown in SEQ ID NO.1 and SEQ ID NO.2, and a primer pair B consisting of sequence primers shown in SEQ ID NO.1 and SEQ ID NO.3.

[0009] The present invention also provides a kit for detecting homozygous transgenic corn LD05, characterized in that it comprises the primer pair combination according to claim 1.

[0010] The present invention also provides a method for detecting homozygous transgenic corn LD05, characterized in that the method comprises the following steps:

[0011] i) using the genomic DNA of the sample to be tested as a template, the primer pair A and the primer pair B described in claim 1 can both amplify the target band, then the sample to be tested is heterozygous LD05;

[0012] ii) using the genomic DNA of the sample to be tested as a template, the primer pair A described in claim 1 cannot amplify the target band, but the primer pair B described in claim 1 can amplify the target band, then the sample to be tested is homozygous LD05;

[0013] iii) If the primer pair A described in claim 1 can amplify the target band using the genomic DNA of the sample to be tested as a template, but the primer pair B described in claim 1 cannot amplify the target band, then the sample to be tested is not LD05.

[0014] Furthermore, the present invention also provides the use of the primer pair combination of claim 1, the kit of claim 2, and the method of claim 3 in identifying homozygous transgenic corn LD05.

[0015] The beneficial effects of the present invention are as follows: based on common PCR technology, the present invention screens out a group of primer combinations with high amplification efficiency and detection sensitivity, which can quickly, accurately and high-throughput complete the homozygous and heterozygous identification of transgenic corn LD05; the identified homozygous LD05 can be used as a parent to establish a transgenic pure line, thereby accelerating the cultivation of new transgenic corn varieties with excellent insect resistance and herbicide resistance and stable genetic traits.

[0016] In the method of the present invention for detecting the homozygous trait of corn LD05, the following definitions and methods can better define the present invention and guide ordinary technicians in the field to implement the present invention. Unless otherwise specified, the terms are understood according to the conventional usage of ordinary technicians in the field.

[0017] The term "gene" refers to a nucleic acid fragment that expresses a specific protein, including regulatory sequences before the coding sequence (5' non-coding sequences) and regulatory sequences after the coding sequence (3' non-coding sequences). "Native gene" refers to a gene found in nature with its own regulatory sequences. "Chimeric gene" refers to any gene that is not a natural gene, which contains regulatory and coding sequences that are not found in nature. "Endogenous gene" refers to a natural gene that is located in its natural location in the genome of an organism. "Foreign gene" is a foreign gene that is now present in the genome of an organism and did not exist originally, and also refers to a gene introduced into a recipient cell through a transgenic step. Foreign genes can include natural genes inserted into non-natural organisms or chimeric genes. "Transgene" is a gene that has been introduced into the genome through a transformation procedure. The site in the plant genome where the recombinant DNA has been inserted can be called an "insertion site" or "target site."

[0018] "Flanking DNA" can comprise the exogenous (heterologous) DNA naturally present in the genome of the organism of for example plant or introduced by the transformation process, for example the fragment relevant to the transformation event.Therefore, flanking DNA can comprise the combination of natural and exogenous DNA.In the present invention, "flanking region" or "flanking sequence" or "genome border region" or "genome border sequence" refers to at least 3,5,10,11,15,20,50,100,200,300,400,1000,1500,2000,2500 or 5000 base pairs or longer sequence, which is located at the direct upstream or downstream of the initial exogenous insertion DNA molecule and adjacent to the initial exogenous insertion DNA molecule.When this flanking region is located at upstream, it can also be referred to as "left border flank" or "5' flank" or "5' genome flanking region" or "genome 5' flanking sequence" etc. When the flanking region is located downstream, it may also be referred to as the “right border flank” or the “3′ flank” or the “3′ genomic flanking region” or the “genomic 3′ flanking sequence” or the like.

[0019] The transformation program that causes random integration of foreign DNA can lead to transformation events containing different flanking regions, and the different flanking regions are that each transformation event specifically contains.When recombinant DNA is introduced into plants by traditional hybridization, its flanking regions usually do not change.Transformation events also can contain the unique junction between the segment of heterologous insert DNA and genomic DNA or between two sections of genomic DNA or between two sections of heterologous DNA. "Jointation" is the point where two specific DNA fragments connect.For example, the junction is present at the position where the insert DNA connects the flanking DNA.Jointing points are also present in transformed organisms, where two DNA fragments are connected together in a way modified from natural organisms. "Jointing DNA" refers to the DNA comprising a junction.

[0020] The present invention provides a transgenic corn event called LD05 and its progeny, wherein the transgenic corn event LD05 is corn LD05, which includes plants and seeds of the transgenic corn event LD05 and plant cells or their regenerable parts, and the plant parts of the transgenic corn event LD05 include but are not limited to cells, pollen, ovules, flowers, shoots, roots, stems, leaves and products from corn LD05, such as corn seeds, corn oil, corn meal, corn flour, corn cobs and biomass left in corn crop fields.

[0021] The term "primer" is a separate nucleic acid molecule that anneals and binds to a complementary target DNA strand through nucleic acid hybridization, forms a hybrid between the primer and the target DNA strand, and then extends along the target DNA strand under the action of a polymerase (e.g., DNA polymerase). The primer pair of the present invention relates to its application in amplification of a target nucleic acid sequence, for example, by polymerase chain reaction (PCR) or other conventional nucleic acid amplification methods. The length of the primer is generally 11 polynucleotides or more, preferably 18 polynucleotides or more, more preferably 24 polynucleotides or more, and most preferably 30 polynucleotides or more. Such primers specifically hybridize with the target sequence under highly stringent hybridization conditions. Although primers that are different from the target DNA sequence and maintain hybridization ability to the target DNA sequence can be designed by conventional methods, preferably, the primers in the present invention have complete DNA sequence identity with the continuous nucleic acid of the target sequence.

[0022] With respect to amplification of a target nucleic acid sequence using specific amplification primers (e.g., by PCR), "stringent conditions" refer to conditions that only allow the primers to hybridize to the target nucleic acid sequence in a DNA thermal amplification reaction, and primers having a wild-type sequence (or its complementary sequence) corresponding to the target nucleic acid sequence are able to bind to the target nucleic acid sequence and preferably produce a unique amplification product, i.e., an amplicon.

[0023] The term "specifically binds to (target sequence)" means that the primer hybridizes only to the target sequence in a sample containing the target sequence under stringent hybridization conditions.

[0024] As used herein, "amplified DNA" or "amplicon" refers to the nucleic acid amplification product of a target nucleic acid sequence that is part of a nucleic acid template. For example, to determine whether a corn plant is produced by sexual hybridization containing the transgenic corn event LD05 of the present invention, or whether a corn sample collected from a field contains the transgenic corn event LD05, or whether a corn extract, such as corn oil, contains the transgenic corn event LD05, DNA extracted from a corn plant tissue sample or extract can be used by a nucleic acid amplification method using a primer pair to produce an amplicon that is diagnostic for the presence of DNA of the transgenic corn event LD05. The primer pair includes a first primer derived from a flanking sequence adjacent to the inserted exogenous DNA insertion site in the plant genome, and a second primer derived from the inserted exogenous DNA. The amplicon has a certain length and sequence, and the sequence is also diagnostic for the transgenic corn event LD05.

[0025] The length of the amplicon can range from the combined length of the primer pair plus one nucleotide base pair, preferably plus about fifty nucleotide base pairs, more preferably plus about two hundred and fifty nucleotide base pairs, and most preferably plus about four hundred and fifty nucleotide base pairs or more.

[0026] Alternatively, the primer pairs can be derived from flanking genomic sequences on both sides of the inserted DNA to produce an amplicon comprising the entire inserted nucleotide sequence. One of the primer pairs derived from the plant genomic sequence can be located at a distance from the inserted DNA sequence, the distance ranging from one nucleotide base pair to about 20,000 nucleotide base pairs. The use of the term "amplicon" specifically excludes primer dimers formed in a DNA thermal amplification reaction.

[0027] The technical solution of the present invention is further described in detail below through the accompanying drawings and embodiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 Schematic diagram of PCR primers specific for transgenic maize LD05.

[0029] Figure 2 Electropherogram of screening primer pair B

[0030] M: DL2000Maker, the molecular weights from top to bottom are 2000bp, 1000bp, 750bp, 500bp, 250bp and 100bp;

[0031] 1: amplification product of primers LC937 and LC936; 2: amplification product of primers LC937 and LC938;

[0032] 3: amplification product of primers LC937 and LC944; 4: amplification product of primers LC937 and LC948;

[0033] 5: amplification product of primers LC947 and LC936; 6: amplification product of primers LC947 and LC938;

[0034] 7: amplification product of primers LC947 and LC944; 8: amplification product of primers LC947 and LC948;

[0035] 9: amplification product of primers LC917 and LC936; 10: amplification product of primers LC917 and LC938;

[0036] 11: amplification product of primers LC917 and LC944; 12: amplification product of primers LC917 and LC948;

[0037] 13: amplification product of primers LC913 and LC936; 14: amplification product of primers LC913 and LC938;

[0038] 15: amplification product of primers LC913 and LC944; 16: amplification product of primers LC913 and LC948;

[0039] 17: amplification product of primers LC915 and LC936; 18: amplification product of primers LC915 and LC938;

[0040] 19: amplification product of primers LC915 and LC944; 20: amplification product of primers LC915 and LC948;

[0041] 21: amplification product of primers LC928 and LC929; 22: amplification product of primers LC931 and LC962;

[0042] 23: amplification product of primers LC941 and LC962; 24: amplification product of primers LC951 and LC962;

[0043] 25: amplification product of primers LC953 and LC962; 26: amplification product of primers LC931 and LC964;

[0044] 27: amplification product of primers LC941 and LC964; 28: amplification product of primers LC951 and LC964;

[0045] 29: amplification product of primers LC953 and LC964; 30: amplification product of primers LC931 and LC966;

[0046] 31: amplification product of primers LC941 and LC966; 32: amplification product of primers LC951 and LC966;

[0047] 33: amplification product of primers LC953 and LC966; 34: amplification product of primers LC931 and LC968;

[0048] 35: amplification product of primers LC941 and LC968; 36: amplification product of primers LC951 and LC968;

[0049] 37: amplification product of primers LC953 and LC968; 38: amplification product of primers LC931 and LC958;

[0050] 39: amplification product of primers LC941 and LC958; 40: amplification product of primers LC951 and LC958;

[0051] 41: amplification product of primers LC953 and LC958; 42: amplification product of primers LC933 and LC934.

[0052] Figure 3 Electropherogram of screening primer set A

[0053] M: DL2000Maker, the molecular weights from top to bottom are 2000bp, 1000bp, 750bp, 500bp, 250bp and 100bp;

[0054] 1: homozygous LD05 genomic DNA; 2: heterozygous LD05 genome; 3: Zheng 58 genomic DNA.

[0055] Figure 4 Test the amplification efficiency of 5 sets of primer pairs A (different cycle numbers)

[0056] M: DL2000Maker, the molecular weights from top to bottom are 2000bp, 1000bp, 750bp, 500bp, 250bp and 100bp;

[0057] 1, 7, 13, 19, 25, 31: number of cycles 20;

[0058] 2, 8, 14, 20, 26, 32: number of cycles 22;

[0059] 3, 9, 15, 21, 27, 33: cycle number 24;

[0060] 4, 10, 16, 22, 28, 34: number of cycles 26;

[0061] 5, 11, 17, 23, 29, 35: cycle number 28;

[0062] 6, 12, 18, 24, 30, 36: 30 cycles.

[0063] Figure 5 Test the detection limit of 5 primer pairs A

[0064] M: DL2000Maker, the molecular weights from top to bottom are 2000bp, 1000bp, 750bp, 500bp, 250bp and 100bp;

[0065] 1, 7, 13, 19, 25, 31: template concentration 100 ng / μL;

[0066] 2, 8, 14, 20, 26, 32: template concentration 10 ng / μL;

[0067] 3, 9, 15, 21, 27, 33: template concentration 1 ng / μL;

[0068] 4, 10, 16, 22, 28, 34: template concentration 0.1 ng / μL;

[0069] 5, 11, 17, 23, 29, 35: template concentration 0.01 ng / μL;

[0070] 6, 12, 18, 24, 30, 36: Template concentration is 0.001 ng / μL.

[0071] Figure 6 PCR amplification results of primer pair A (LC915+LC966)

[0072] M: DL2000Maker, the molecular weights from top to bottom are 2000bp, 1000bp, 750bp, 500bp, 250bp and 100bp;

[0073] 1-93: 93 random BC6F2 generation LD05 genomic DNAs; 94: blank control water; 95: Zheng 58 genomic DNA; 96: homozygous LD05 genomic DNA.

[0074] Figure 7 PCR amplification results of primer pair B (LC915+LC948)

[0075] M: DL2000Maker, the molecular weights from top to bottom are 2000bp, 1000bp, 750bp, 500bp, 250bp and 100bp;

[0076] 1-93: 93 random BC6F2 generation LD05 genomic DNAs; 94: blank control water; 95: Zheng 58 genomic DNA; 96: homozygous LD05 genomic DNA.

[0077] Figure 8 qRT-PCR analysis of m2cryAb-vip3A and bar in pure heterozygous plants

[0078] A: Detection of m2cryAb-vip3A; B: Detection of bar.

[0079] 3, 7, and 17: BC6F2 generation homozygous LD05 genomic DNA;

[0080] 1, 2, 5, 6: genomic DNA of heterozygous LD05 of the BC6F2 generation. DETAILED DESCRIPTION

[0081] The insect-resistant and herbicide-resistant transgenic corn LD05 involved in the present application is a new transgenic corn material that is insect-resistant and herbicide-resistant and has excellent agronomic traits, obtained by introducing the insect-resistant fusion gene m2cryAb-vip3A and the herbicide-resistant gene bar in series into the receptor material by Agrobacterium-mediated method, and backcrossing is carried out with the backbone inbred line Zheng 58 as the recurrent parent. After backcrossing, the BC6F1 generation transgenic backcross material obtained has excellent insect-resistant and herbicide-resistant traits, and the agronomic traits such as yield are comparable to those of the recurrent parent Zheng 58. In the BC6F2 material obtained by self-pollination, the target trait is separated. The present invention can screen out homozygous LD05 plants in the present generation by establishing a pure heterozygous detection method, thereby shortening the breeding time.

[0082] Example 1 Primer screening for pure heterozygous identification of transgenic maize LD05

[0083] Primer screening and homozygous identification strategy of LD05

[0084] First, PCR primers were designed at the left and right borders of the LD05 transgenic insertion site, and primer pair B that can specifically amplify LD05 was screened out; then, the upstream primer of primer pair B at the left border and the downstream primer of primer pair B at the right border were combined into a new primer pair, and primer pair A that can specifically amplify wild-type Zheng 58 was screened out.

[0085] If primer pair B amplifies the target band and primer pair A does not amplify the target band, it is a homozygous LD05 plant; if primer pair A and primer pair B both amplify the target band, it is a heterozygous LD05 plant; if primer pair B does not amplify the target band, but primer pair A amplifies the target band, it is not an LD05 plant.

[0086] Extraction of genomic DNA

[0087] Young leaves of BC6F2 transgenic corn LD05 and Zheng 58 were taken to extract genomic DNA. The specific method is as follows:

[0088] Weigh a small amount of leaves and place them in a 2mL centrifuge tube. Quickly freeze them with liquid nitrogen and then place them in a sample grinder to fully grind them into powder; add 400μL 2% CTAB to the centrifuge tube, shake it slightly to mix, place it in a floating plate, and place it in a 65℃ water bath for 30min, inverting it 3-4 times to mix; after cooling, add 400μL chloroform, shake it slowly for 10min, and centrifuge it at 12000rpm for 10min; use a tipless pipette to draw 400μL of supernatant, add 2 times the volume of anhydrous ethanol, invert it upside down, and let it stand at -20℃ for 2h; centrifuge it at 12000rpm for 10min, discard the supernatant, and keep the precipitate; add 75% alcohol to wash, centrifuge it at 12000rpm for 1min, discard the alcohol, and invert it to absorb water and dry it; dissolve the DNA in 80μL of pure water, determine the DNA quality and concentration with NanoDrop2000 ultra-micro spectrophotometer, and dilute the DNA to about 50ng / μL with pure water for use.

[0089] Design of candidate primers for specific PCR amplification

[0090] Using the left border sequence (SEQ ID NO.4) and the right border sequence (SEQ ID NO.5) of the LD05 transgenic insertion site as templates, a total of 22 LD05-specific PCR amplification candidate primers were designed and synthesized using the primer design software Primer Premier 5 ( Figure 1 ). The primer sequence information and positions of these candidate primers and maize internal reference genes are shown in Table 1.

[0091] Table 1 Sequence information of candidate primers for LD05 specific amplification and primers for maize internal reference gene zSSIIb

[0092]

[0093]

[0094] PCR amplification method

[0095] PCR amplification used a 20 μL universal reaction system: 10 μL 2× Phanta Max Master Mix, 0.4 μL 10 μmol / L primer (final concentration 0.2 μmol / L), 1 μL (50 ng) of template DNA, and ddH2O supplemented to 20 μL.

[0096] PCR reaction conditions were: 95°C pre-denaturation for 5 min; 95°C denaturation for 30 s, 58°C annealing for 30 s, 72°C

[0097] Extension was performed for 35 seconds for a total of 32 cycles; post-extension was performed at 72°C for 7 minutes.

[0098] The PCR products were detected by 1% agarose gel electrophoresis.

[0099] Screening of primer pair B

[0100] Using the DNA extracted from each leaf as a template, PCR amplification was performed using the left border primer pair (6 upstream primers and 5 downstream primers freely combined) and the right border primer pair (5 upstream primers and 6 downstream primers freely combined) in Table 1 to screen primer pair B that can be used for specific identification of LD05.

[0101] The results showed that the amplification products of the left border primer pairs LC937+LC944, LC937+LC948, LC947+LC944, LC947+LC948, LC917+LC944, LC917+LC948, LC915+LC944, LC915+LC948, LC928+LC929 and the right border primer pairs LC941+LC958, LC953+LC958, LC941+LC962, LC953+LC962, LC941+LC964, LC953+LC964, LC941+LC966, LC953+LC966, LC941+LC968, LC953+LC968, LC933+LC934 were in accordance with the expected size, with clear bands, no primer dimers, and no mixed bands ( Figure 2 ), all of which are primer pair B that meet the requirements. The specific amplification results are shown in Table 2.

[0102] Table 2 Amplification results of LD05 specific PCR primers

[0103]

[0104]

[0105] Screening of primer pair A

[0106] Primer pair A is composed of a combination of a left border upstream primer and a right border downstream primer of primer pair B that meet the requirements. The genome sequence of the receptor Zheng 58 at the insertion site is shown in SEQ ID NO:6.

[0107] In order to further screen primer pairs A that can be used for LD05 homozygous heterozygous identification, PCR amplification was performed using the genomic DNA of homozygous LD05 with no target trait segregation in self-pollinated progenies, heterozygous LD05 with trait segregation in self-pollinated progenies, and wild-type Zheng 58 as templates. A total of 21 sets of primer pairs A were tested in the experiment (see Table 3). The electrophoresis results of the PCR products are shown in Figure 3. Figure 3 shown.

[0108] The five primer pairs A, LC917+LC966, LC915+LC966, LC915+LC968, LC917+LC958 and LC928+LC934, could not amplify bands in homozygous plants, but could amplify bands in heterozygous plants and wild-type plants, and the PCR products were in line with the expected size, with clear bands and no mixed bands, which were primer pairs A that met the requirements for LD05 homozygous identification. The analysis of PCR amplification products is shown in Table 3.

[0109] Table 3 Pure heterozygous identification results of primer pair A

[0110]

[0111]

[0112] Analysis of amplification cycle numbers of five primer pairs A

[0113] In order to screen primers with better specificity and higher sensitivity, the five primer pairs A were further tested and compared in terms of amplification cycle number and template detection limit. Using genomic DNA (50 ng / μL) of LD05 heterozygous plants as template, the five primer pairs A, LC917+LC966, LC915+LC966, LC915+LC968, LC917+LC958 and LC928+LC934, were tested for amplification efficiency at different amplification cycle numbers. The internal reference gene was used as a control.

[0114] like Figure 4 As shown in the figure, the target band could not be amplified when the cycle number was 20, only the LC915+LC966 primer combination could amplify the target band when the cycle number was 22, and the four primer combinations of LC917+LC966, LC915+LC966, C915+LC968 and LC917+LC958 could amplify the target band when the cycle numbers were 24, 26, 28, and 30. LC928+LC934 and the internal reference gene amplification primers could not amplify the target band when the cycle numbers were 20, 22, and 24, but could amplify the target band when the cycle numbers were 26, 28, and 30. The specific test results are shown in Table 4.

[0115] Table 4 Amplification cycle test results of 5 primer pairs A

[0116]

[0117] √ indicates that there is a target band that meets the expectations; × indicates that there is no band

[0118] Analysis of detection limits of five primer pairs A

[0119] The genomic DNA concentration of the transformant LD05 heterozygous plant was determined by spectrophotometer to be 100 ng / μL, and the concentration was 10.0 ng / μL (diluted 10 times), 1.0 ng / μL (diluted 10 times), and 1.1 ng / μL (diluted 10 times). 2 times), 0.10ng / μL (diluted 10 3 times), 0.010ng / μL (diluted 10 4 times), 0.0010ng / μL (diluted 10 5 The same volume (1 uL) of template was added to perform PCR amplification. At the same time, the internal reference gene zSSIIb was amplified by PCR using the same method.

[0120] The results are as follows Figure 5 As shown, the LC915+LC966 primer combination was used in the template dilution 10 4 When the concentration was 0.010 ng / μL, the target fragment could no longer be amplified, indicating that the detection limit of the target fragment using ordinary PCR was 0.1 ng / μL; the three primer combinations of LC917+LC966, LC915+LC968, and LC917+LC958 were 10 3 When the concentration was 0.1 ng / μL, the target fragment could no longer be amplified, indicating that the detection limit of the target fragment using ordinary PCR was 1 ng / μL. 3 When the concentration was 0.1 ng / μL, the target fragment could no longer be amplified, indicating that the detection limit of the target fragment using ordinary PCR was 1 ng / μL.

[0121] Table 5 Detection limit analysis results of 5 primer pairs A

[0122]

[0123] √ indicates that there is a target band that meets the expectations; × indicates that there is no band

[0124] Taking into account factors such as the number of amplification cycles and the limit of detection, the optimal primer pair A for pure heterozygous identification of LD05 was finally determined to be LC915+LC966, and the number of amplification cycles and the limit of detection were lower than those of the other four primer pairs A, such as LC928+LC934.

[0125] Example 2 Method for Identifying Pure Heterozygous Genetically Modified Maize LD05 of BC6F2 Generation

[0126] 93 BC6F2 LD05 plants were randomly selected, young leaves were taken, genomic DNA was extracted using the method in Example 1, primer pair A (LC915+LC948) and primer pair B (LC915+LC966), and the PCR reaction system and reaction procedure in Example 1 were used for pure heterozygous identification.

[0127] The electrophoresis results of PCR amplification products are as follows Figure 6 and Figure 7 The results analysis is shown in Table 6.

[0128] Among them, the target bands could not be amplified by LC915+LC966, while the target bands that could be amplified by LC915+LC948 were 3, 7, 17, 18, 22, 25, 31, 34, 40, 42, 45, 51, 53, 59, 64, 65, 68, 72, 73, 75, 79, 88, and 92, for a total of 23 homozygous LD05 samples.

[0129] The target bands that could be amplified by LC915+LC966 and LC915+LC948 were 1, 2, 5, 6, 8, 9, 11, 13, 14, 15, 16, 20, 21, 23, 26, 28, 29, 30, 32, 36, 37, 38, 39, 41, 43, 44, 46, 47, 50, 52, 55, 56, 57, 60, 61, 63, 67, 69, 71, 74, 76, 78, 81, 82, 83, 85, 86, 89, 90, 91, and 93, a total of 51 heterozygous LD05 samples.

[0130] The target bands that could be amplified by LC915+LC966 but not by LC915+LC948 were 4, 10, 12, 19, 24, 27, 33, 35, 48, 49, 54, 58, 62, 66, 70, 77, 80, 84, and 87. These 19 samples were wild-type samples, not LD05.

[0131] Table 6 Analysis of pure heterozygous identification results of BC6F2 generation LD05 plant samples

[0132]

[0133]

[0134] √ indicates that there is a target band that meets the expectations; × indicates that there is no band

[0135] Analysis showed that among the 93 randomly selected BC6F2 generation LD05 plants, the ratio of homozygous: heterozygous: wild type was 23:51:19, which basically conformed to the Mendelian segregation ratio of 1:2:1.

[0136] The results showed that the combination of LC915+LC948 as primer pair A and LC915+LC966 as primer pair B was reliable for the identification of LD05 homozygous samples.

[0137] Example 3 Detection of the expression levels of m2cryAb-vip3A and bar genes in pure heterozygous plants of LD05

[0138] In order to further verify the effectiveness of the identification method of the present invention, the inventors used real-time fluorescence quantitative PCR to detect the expression levels of the target genes m2cryAb-vip3A and bar in the homozygous and heterozygous plants of LD05 identified in Example 2. The results are as follows: Figure 8 As shown, the expression levels of m2cryAb-vip3A and bar genes at the transcriptional level in homozygous plants No. 3, 7, and 17 were significantly higher than those in heterozygous plants No. 1, 2, 5, and 6. However, the expression of m2cryAb-vip3A and bar genes was not detected in the wild-type control Zheng 58.

[0139] The difference in the expression levels of the target genes in the pure heterozygous plants demonstrates the effectiveness of the identification method of the present invention.

[0140] In summary, the primer pair A (LC915+LC948) and primer pair B (LC915+LC966) screened by the present invention can be used for the identification of LD05 homozygous samples, thereby greatly shortening the breeding time and accelerating the biological breeding process.

[0141] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solution of the present invention can be modified or replaced by equivalents without departing from the spirit and scope of the technical solution of the present invention.

Claims

1. A primer pair combination, characterized in that: The primer pair includes a primer pair A consisting of primers with sequences shown as SEQ ID NO.1 and SEQ ID NO.2, and a primer pair B consisting of primers with sequences shown as SEQ ID NO.1 and SEQ ID NO.

3.

2. A kit, characterized in that: Comprising the primer pair combination of claim 1.

3. A method for detecting the homozygous characteristics of transgenic corn LD05, characterized in that: The method comprises the following steps: i) using the genomic DNA of the sample to be tested as a template, the primer pair A and the primer pair B described in claim 1 can both amplify the target band, then the sample to be tested is heterozygous LD05; ii) using the genomic DNA of the sample to be tested as a template, the primer pair A described in claim 1 cannot amplify the target band, but the primer pair B described in claim 1 can amplify the target band, then the sample to be tested is homozygous LD05; iii) If the genomic DNA of the sample to be tested is used as a template and the primer pair A described in claim 1 can amplify the target band, but the primer pair B described in claim 1 cannot amplify the target band, then the sample to be tested is not LD05.

4. Use of the primer pair combination according to claim 1, the kit according to claim 2, and the method according to claim 3 in identifying transgenic corn LD05.

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