Primer group, kit and method for homozygosity and heterozygosity identification of transgenic papaya 55-1 transformant

Abstract

The invention provides a primer group, a kit and a method for homozygosity and heterozygosity identification of a transgenic papaya 55-1 transformant, and belongs to the technical field of molecular biology, the primer group comprises three primers, namely 55-1-SSF: 5 '-CACTTTGATCAATGCAGAGTTGC-3', 55-1-SUT: 5 '-CAGGATTCGAACCTACGTA-3' and 55-1-SSR: 5 '-AGTACAAGCTGAACCGAAATTTA-3', and the primer group comprises three primers, namely a primer group, a primer group, a primer group and a primer group, the kit comprises the primer group. According to the method, the primer group is adopted to carry out PCR amplification on a papaya sample to be detected so as to judge the homozygous and heterozygous state of the papaya 55-1 transformant to be detected. The method can be used for rapidly identifying the homozygous and heterozygous states of the 55-1 transformant in the papaya, and is suitable for homozygous degree detection and breeding and seed selection.

Description

Technical Field

[0001] The invention belongs to the field of molecular biology, and relates to a primer set, a kit and a method for identifying homozygous and heterozygous transgenic papaya 55-1 transformants, and specifically relates to a primer set, a kit and a method for identifying homozygous and heterozygous transgenic papaya 55-1 transformants. Background Art

[0002] Papaya ringspot virus (PRSV) was first discovered in Hawaii in 1949. The PRSV pandemic in 1992 caused devastating damage to the papaya industry in Hawaii. The 55-1 strain was a transgenic papaya strain resistant to PRSV that was bred by Cornell University and the University of Hawaii in the United States, using a gene gun to transfer the coat protein (CP) gene of the PRSV HA5-1 strain into papaya. The transgenic varieties such as "SunUp" and "Rainbow" bred on this basis have better disease resistance and ecological adaptability than the parent "Sunset", and became the first commercially grown transgenic fruit, directly saving the papaya industry in the United States. In 2022, the research team of the Genome Center of the Straits Joint Research Institute deciphered the high-quality genome maps of the transgenic papaya variety "SunUp" and the non-transgenic variety "Sunset". The team found that the exogenous gene fragment was located on a 1.6Mb insertion fragment on chromosome 5 of "SunUp".

[0003] In 2019, my country approved the GMO safety certificate for the 55-1 strain as a food processing raw material, but currently there is only a specific PCR detection method for identifying the 55-1 transformation event, and there is no method for identifying homozygous and heterozygous from a breeding perspective. Screening homozygous plants can speed up the breeding process of transgenic plants, so the method for identifying pure and heterozygous transgenic plants has important application value. The present invention has created a set of methods for quickly and efficiently screening pure and heterozygous transgenic papaya 55-1 transformants. Summary of the invention

[0004] In view of the above problems, the present invention provides a primer set, a kit and a method for identifying the homozygous or heterozygous state of transgenic papaya 55-1 transformants. The primer set is designed and verified based on the 1.6 Mb insert fragment on chromosome 5 of "SunUp" and the sequence comparison of the "SunUp" variety and the "Sunset" variety, and is a specific primer set for identifying the homozygous or heterozygous state of papaya 55-1 transformants.

[0005] A primer set for identifying pure heterozygous transformants of transgenic papaya 55-1, the primer set comprising the following three primers: 55-1-SSF: 5'-CACTTTGATCAATGCAGAGTTGC-3'; 55-1-SUT:5'-CAGGATTCGAACCTACGTAG-3'; 55-1-SSR: 5'-AGTACAAGCTGAACCGAAATTTA-3'.

[0006] A kit for identifying pure heterozygous transgenic papaya 55-1 transformants, comprising the above primer set.

[0007] Furthermore, the kit also includes conventional reagents for PCR amplification.

[0008] Furthermore, conventional reagents used for PCR amplification include: GO Taq DNA polymerase, 5×Green GO TaqReaction buffer, dNTPs mix and sterile water.

[0009] A method for identifying pure heterozygous transgenic papaya 55-1 transformants, the method comprises using the above primer set to perform PCR amplification on the DNA of the papaya to be tested, so as to determine the pure heterozygous state of the papaya 55-1 transformants to be tested.

[0010] Further, the pure heterozygous state of the papaya 55-1 transformant to be tested is determined by electrophoresis of the PCR amplification product, and the pure heterozygous state of the papaya to be tested is determined according to the electrophoresis detection result, specifically: When only a 694 bp band appears in the electrophoresis test results, the papaya material to be tested contains a homozygous 55-1 transformant; When the electrophoresis results show both 694 bp and 282 bp bands, the tested papaya contains a heterozygous 55-1 transformant; When only a 282 bp band appears in the electrophoresis detection result, the tested papaya does not contain the 55-1 transformant.

[0011] Furthermore, the electrophoresis is to place the PCR amplification product on a 2% agarose gel with a mass volume ratio, and perform electrophoresis at a voltage of 100V for 15 minutes to obtain the electrophoresis detection result.

[0012] Furthermore, the PCR amplification system includes: GO Taq DNA polymerase, 5×Green GO Taq Reaction buffer, dNTPs mix, primers 55-1-SSF, 55-1-SUT and 55-1-SSR, a DNA template of the papaya to be tested and sterile water.

[0013] Furthermore, the PCR amplification system is as follows: 0.125 μL of GO Taq DNA polymerase with a concentration of 5 U / μL, 5 μL of 5×Green GO Taq Reaction buffer, 0.5 μL of dNTPs mix with a concentration of 10 mM, 1 μL each of primers 55-1-SSF, 55-1-SUT and 55-1-SSR with a concentration of 10 μmol / L, 2 μL of the DNA template of the papaya to be tested with a concentration of 100 ng / μL, and sterile water is added to 25 μL.

[0014] Furthermore, the PCR amplification program was as follows: pre-denaturation at 94°C for 5 min; denaturation at 94°C for 30 s, annealing at 55°C for 30 s, and extension at 72°C for 30 s, for 35 cycles; and extension at 72°C for 5 min.

[0015] The primer set, kit and method for identifying pure heterozygous transgenic papaya 55-1 transformants of the present invention have the following beneficial effects: The conventional 55-1 transformant detection method in the prior art can only detect whether a 55-1 transformant is present, but cannot further determine the pure heterozygous state of the 55-1 transformant. The present invention uses a primer set for pure heterozygous identification of transgenic papaya 55-1 transformants to amplify the DNA template of the papaya to be tested, and performs electrophoresis detection. The pure heterozygous state of the papaya 55-1 transformant to be tested can be intuitively and quickly identified through the electrophoresis detection result, without the need to conventionally determine whether it is homozygous or heterozygous through the separation of offspring produced by self-pollination or hybridization of papaya materials. The whole process is simple, convenient, time-saving and labor-saving. By using the primer set of the present invention, DNA can be sampled and extracted at any stage of papaya plant growth, and PCR and electrophoresis can be used to quickly and conveniently determine whether the 55-1 transformant exists and the pure heterozygous state, which is very convenient, and the whole process is fast, accurate, and efficient, and the required experimental conditions are not high, and it has very high practical value; Since transgenic papaya 55-1 is a transgenic event mediated by gene gun bombardment, a specific primer set was designed based on a 1.6 Mb insertion variation structure on chromosome 5 of its commercial variety "SunUp". PCR amplification was performed on the papaya samples to be tested, which can quickly identify the pure heterozygous state of 55-1 transformants in papaya, and is suitable for homozygosity detection and breeding selection. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 Schematic diagram of the positions of primers for pure and heterozygous PCR detection of 55-1 transformants in the embodiment of the present invention; Figure 2It is the electrophoresis diagram of pure and heterozygous PCR detection of 55-1 transformants in Example 2 of the present invention; wherein 1 represents a homozygous single plant of 55-1 transformants; 2 represents a heterozygous single plant of 55-1 transformants; 3 represents a single plant without 55-1 transformants; M represents a molecular weight marker DL2000; Figure 3 is the papaya blind sample identification spectrum in Example 3 of the present invention; wherein 1 to 20 represent 20 homemade papaya blind samples respectively; M represents the molecular weight marker DL2000; Figure 4 Electrophoresis diagram of specific PCR detection of different papaya strains in Example 4 of the present invention; wherein, 1 represents the transgenic papaya 55-1 homozygous strain; 2 represents the transgenic papaya 55-1 heterozygous strain; 3 to 7 represent the transgenic papaya YK16-0-1, the transgenic papaya "Huanong No. 1", the non-transgenic papaya SunSet, the non-transgenic papaya Suizhonghong, and the non-transgenic papaya Guangmi, respectively; M represents the molecular weight marker DL2000. DETAILED DESCRIPTION

[0017] The technical solutions in the embodiments of the present invention are described clearly and completely below. In the following description, many specific details are set forth to facilitate a full understanding of the present invention, but the present invention can also be implemented in other ways different from those described herein, and those skilled in the art can make similar generalizations without violating the connotation of the present invention, so the present invention is not limited by the specific embodiments disclosed below. The present invention is further described in detail below in conjunction with specific embodiments so that those skilled in the art can understand.

[0018] Plant materials: All papaya leaf samples used in the examples of the present invention were preserved by this laboratory.

[0019] Enzymes and reagents: Molecular biology reagents, such as dNTPs, Taq DNA polymerase, and 5×Taq Buffer, were purchased from Promega Biotechnology Co., Ltd. in Beijing. DNA extraction kits were purchased from Guangzhou MAGEN Biotechnology Co., Ltd. Primer set synthesis and sequencing were completed by Nanshan Gene Biotechnology Co., Ltd. in Haikou. The remaining biochemical reagents were all domestic analytical grade.

[0020] Experimental instruments: PCR amplification instrument: T1, Biometra, Germany; electrophoresis instrument: DYY-6C, Beijing Liuyi Instrument Factory; gel imaging system: Tanon-4300SF, Shanghai Tianneng Technology Co., Ltd.; ultra-micro-volume UV spectrophotometer: NanoDrop2000, Thermo Fisher Scientific. Other instruments include: centrifuge, water bath, electronic balance, etc.

[0021] Example 1 Primer set and kit for identifying pure heterozygous transgenic papaya 55-1 transformants Transgenic papaya 55-1 is a transgenic event mediated by gene gun bombardment. Based on a 1.6 Mb insertion variant structure on chromosome 5 of its commercial variety SunUp, the forward primer 55-1-SSF was selected from the 5' flanking sequence of the insert fragment, the reverse primer 55-1-SUT was selected from the insert fragment, and the reverse primer 55-1-SSR was selected from the 3' flanking sequence of the insert fragment (such as Figure 1 as shown).

[0022] The specific three primer sequences are: 55-1-SSF: 5'-CACTTTGATCAATGCAGAGTTGC-3'; 55-1-SUT:5'-CAGGATTCGAACCTACGTAG-3'; 55-1-SSR: 5'-AGTACAAGCTGAACCGAAATTTA-3'.

[0023] The region amplified by primer pair 55-1-SSF / 55-1-SUT is the connection region between the papaya genome and the 3' short sequence of papaya 55-1 transformant. The amplified sequence of papaya 55-1 transformant is 694 bp, and the sequence is shown in SEQ ID NO: 1. The region amplified by primer pair 55-1-SSF / 55-1-SSR is the papaya genome. The amplified sequence of papaya 55-1 transformant is 282 bp, and the sequence is shown in SEQ ID NO: 2 When designing primers, in order to enhance the applicability and sensitivity of the primers, the designed primer lengths are between 18 and 25 bp, and the primers do not interfere with each other. The above three primers can be obtained by artificial synthesis.

[0024] This embodiment also provides a kit for identifying pure heterozygous transgenic papaya 55-1 transformants. The kit includes the above primer set.

[0025] The kit also includes common reagents for PCR amplification, such as GO Taq DNA polymerase, 5×Green GOTaq Reaction buffer, dNTPs mix, and sterile water.

[0026] Example 2 Method for Identifying Pure Heterozygous Transformants of Transgenic Papaya 55-1 This example provides a method for identifying pure heterozygous transformants resistant to papaya ringspot virus 55-1, and the specific method comprises the following steps: S1, extracting papaya DNA to be tested as a DNA template; S2. Use the primer set in Example 1 to perform PCR amplification on the papaya DNA to be tested.

[0027] The PCR amplification system was as follows: 0.125 μL of GO Taq DNA polymerase at a concentration of 5 U / μL, 5 μL of 5×GreenGO Taq Reaction buffer, 0.5 μL of dNTPs mix at a concentration of 10 mM, 1 μL each of primers 55-1-SSF, 55-1-SUT and 55-1-SSR at a concentration of 10 μmol / L, 2 μL of DNA template at a concentration of 100 ng / μL, and the volume was filled to 25 μL with sterile water.

[0028] The PCR amplification program was as follows: pre-denaturation at 94°C for 5 min; denaturation at 94°C for 30 s, annealing at 55°C for 30 s, extension at 72°C for 30 s, 35 cycles; and extension at 72°C for 5 min.

[0029] S3, performing electrophoresis detection on the obtained PCR amplification product, and interpreting the electrophoresis detection result, specifically as follows: placing the PCR amplification product on a 2% agarose gel with a mass volume ratio, and performing electrophoresis at a voltage of 100 V for 15 minutes to obtain the electrophoresis detection result, and then judging the pure heterozygous state of the tested papaya according to the electrophoresis detection result; Among them, when the electrophoresis detection result shows only a 694bp band, the papaya material to be tested contains a homozygous 55-1 transformant, that is, a homozygous single plant of the 55-1 transformant. Figure 2 As shown in strip 1; When the electrophoresis test results show both 694bp and 282bp bands, the papaya to be tested contains a heterozygous 55-1 transformant, that is, a 55-1 transformant heterozygous single plant. Figure 2 As shown in strip 2; When only a 282 bp band appears in the electrophoresis test results, the papaya to be tested does not contain the 55-1 transformant, that is, it does not contain a 55-1 transformant single plant. Figure 2 As shown in strip 3 in FIG.

[0030] Example 3 Qualitative PCR Detection of 55-1 Transformants in Papaya Leaf Samples Twenty blind samples were prepared from papaya samples stored in the laboratory and identified using the method for pure heterozygous identification of transgenic papaya 55-1 transformants in Example 2. The results are as follows: Figure 3As shown, it is shown that among the 20 samples, 5, 2 and 13 samples were homozygous for 55-1 transformants, heterozygous for 55-1 transformants and did not contain 55-1 transformants, respectively. It shows that the method of the present invention can accurately identify whether a single papaya material contains 55-1 transformants, and simultaneously identify the pure and heterozygous state of 55-1 transformants, and can also be used to identify the proportion of homozygous or heterozygous individuals in a population.

[0031] Example 4 Strain-specific PCR detection of pure and heterozygous primer sets for transgenic papaya 55-1 transformants The specificity of the primer set was detected for different papaya strains. The experimental materials included transgenic papaya 55-1 homozygous strain, transgenic papaya 55-1 heterozygous strain, transgenic papaya YK16-0-1, transgenic papaya "Huanong No. 1", non-transgenic papaya SunSet, non-transgenic papaya Suizhonghong, and non-transgenic papaya Guangmi, all of which were preserved in this laboratory. The corresponding genomic DNA was extracted using the MAGEN plant genomic DNA rapid extraction kit, and the extracted genomic DNA was used as a template to amplify using the PCR system and program in Example 2, and the PCR amplification product was detected by agarose gel electrophoresis. The results are as follows: Figure 4 shown.

[0032] In this example, the pure and heterozygous identification primers of transgenic papaya 55-1 transformants were used to perform specific detection on multiple papaya varieties. The results showed that among the above-mentioned papaya varieties tested, the homozygous transgenic papaya 55-1 strain only amplified a 694 bp fragment. Figure 4 As shown in band 1 in the figure; the transgenic papaya 55-1 heterozygous strain simultaneously amplified 694 bp and 282 bp fragments, as shown in Figure 4 As shown in band 2 in the figure; transgenic papaya YK16-0-1, transgenic papaya "Huanong No. 1", non-transgenic papaya SunSet, non-transgenic papaya Suizhonghong, and non-transgenic papaya Guangmi all amplified only 282bp fragments, such as Figure 4 As shown in bands 3 to 7 in the figure, it is shown that the method for identifying pure heterozygous transgenic papaya 55-1 transformants of the present invention is specific.

[0033] Although the above embodiments have been described in detail, they are only a part of the embodiments of the present invention, rather than all the embodiments. A person skilled in the art can also obtain other embodiments based on the embodiments without inventiveness, and these embodiments all belong to the protection scope of the present invention.

Claims

1. A primer set for identifying pure heterozygous transformants of transgenic papaya 55-1, characterized in that: The primer set includes the following three primers: 55-1-SSF: 5'-CACTTTGATCAATGCAGAGTTGC-3'; 55-1-SUT:5'-CAGGATTCGAACCTACGTAG-3'; 55-1-SSR: 5'-AGTACAAGCTGAACCGAAATTTA-3'.

2. A kit for identifying pure heterozygous transgenic papaya 55-1 transformants, characterized in that: The kit comprises the primer set according to claim 1.

3. The kit according to claim 2, characterized in that The kit also includes conventional reagents for PCR amplification.

4. The kit according to claim 2 or 3, characterized in that Conventional reagents used for PCR amplification include: GOTaq DNA polymerase, 5×Green GO Taq Reaction buffer, dNTPs mix and sterile water.

5. A method for identifying pure heterozygous transgenic papaya 55-1 transformants, characterized in that: The method comprises using the primer set of claim 1 to perform PCR amplification on the DNA of the papaya to be tested, so as to determine the pure heterozygous state of the papaya 55-1 transformant to be tested.

6. The method for identifying pure heterozygous transgenic papaya 55-1 transformants according to claim 5, characterized in that: The pure heterozygous state of the papaya 55-1 transformant to be tested is determined by electrophoresis of the PCR amplification product, and the pure heterozygous state of the papaya to be tested is determined according to the electrophoresis detection result, specifically: When only a 694 bp band appears in the electrophoresis test results, the papaya material to be tested contains a homozygous 55-1 transformant; When the electrophoresis results show both 694 bp and 282 bp bands, the tested papaya contains a heterozygous 55-1 transformant; When only a 282 bp band appears in the electrophoresis detection result, the tested papaya does not contain the 55-1 transformant.

7. The method for identifying pure heterozygous transgenic papaya 55-1 transformants according to claim 6, characterized in that: Electrophoresis is the process of placing the PCR amplification product on an agarose gel and performing electrophoresis to obtain the electrophoresis test results.

8. The method for identifying pure heterozygous transgenic papaya 55-1 transformants according to any one of claims 5 to 7, characterized in that: The PCR amplification system includes: GO Taq DNA polymerase, 5×Green GO Taq Reaction buffer, dNTPs mix, primers 55-1-SSF, 55-1-SUT and 55-1-SSR, a DNA template of the papaya to be tested and sterile water.

9. The method for identifying pure heterozygous transgenic papaya 55-1 transformants according to claim 8, characterized in that: The specific PCR amplification system is as follows: 0.125 μL of GO Taq DNA polymerase with a concentration of 5U / μL, 5 μL of 5×Green GO TaqReaction buffer, 0.5 μL of dNTPs mix with a concentration of 10 mM, 1 μL each of primers 55-1-SSF, 55-1-SUT and 55-1-SSR with a concentration of 10 μmol / L, 2 μL of the DNA template of the papaya to be tested with a concentration of 100 ng / μL, and sterile water is added to 25 μL.

10. The method for identifying pure heterozygous transgenic papaya 55-1 transformants according to claim 5, 6, 7 or 9, characterized in that: The PCR amplification program was as follows: pre-denaturation at 94°C for 5 min; denaturation at 94°C for 30 s, annealing at 55°C for 30 s, extension at 72°C for 30 s, 35 cycles; and extension at 72°C for 5 min.

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