CN114891909B - Flanking sequences of exogenous inserted fragments of transgenic maize VP3C1 and their application - Google Patents

Abstract

The present invention relates to the flanking sequence and application of the exogenous insertion fragment of transgenic corn VP3C1. The present invention screens a transgenic event VP3C1 with significant anti-spodoptera frugiperda effect from corn plants transformed with Bt vip3Aa-m3 gene; the left border flanking sequence and the right border flanking sequence thereof are obtained by the chromosome walking method, as shown in the 1-756th position in SEQ ID NO.1 and the 359-932th position in SEQ ID NO.2, respectively; the VP3C1 exogenous fragment is a double copy unit site reverse insertion, and the 5' flanking sequence, the exogenous insertion sequence and the 3' flanking sequence on the corn genome are shown in SEQ ID NO.3. The flanking sequences at both ends can be used as specific detection sequences of the transformation event, and primers can be designed by the two flanking sequences to specifically detect the transgenic event VP3C1, and are applied to the development of a detection kit.

Description

Flanking sequences of exogenous inserted fragments of transgenic maize VP3C1 and their application

Technical Field

The invention belongs to the field of biotechnology, and particularly relates to transgenic insect-resistant corn plants and detection thereof.

Background Art

Corn (Zea mays L.) is native to Mexico and is commonly known as corn. It is an important subspecies of the genus Gemus Zea in the Poaceae family. Corn is a staple food crop and an important feed and industrial raw material. In recent years, the planting area and output of corn have gradually surpassed rice and wheat to become the largest in the country. However, corn insect pests have always been one of the important factors restricting corn production. Due to the occurrence of insect pests, corn production has been severely reduced or even completely lost, causing huge losses. Therefore, effective pest control is of great significance to agricultural production in my country and even the world.

There are many methods for pest control, such as physical control, chemical control, biological control, agricultural control, etc. At present, my country adopts chemical control methods on a large scale, that is, spraying chemical pesticides during plant growth. However, a series of environmental problems caused by excessive spraying of pesticides are particularly serious, such as land pollution, pesticide residues, pest resistance and ecological imbalance. At the same time, the use of pesticides has seriously affected food safety. In the 1990s, the rapid development of transgenic technology transferred exogenous insect-resistant Bt genes into corn to cultivate transgenic insect-resistant corn, providing a new way for pest control. Since the first Bt transgenic insect-resistant corn was approved for commercial planting in the United States in 1996, the global planting area of insect-resistant transgenic corn has increased year by year. The main insect-resistant genes used are cry1Ab, cry1Ac, cry9C, cry1F and vip3Aa20. The application of insect-resistant transgenic corn plays an important role in pest control. The domestic Bt transgenic insect-resistant corn started late, developed relatively slowly, has not yet been industrialized, and has urgent needs. At present, many domestic Bt corn products have obtained production and application safety certificates, which is a major step forward for the research and development and application of Bt corn in my country.

The fall armyworm (Spodoptera frugiperda) belongs to the genus Spodoptera of the family Noctuidae in the order Lepidoptera. It is a migratory agricultural pest with the characteristics of "international, explosive, migratory and destructive". Its larvae feed on corn and can eat up all the corn leaves in severe cases, causing corn yield reduction or even crop failure. The fall armyworm originated in the Americas and spread to Asia via Africa, where it developed resistance to a variety of insecticides. In December 2018, the fall armyworm was first discovered in Yunnan Province. In 2019, it advanced from south to north, invading the Yangtze River Basin, the Huanghuaihai Basin and the Northwest China. By October 2019, it had migrated and spread to 26 provinces, and the area of fall armyworm occurrence reached 1.065 million hectares [Wu Qiulin, Jiang Yuying, Wu Kongming. Analysis of the path of fall armyworm migration from Myanmar to China [J]. Plant Protection, 2019, 45(02): 1-6]. The fall armyworm has a particularly strong migratory ability, a wide range of spread, strong reproductive capacity, severe overeating damage, and is difficult to control, causing serious losses to my country's corn production. Few studies have been conducted on the control of the fall armyworm in China. Zhang Lei et al. conducted molecular identification of the fall armyworm invading Yunnan and found that the fall armyworm invading my country were all "corn-type" [Zhang Lei, Jin Minghui, Zhang Dandan, et al. Molecular identification of the fall armyworm invading Yunnan [J]. Plant Protection, 2019(2):7.]. Li Guoping et al. determined the sensitivity of fall armyworm larvae to five Bt proteins, including Cry1Ab, Cry1Ac, Cry1F, Cry2Ab and Vip3A, by the feed surface smear method and found that Vip3A protein ranked first in the lethal effect on fall armyworm [Li Guoping, Ji Tingjie, Sun Xiaoxu, et al. Evaluation of the sensitivity of the fall armyworm population invading Yunnan to five commonly used Bt proteins [J]. Plant Protection, 2019, 45(3):6.]. Existing research shows that Vip3A protein has a good insecticidal effect on the fall armyworm.

In previous studies, the vip3Aa11 gene derived from the Bt C9 strain was codon-optimized using corn-preferred codons, and a new gene was synthesized and named vip3Aa-m3 gene. A plant expression vector was constructed to transform corn to obtain vip3Aa-m3 gene-transgenic corn, and insect-resistant corn transformants with high resistance to the fall armyworm were obtained through insect resistance screening.

Since the position where the foreign gene is integrated into the corn genome will affect the expression of the foreign gene, if you want to obtain transgenic plants with high expression and good insect resistance, you need to screen from a large number of transformation events and undergo multiple generations of genetic stability testing to obtain insect-resistant corn with industrial prospects. At the same time, the flanking sequence of the transgenic corn event inserted into the corn genome can be used as an identity tag for the transgenic material, and an insertion site on the chromosome can be used as an independent transformation event, which can be detected using specific primers. The transgenic corn VP3C1 with the vip3Aa-m3 gene obtained by the present invention is a new type of transformation event, and its insertion position in the corn genome is different from other transgenic events, and its flanking sequence can be used as an identity tag for specific identification.

Summary of the invention

The present invention constructs a plant expression vector pBRI-IRGT5901 containing the Bt vip3Aa-m3 gene (a highly toxic insecticidal gene and application against fall armyworm artificially synthesized by Lang Zhihong et al., a national invention patent, application number: 202110008895.3), transforms corn to obtain corn plants containing the Bt vip3Aa-m3 gene, and screens out a transgenic event VP3C1 with significant resistance to fall armyworm; the position of the inserted fragment on the corn genome is obtained by the chromosome walking method, including the left border flanking sequence and the right border flanking sequence, and the flanking sequences at both ends can be used as specific detection sequences for this transformation event. Primers are designed by the two flanking sequences, and the transgenic event VP3C1 can be specifically detected, and applied to the development of detection kits.

The 5' flanking sequence of the exogenous insertion fragment of transgenic insect-resistant corn VP3C1 is shown at positions 1-756 in SEQ ID NO.1.

The 3’ flanking sequence of the exogenous inserted fragment of transgenic insect-resistant corn VP3C1 is shown at positions 359-932 in SEQ ID NO.2.

Specific primers for PCR reaction detection were designed based on the 5' flanking sequence of the exogenous insertion fragment of transgenic insect-resistant corn VP3C1.

The specific primers for the 5' flanking sequence are:

VP3C1-Left-F1:5'-GGCAAAGGACGGCAAAAGTA-3',

VP3C1-NOS-R1: 5'-TGAATCCTGTTGCCGGTCTT-3'.

Specific primers for PCR reaction detection were designed based on the 3’-end flanking sequence of the exogenous inserted fragment of transgenic insect-resistant corn VP3C1.

The specific primers for the 3' end flanking sequence are:

VP3C1-Ubi-F1: 5’-CCTGTTGTCAAAATACTCAATTGTCC-3’,

VP3C1-Right-R1: 5’-CCGTTCATGTGGTTTGTGCA-3’.

The PCR reaction detection method of transgenic insect-resistant corn VP3C1 is characterized in that the primers in the PCR reaction are the above-mentioned specific primers.

The specific primers are:

VP3C1-Left-F1:5'-GGCAAAGGACGGCAAAAGTA-3',

VP3C1-NOS-R1: 5'-TGAATCCTGTTGCCGGTCTT-3',

The amplified fragment size of transgenic insect-resistant corn VP3C1 is 1067 bp; or

VP3C1-Ubi-F1: 5’-CCTGTTGTCAAAATACTCAATTGTCC-3’,

VP3C1-Right-R1: 5’-CCGTTCATGTGGTTTGTGCA-3’,

The amplified fragment size of transgenic insect-resistant corn VP3C1 was 932 bp.

A kit for detecting insect-resistant corn, characterized by containing a specific primer for a 5' end flanking sequence and/or a specific primer for a 3' end flanking sequence.

The specific primers for the 5' flanking sequence are:

VP3C1-Left-F1：5'-GGCAAAGGACGGCAAAAGTA-3'

VP3C1-NOS-R1: 5'-TGAATCCTGTTGCCGGTCTT-3'

The specific primers for the 3' end flanking sequence are:

VP3C1-Ubi-F1: 5’-CCTGTTGTCAAAATACTCAATTGTCC-3’

VP3C1-Right-R1: 5’-CCGTTCATGTGGTTTGTGCA-3’

The above-mentioned flanking sequences, flanking sequence-specific primers, and insect-resistant corn detection kit are used in detecting genetically modified corn.

The present invention transforms the vector pBRI-IRGT5901 (structure shown in FIG1 ) into Agrobacterium EHA105 by freeze-thaw method, and performs PCR identification. Freshly peeled corn immature embryos of about 1.2 mm are used as materials. After the immature embryos are placed in an infection medium for one hour, they are washed once with the infection medium, and then immersed in an Agrobacterium bacterial solution added with 100 μM acetosyringone, and placed for 5 minutes. The immature embryos are taken out and blotted dry with sterile filter paper, placed on a co-culture medium, and co-cultured for 3 days under dark conditions at 26° C., and a control is set. The young embryos are then transferred to the recovery medium and cultured for 10 days until callus tissue is induced. The callus tissue is then removed from the buds and then transferred to the screening medium containing glyphosate. The callus tissue is subcultured every two weeks. After 6 weeks of screening, the resistant callus tissue is transferred to the regeneration medium for light differentiation. After about one week of light exposure, green buds begin to appear. The callus blocks are cut to separate the green buds and transferred to the regeneration medium for culture, which is conducive to the growth of the main stem. When the main stem is extended to 3-4 cm, it is transferred to the regeneration medium for rooting induction. When the corn plant grows strong and has a well-developed root system, it is transferred to a small flower pot in the greenhouse for growth. After two weeks of continuous culture, the transformed seedlings are transferred to the greenhouse ground after they grow well. After the female ear spins silk, it is covered with a paper bag. After the male ear scatters pollen, it is pollinated and the fruit is harvested.

The harvested T0 generation transgenic corn was planted in the field, and samples were taken when the corn grew to the six-leaf stage for indoor biological activity detection. The 4-day survival rate of transgenic corn VP3C1 against fall armyworm larvae was 0%, and the anti-fall armyworm effect was significant. The 5' end flanking sequence and 3' end flanking sequence of the exogenous insertion fragment of transgenic corn VP3C1 in the corn genome were obtained by chromosome walking. The flanking sequences at both ends can be used as specific detection sequences for this transformation event. Primers designed by the two flanking sequences can be used to specifically detect the transgenic event VP3C1 and are used in the development of detection kits.

Genetically modified maize VP3C1, classified as Zea mays

The accession number is CGMCC No.23074

Date of deposit: December 16, 2021

Depository: China National Microbiological Culture Collection Administration General Microbiology Center

Deposit address: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, 100101, China

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 Schematic diagram of the plant expression vector pBRI-IRGT5901 used for corn transformation,

Figure 2 PCR test results of maize transformed plants, where M is Trans5K DNA Marker; CK+ is a positive control, amplified using plasmid pBRI-IRGT5901 as a template; CK- is amplified using genomic DNA of non-transgenic plants as a template; Blank is a blank control, amplified using water as a template; 1-9 is amplified using genomic DNA of maize transformed with vip3Aa-m3 gene as a template,

Figure 3 Indoor insect resistance evaluation of transgenic corn VP3C1, a: Indoor test of non-transgenic control corn against fall armyworm, b: Indoor test of VP3C1 transgenic corn against fall armyworm,

Figure 4 Schematic diagram of restriction enzyme cleavage sites for inserted fragments in plant expression vectors,

Figure 5 Southern blot hybridization results of transgenic corn VP3C1, where CK+ is the vip3Aa-m3 expression cassette, VP3C1 transformant genomic DNA was digested with Hind III, Kpn I and Nco I, CK- is non-transgenic corn digested with Hind III, Blank is the blank control,

Figure 6 Schematic diagram of the structure of the exogenous inserted fragment of transgenic corn VP3C1,

Figure 7 The location of the exogenous insertion fragment of transgenic corn VP3C1 in the corn genome.

Figure 8 is a PCR detection diagram of the flanking sequence at the 5' end of the inserted fragment, M is the Trans5K DNA Marker, and 1 is the PCR amplification product. Figure 9 is a PCR detection diagram of the flanking sequence at the 3' end of the inserted fragment, M is the Trans5K DNA Marker, and 1 is the PCR amplification product.

DETAILED DESCRIPTION

The present invention is further described in detail below in conjunction with the embodiments.

Example 1. Obtaining transgenic maize event VP3C1

1. Construction of plant expression vector for corn transformation

The plant expression vector used in this study is pBRI-IRGT5901 (deposited at the Institute of Biotechnology, Chinese Academy of Agricultural Sciences, and available for public release), as shown in Figure 1. The backbone of the plant expression vector is pPZP200, and the ubiquitin promoter from maize ubiquitin protein, the modified Bt vip3Aa-m3 gene, and the NOS terminator are inserted into the multiple cloning site of the vector. The gene sequence is shown in the patent application "Artificially synthesized highly toxic insecticidal genes against fall armyworm and their applications" (application number: 202110008895.3); the herbicide-resistant gene is the cp4-epsps gene, which encodes 5-enolpyruvyl-shikimate-3-phosphate synthase, with a gene size of 1368 bp and encoding 455 amino acids. Its encoded product, CP4-EPSPS protein, makes glyphosate non-toxic to plants, and obtains transgenic corn tolerant to glyphosate.

2. Agrobacterium transformation of maize embryos to obtain transformed plants

The vector pBRI-IRGT5901 was transformed into Agrobacterium EHA105 by freeze-thaw method, and positive Agrobacterium colonies were obtained by PCR identification. Freshly peeled maize embryos of about 1.2 mm were used as materials. After the embryos were placed in the infection medium for one hour, they were washed once with the infection medium, and then immersed in the Agrobacterium liquid supplemented with 100 μM acetosyringone, and left for 5 minutes. Take out and dry with sterile filter paper, place on the co-culture medium, and co-culture at 26°C in the dark for 3 days, and set up a control. The young embryos are then transferred to the recovery medium and cultured for 10 days until callus tissue is induced. The callus tissue is then removed from the buds and then transferred to the screening medium containing glyphosate. The callus tissue is subcultured every two weeks. After 6 weeks of screening, the resistant callus tissue is transferred to the regeneration medium for light differentiation. After about one week of light exposure, green buds begin to appear. The callus blocks are cut to separate the green buds and transferred to the regeneration medium for culture, which is conducive to the growth of the main stem. When the main stem is extended to 3-4 cm, it is transferred to the regeneration medium for rooting induction. When the corn plant grows strong and has a well-developed root system, it is transferred to a small flower pot in the greenhouse for growth. After two weeks of continuous culture, the transformed seedlings are transferred to the greenhouse ground after they grow well. After the female ear spins silk, it is covered with a paper bag. After the male ear scatters pollen, it is pollinated and the fruit is harvested.

3. PCR detection of transformed plants

3.1 Extraction of small amount of corn genomic DNA (the kit was purchased from Beijing Puboxin Biological Company):

(1) Take about 100 mg of fresh plant tissue or about 30 mg of dry weight tissue, add liquid nitrogen and grind thoroughly.

(2) The ground powder was quickly transferred to a centrifuge tube pre-filled with 700 μL of 65°C preheated buffer GP1 (mercaptoethanol was added to the preheated GP1 before the experiment to make the final concentration 0.1%), and the centrifuge tube was quickly inverted to mix. Then, the centrifuge tube was placed in a 65°C water bath for 20 min. During the water bath, the centrifuge tube was inverted several times to mix the sample.

(3) Add 700 μL of chloroform, mix thoroughly, and centrifuge at 12,000 rpm for 5 min.

(4) Carefully transfer the upper aqueous phase obtained in the previous step into a new centrifuge tube, add 700 μL of buffer GP2, and mix thoroughly.

(5) Transfer the mixed liquid into adsorption column CB3, centrifuge at 12,000 rpm for 30 seconds, and discard the waste liquid.

(6) Add 500 μL of buffer GD to the adsorption column CB3 (please check whether anhydrous ethanol has been added before use), centrifuge at 12,000 rpm for 30 s, discard the waste liquid, and place the adsorption column CB3 in a collection tube.

(7) Add 600 μL of rinse solution PW to the adsorption column CB3 (please check whether anhydrous ethanol has been added before use), centrifuge at 12,000 rpm for 30 s, discard the waste liquid, and place the adsorption column CB3 in a collection tube.

(8) Repeat step 7.

(9) Place the adsorption column CB3 back into the collection tube, centrifuge at 12,000 rpm for 2 min, and discard the waste liquid. Place the adsorption column CB3 at room temperature for several minutes to completely dry the residual rinse liquid in the adsorption material, and add 40 μL ddH ₂ O to elute the DNA.

3.2 Primers for amplifying the vip3Aa-m3 gene fragment:

vip3Aa-F1:5'-AAATCACTCCCGCCTACCAA-3'

vip3Aa-R1:5'-TGATTGGGGTCAGGAAGGTC-3'

Target fragment size: 966bp

3.3 PCR reaction system is as follows:

The amplification conditions were as follows: pre-denaturation at 94°C for 5 min; denaturation at 94°C for 20 s, annealing at 56°C for 20 s, extension at 72°C for 1 min, and 30 amplification cycles; and finally extension at 72°C for 5 min.

The PCR-positive plants can amplify a band with the same size as the positive control, and the test results are shown in Figure 2. All the 9 transformed plants tested were transgenic plants.

4. Identification of insect resistance of transgenic plants

The insect resistance of transgenic corn with vip3Aa-m3 gene was tested, and the VP3C1 transgenic event had high insecticidal activity against fall armyworm. VP3C1 transgenic corn at the heart leaf stage (6-8 leaves) and the corresponding non-transgenic corn Zheng 58 were taken from the field, and their heart leaves were cut into ^1cm2 size and placed in a 24-well insect inoculation plate. A 1-day-old newly hatched larva was placed in each hole, and three biological replicates were performed for each treatment. The 24-well insect inoculation plate was placed in a culture room with a temperature (26±1°), humidity of 60%±10%, and a photoperiod (L//D=16h//8h). After the heart leaf stage, the feeding of fall armyworm on leaves was investigated for 6 consecutive days, the survival of fall armyworm was recorded, and insect resistance was graded. As can be seen from Figure 3, Figure 3a shows non-transgenic corn leaves fed to fall armyworms. The leaves were severely damaged by fall armyworms, but all the fall armyworms survived and grew well. Figure 3b shows VP3C1 transgenic corn leaves fed to fall armyworms. The leaves were not damaged, but the insect bodies turned black and died.

The results of the investigation on the resistance of fall armyworm at the heart leaf stage showed (Table 1) that all the fall armyworm larvae that fed on transgenic corn VP3C1 died on the 4th day, and the larval survival rate was significantly different from that of the control material.

Table 1 Insecticidal effect of transgenic corn VP3C1 on fall armyworm larvae (larvae survival rate %)

Based on the survival rates of the fall armyworm of transgenic corn VP3C1 and the control material Zheng 58, the in vitro resistance level of the transgenic corn VP3C1 was qualitatively judged. The results showed that the resistance level of the transgenic corn VP3C1 to the fall armyworm reached "high resistance", while Zheng 58 was "highly susceptible".

Table 2 Determination of resistance level in vitro

Example 2: Southern blot detection of the copy number of the exogenous gene in transgenic maize VP3C1

1. Large-scale extraction of corn genomic DNA (slightly improved CTAB method):

(1) Weigh 5 g of leaves, grind them into powder with liquid nitrogen, and transfer them into a 50 mL centrifuge tube;

(2) Add 15 mL of 2×CTAB buffer (Tris 100 mM, NaCl 1.4 M, 20 mM EDTA, 2% CTAB, 0.1% mercaptoethanol), mix thoroughly, and incubate in a water bath at 65°C for 1 hour;

(3) After cooling to room temperature, add 15 mL of chloroform/isoamyl alcohol (24:1), mix by inverting until an emulsion is obtained, and leave at room temperature for 15 to 60 minutes;

(4) Centrifugation at 12,000 rpm for 15 min at room temperature;

(5) After transferring the supernatant into a clean centrifuge tube, add 2/3 volume of isopropanol, invert the tube several times, pick out the DNA, and place it into a clean 1.5 mL centrifuge tube;

(6) Wash the DNA twice with 70% ethanol and air dry for 1 hour;

(7) After dissolving the DNA in 500 μL TE, add 5 μL RNase A (10 mg/mL) and dissolve it at 4°C overnight or at 37°C for 1 hour;

(8) Add 500 μL of phenol, mix thoroughly by inversion, centrifuge at 12,000 rpm for 5 min, and transfer the supernatant to another centrifuge tube;

(9) Add 250 μL of phenol and chloroform respectively, mix thoroughly by inversion, centrifuge at 12,000 rpm for 5 min, and transfer the supernatant to another centrifuge tube;

(10) Add 500 μL of chloroform, mix thoroughly by inversion, centrifuge at 12,000 rpm for 5 min, and transfer the supernatant to a 10 mL centrifuge tube;

(11) Add TE to 3 mL, then add 2 volumes of anhydrous ethanol and 1/10 volume of 3 M NaAc, and invert several times;

(12) Wash the precipitated DNA twice with 70% ethanol; transfer the DNA to a 1.5 mL centrifuge tube, air dry and dissolve in 500 μL TE, quantify the DNA and set aside.

2. Preliminary experiment of small amount of genomic DNA digestion:

After mixing, digest at 37°C for 2-3 hours; the digestion reaction products were separated by electrophoresis on 0.7% agarose to check the digestion effect.

3. Large-scale digestion of genomic DNA:

Genomic DNA 100 μg

Enzyme (10U/μL) 5μL

(This experiment uses Hind III, Kpn I and Nco I enzymes for digestion)

10×Buffer 40μL

Total 400μL

After mixing, digest at 37℃ for 10 hours; take 2μL of the digestion product for electrophoresis separation to check the digestion effect; after the digestion is complete, precipitate the digestion product, add 1/10 volume of 3M NaAc and 2 volumes of anhydrous ethanol (pre-cooled at -20℃), mix well, and place at -20℃ for 2 hours; centrifuge at 12,000rpm, 4℃ for 20min, discard the supernatant, add 1ml 70% ethanol to the precipitate, centrifuge at 12,000rpm for 2min, discard the supernatant, blow dry the precipitate and dissolve it in 30μL _ddH2O for later use.

4. Preparation of probe

The probe was prepared according to the instructions of PCR DIG Probe Synthesis Kit.

Primers:

Vip3A-probe-F1:5'-AAATCACTCCCGCCTACCAA-3'

Vip3A-probe-R1:5'-TGATTGGGGTCAGGAAGGTC-3'

Target fragment size: 966bp

The PCR reaction system is as follows:

The PCR reaction conditions are as follows:

After PCR, the DIG-labeled probe was detected by electrophoresis and its concentration was determined.

5. Southern blot hybridization

(1) Prepare 0.7% agarose gel, add 6 μL 6× loading buffer to 30 μL genomic DNA digestion product for electrophoresis separation, let stand for 10 min after loading, use low voltage at the beginning, and increase the voltage to 50 V after bromophenol blue runs out of the sample well for 2 to 3 cm, and perform electrophoresis for 5 to 6 hours;

(2) After electrophoresis, the gel was treated in the following order: soak the gel in 0.125 M hydrochloric acid for 10 min, so that the bromophenol blue in the gel turns yellow; treat the gel with distilled water for 5 min; soak the gel in neutralizing solution for 30 min;

(3) Transfer the DNA to the nylon membrane using the capillary transfer method (for detailed procedures, see the Molecular Cloning Experiment Manual);

(4) After the transfer, soak the nylon membrane in 6×SSC for 5 min and place the nylon membrane in a clean bench to air dry or air dry at room temperature;

(5) Baking the film at 80°C for 2 hr to fix the DNA sample;

(6) Preparation of DIG-labeled probe: The probe preparation method in the PCR DIG Probe Synthesis Kit (purchased from Roche) was used, using plasmid pBRI-IRGT5901 as a template, Vip3A-probe-F1 and Vip3A-probe-R1 as primers, and the probe size was 966 bp. The concentration of the probe was determined;

(7) Prehybridization: Use tweezers to carefully place the nylon membrane into the hybridization tube, being careful not to create bubbles, and then add 10 mL of DIG Easy Hyb hybridization solution (Digoxigenin Labeling and Detection Kit II purchased from Roche) preheated at 42°C, and prehybridize at 42°C for 3 hours;

(8) Hybridization: First, treat the probe. Denature the labeled probe at 99°C for 6 min and immediately cool it in ice for 2 min. Take 10 mL of DIG Easy Hyb hybridization solution, add the treated probe (25 ng/ml Hyb hybridization solution), mix gently and be careful not to generate bubbles, place in a hybridization oven, and hybridize at 46°C for 16 to 20 hours;

(9) Wash the membrane: Wash twice with 50 mL of 2×SSC, 0.1% SDS solution at room temperature, 15 min each time. Then wash twice with 50 mL of 0.5×SSC, 0.1% SDS solution at 65°C, 30 min each time. Use tweezers to carefully remove the membrane and transfer it to a plate containing 50 mL of washing buffer and wash with shaking for 1 to 5 min.

(10) Incubate with 100 ml of 1× Blocking solution at room temperature for 60 min.

(11) Incubate with 20 ml of Antibody solution for 30 min;

(12) Wash twice with 50 ml washing buffer, 30 min each time;

(13) Equilibrate in 20 ml detection buffer for 2–5 min.

(14) Use tweezers to place the membrane flat between two layers of plastic wrap. Lift the upper layer of plastic wrap first, then add 1 ml of CSPD substrate and slowly lower the upper layer of plastic wrap from one end so that the substrate evenly covers the surface of the membrane. Let it stand at room temperature for 5 min.

(15) Use a glass rod to remove excess liquid, absorb the substrate outside the membrane with filter paper, and incubate at 37°C for 10 min.

(16) Place the sealed nylon film in a photo folder, press it with X-ray film in a dark room, and perform exposure, development, and fixing.

The Southern blot results of the transgenic corn event VP3C1 were analyzed: according to the restriction sites of the inserted fragment in Figure 4, three restriction endonucleases HindIII, KpnI, and NcoI were selected to digest the corn genomic DNA. Among them, HindIII has only one restriction site on the vector, which can be used as a restriction endonuclease to identify the number of vip3Aa-m3 copies; although KpnI has multiple restriction sites, they are all on the side of the vip3Aa-m3 gene, which can be used as a restriction endonuclease to identify the number of vip3Aa-m3 copies. The vip3Aa-m3 gene fragment labeled with digoxin of 966bp was hybridized as a probe, and the results showed that the restriction products of Hind III and Nco I hybridized to 1 band, and the restriction product of KpnI hybridized to two bands (Figure 5). The preliminary results showed that the vip3Aa-m3 gene may be inserted into the corn genome in 2 copies.

Example 3: Genetic analysis of VP3C1 transgenic corn

Genetic analysis of VP3C1 transgenic corn: VP3C1 was backcrossed with Zheng 58 inbred line for two generations for segregation identification. Among the 277 BC2F1 offspring tested, 140 were positive plants, and the ratio of positive plants to negative plants was 1:1, proving that the exogenous gene unit site was inserted on the same chromosome. The exogenous gene was positively identified in the three generations of backcross offspring. Among the 372 BC3F1 offspring tested, 185 were positive, and the ratio of positive to negative plants was in line with the expected 1:1, which was in line with Mendel's law of inheritance, proving that the exogenous gene unit site was inserted on the same chromosome (Table 3).

Table 3 Genetic analysis of VP3C1 transgenic maize

Example 4: Obtaining the left and right border flanking sequences of the transgenic event VP3C1 by chromosome walking

Southern blot and genetic analysis showed that the exogenous fragment inserted into the maize genome was a double copy insertion at a single site, and the two copies were inserted in an end-to-end manner (Figure 6). Because the ubiquitin promoter comes from the maize ubiquitin protein gene, which is contained in the maize genome, it is not easy to obtain the right border flanking sequence using the chromosome walking method. Therefore, primers were designed starting from the cp4-epsps gene to amplify the left border flanking sequence from the 5' end to the 3' end of the cp4-epsps gene.

1. Obtaining the left border flanking sequence of the transgenic event VP3C1 by chromosome walking

The specific primers for amplifying the left border flanking sequence were designed as follows:

CP4-SP1: 5'-GCCAACCTTACCGTCGAGA-3' (located on the cp4-epsps gene)

CP4-SP2: 5'-CTCAACGTGCTGATGAACCC-3' (located on the cp4-epsps gene)

NOS-SP3: 5'-CATTTAATACGCGACGCGATAGAA-3' (located on the NOS terminator)

The Genome Walking Kit was purchased from TaKaRa. The kit contains four degenerate primers. Two rounds of amplification were performed using CP4-SP1 and CP4-SP2 and four degenerate primers (AP1, AP2, AP3, and AP4), respectively. Based on the amplification effect, the AP2 primer was finally selected. The third round of amplification was performed using the AP2 primer and NOS-SP3, and the PCR products were sequenced.

(1) First round of PCR reaction

Using CP4-SP1 as the upstream primer and the four degenerate primers as the downstream primers, AP2 was used as an example to carry out the first round of PCR reaction.

Reaction system:

Reaction conditions:

(2) Second round of PCR reaction

Take 5 μL of the first round PCR reaction product for electrophoresis, and select the dilution multiple according to the brightness of the first round electrophoresis band. Take 1 μL of the first round PCR reaction dilution product as a template for the second round of PCR reaction, using CP4-SP2 as the upstream primer and the four degenerate primers as the downstream primers, and take AP2 as an example to perform the second round of PCR reaction.

Reaction system:

Reaction conditions:

(3) The third round of PCR reaction

Take 5 μL of the second round PCR reaction product for electrophoresis, and select the dilution multiple according to the brightness of the electrophoresis band in round 2. Take 1 μL of the second round PCR reaction diluted product as a template for the third round of PCR reaction, using NOS-SP3 as the upstream primer and AP2 as the downstream primer for the third round of PCR reaction.

Reaction system:

Reaction conditions:

(4) The third-round PCR reaction products were subjected to electrophoresis and clear electrophoresis bands were recovered. The third-round PCR products were sequenced using NOS-SP3 as primers.

By comparison, the exogenous insertion fragment was reversely inserted, and the flanking sequence of the exogenous insertion fragment at the 5' end of the corn genome was obtained. The flanking sequence of the left border of the exogenous insertion fragment is shown in SEQ ID NO.1, 1-756 is the chromosome 6 of the corn genome Chr6: 172146256-171147011 (Zm-B73-REFERENCE-NAM-5.0), 757-913 is the vector sequence (reverse complementary chain), and the left border (LB) of the vector sequence is missing 26bp at the 5' end.

2. Obtaining the right border flanking sequence of the inserted fragment

According to the obtained left border flanking sequence, the known maize genome sequence was retrieved, and specific primers were designed to perform PCR amplification on the ubiquitin promoter sequence of the vector and the inferred right border flanking sequence.

Specific primers:

VP3C1-Ubi-F1: 5’-CCTGTTGTCAAAATACTCAATTGTCC-3’ (on the ubiquitin promoter sequence)

VP3C1-Right-R1: 5'-CCTGTCATGTGGTTTGTGCA-3' (on the maize genome)

Reaction system:

Reaction conditions:

The PCR product was subjected to DNA sequencing, and the sequencing result is shown in SEQ ID NO.2. After sequence alignment, 1-358 of SEQ ID NO.2 is a vector sequence, and 39 bp is missing at the 3' end of the right border of the vector sequence (including the right border, RB), and 359-932 of SEQ ID NO.2 is a maize genome sequence, and the maize genome sequence is chromosome 6 Chr6:172147036-172147609 (Zea mays (B73_RefGen_v5). Due to the insertion of exogenous fragments, there is a 24 bp deletion in the maize genome, corresponding to chr6:172147012-172147035 (Zea mays (B73_RefGen_v5)).

The position of the inserted fragment in corn is shown in Figure 7. The full sequence of the transgenic corn VP3C1 exogenous inserted gene sequence and the 5' flanking sequence and 3' flanking sequence of the corn genome is shown in SEQ ID NO.3, where positions 1-756 are the corn genome sequence (5' flanking sequence) and positions 19178-19751 are the corn genome sequence (3' flanking sequence).

Example 5: Application of transgenic maize event VP3C1 flanking sequences

In order to specifically detect VP3C1 transgenic maize events, specific primers were designed according to the 5' flanking sequence and the inserted fragment sequence. The designed primers are as follows:

VP3C1-Left-F1: 5'-GGCAAAGGACGGCAAAAGTA-3' (maize genome)

VP3C1-NOS-R1: 5'-TGAATCCTGTTGCCGGTCTT-3' (vector NOS sequence)

Fragment size: 1067 bp

Reaction system:

Reaction conditions:

The PCR product was detected by 1% agarose gel electrophoresis. The detection result is shown in FIG8 , and a band of 1067 bp was amplified.

Design specific primers according to the 3' flanking sequence and the inserted fragment sequence. The primers are designed as follows:

VP3C1-Ubi-F1: 5’-CCTGTTGTCAAAATACTCAATTGTCC-3’ (on the ubiquitin promoter)

VP3C1-Right-R1: 5'-CCTGTCATGTGGTTTGTGCA-3' (on the maize genome)

Fragment size: 932 bp

Reaction system:

Reaction conditions:

The PCR product was detected by 1% agarose gel electrophoresis. The result is shown in FIG9 , and a band of 932 bp was amplified.

Because the insertion site of the transgenic event in this study is specific, specific primers can be designed using the 5’ flanking sequence and the vector sequence and the 3’ flanking sequence and the vector sequence to detect the transformation event, and a detection kit can be developed using the specific primers.

Sequence Listing

<110> Institute of Biotechnology, Chinese Academy of Agricultural Sciences

Sanya National South China Agricultural University

<120> Flanking sequences of exogenous inserted fragments of transgenic corn VP3C1 and their application

<130> PP22002-SWJ

<141> 2022-02-21

<160> 3

<170> SIPOSequenceListing 1.0

<210> 1

<211> 913

<212> DNA

<213> Artificial Sequence

<400> 1

tcttccccaa cctcggccac aagtgcctca tggcaaagga cggcaaaagt aagaaggtta 60

aatctaaatc ctccactaga tatgagtcct ctagtgatgt taatgctagt gatgaggaag 120

ataatttgcg tacccttttt gccaacctta acatggaaca aaaggaaaaa ttaaatgaat 180

taattagtgc tattcatgaa aaggatgacc ttttggattc tcaagaggac ttcctaatta 240

aggaaaataa gaaacatgtt aaggttaaaa atgctttatgc cctagaaatt gagaaatgtg 300

aaaaattatc tagtgagcta agcacttgcc atgagacaat agacaacctt agaaatgaaa 360

atgctaattt gttagctaag gttgattctc atgtttgcaa tgtttcaact accaattcta 420

gaaataatga tgatgattta cttgctagaa ttgaagaatt gaacatttct cttgctagcc 480

ttaggattga aaatgaaaaa ttacttgcta aggctaaaga ttttgatgtt tgcaatgctg 540

ctatttccga ccttagaact aagaatgata tgttacatgc taaggttgta gaactaaaat 600

cttgcaaaac ctctacatct aatgttgagc atgtttctat ttgtactaga tgtagagatg 660

ttgatatcaa tgctattcat gatcacatgg ttttaattaa acaacaaaat gatcatatag 720

caaaattaga tgctaaaatt gccgagcata acttagaaat tgacgcttag acaacttaat 780

aacacattgc ggacgttttt aatgtactga attaacgccg aattgaggta cccaccatcc 840

gcggggatatc accactttgt acaagaaagc tgggtccgat ctagtaacat agatgacacc 900

gcgcgcgata att 913

<210> 2

<211> 932

<212> DNA

<213> Artificial Sequence

<400> 2

cctgttgtca aaatactcaa ttgtccttta gaccatgtct aactgttcat ttatatgatt 60

ctctaaaaca ctgatattat tgtagtacta tagattat tattcgtaga gtaaagttta 120

aatatatgta taaagataga taaactgcac ttcaaacaag tgtgacaaaa aaaatatgtg 180

gtaatttttt ataacttaga catgcaatgc tcattatctc tagagagggg cacgaccggg 240

tcacgctgca ctgcaggcat gcagcctgct tttttgtaca aacttgtgat atcactagtg 300

cggccaagct tagcttagct tgagcttgga tcagattgtc gtttcccgcc ttcagtttaa 360

gtatgctcta taatgggaga cgccctggca tcaaggatgg cattggcttc caaaggggag 420

acaatgtcaa acttaatgcc cctcctaaga acttgtctaa ctttgttaag ggcaaggctc 480

ccatgcctca ggataacgag ggttacattt tgtaccctgc cggctatcct gagagcaaaa 540

ttaggagaac tcattctagg aagtctcact ctggccctaa ttatgctttt atgtataagg 600

gtgagacatc tagctctagg caaccaaccc gtgccaagtt gcctagaaag aaaactccta 660

gtgcatcaaa tgaacatagc ctttcattta aaacttttga tgcatctttat gtgttgacta 720

acaaatccga caaagtagtt gccaaatatg ttgggggcaa gcacaagggg tcaaagactt 780

gtgtttgggt acccaaagtt ctagtgtcta atgccaaagg acccaaaacc atttgggtac 840

ctaaagtcaa gaactaaaat tgttttgtag gtttatgcat ccgggggctc aagttggatc 900

atcgacagcg ggtgcacaaa ccacatgaca gg 932

<210> 3

<211> 19751

<212> DNA

<213> Artificial Sequence

<400> 3

tcttccccaa cctcggccac aagtgcctca tggcaaagga cggcaaaagt aagaaggtta 60

aatctaaatc ctccactaga tatgagtcct ctagtgatgt taatgctagt gatgaggaag 120

ataatttgcg tacccttttt gccaacctta acatggaaca aaaggaaaaa ttaaatgaat 180

taattagtgc tattcatgaa aaggatgacc ttttggattc tcaagaggac ttcctaatta 240

aggaaaataa gaaacatgtt aaggttaaaa atgctttatgc cctagaaatt gagaaatgtg 300

aaaaattatc tagtgagcta agcacttgcc atgagacaat agacaacctt agaaatgaaa 360

atgctaattt gttagctaag gttgattctc atgtttgcaa tgtttcaact accaattcta 420

gaaataatga tgatgattta cttgctagaa ttgaagaatt gaacatttct cttgctagcc 480

ttaggattga aaatgaaaaa ttacttgcta aggctaaaga ttttgatgtt tgcaatgctg 540

ctatttccga ccttagaact aagaatgata tgttacatgc taaggttgta gaactaaaat 600

cttgcaaaac ctctacatct aatgttgagc atgtttctat ttgtactaga tgtagagatg 660

ttgatatcaa tgctattcat gatcacatgg ttttaattaa acaacaaaat gatcatatag 720

caaaattaga tgctaaaatt gccgagcata acttagaaat tgacgcttag acaacttaat 780

aacacattgc ggacgttttt aatgtactga attaacgccg aattgaggta cccaccatcc 840

gcggggatatc accactttgt acaagaaagc tgggtccgat ctagtaacat agatgacacc 900

gcgcgcgata atttatccta gtttgcgcgc tatattttgt tttctatcgc gtcgcgtatt 960

aaatgtataa ttgcgggact ctaatcataa aaacccatct cataaataac gtcatgcatt 1020

acatgttaat tattacatgc ttaacgtaat tcaacagaaa ttatatgata atcatcgcaa 1080

gaccggcaac aggattcaat cttaagaaac tttattgcca aatgtttgaa cgatcgggga 1140

aattcgagct ctttaaattt tttttttttt tttttttttg ttaaattttt tttttttttt 1200

ttttttttgt taacttgatg tccgaaaaca aaactgaaag aacacagtaa attacaagca 1260

gaacaatggc ttttccaatg ccataatact caaagttaac cttactcagc ctcgaggggg 1320

ggcccggtac ctcatcaggc agccttcgta tcggagagtt cgatcttcgc gcccagcccg 1380

gccatcaggt ccatgaactc cgggaagctc gtggcgatca tcgtggcatc gtccaccgtg 1440

acagggtttt ccgacacgag gcccatgacg aggaagctca tggcgatgcg gtgatcgaga 1500

tgggtggcga cggcggcgcc cgaggcgttg ccgagcccct tgccgtcagg gcggccacgc 1560

acgacgagcg acgtctcgcc ctcatcgcaa tccacgccat tgagcttgag gccattggcg 1620

acggccgaga ggcggtcgct ttccttgacg cggagttctt ccagaccgtt catcacggtc 1680

gccccttccg cgaaggcggc ggcgacagcg agaatcggat attcgtcgat catcgaaggc 1740

gcgcggtctt ccggcaccgt gacgcccttc agcgtggagg agcgaacgcg caggtccgcc 1800

acgtcttcgc cgccggcaag gcgcgggttg atgacttcga tgtcggcgcc catttcctgc 1860

agcgtcagga tgaggccggt gcgggtgggg ttcatcagca cgttgaggat ggtgacgtcg 1920

gagcccggaa caagcagggc cgcaaccagc gggaaggccg tcgaggacgg gtcgcccggc 1980

acgtcgatga cttggccggt gagcttgccg cggccttcca ggcggatggt gcgcacgccg 2040

tccgcatccg tctcgacggt aaggttggcg ccaaagccct gcagcatctt ttccgtatga 2100

tcgcgcgtca tgatcggctc gatgaccgtc gtgatgccgg gcgtgttgag gccggcgagc 2160

agcacggcgg acttcacctg tgcggaggcc atcggcacgc ggtaggtgat cggcgtcggc 2220

gtcttcggcc cgcgcaaggt aacgggaaga cggtcaccgt cttccgattt cacctgcacg 2280

cccattcgc gcagcgggtt caacacgcgg cccatcgggc gctttgtgag cgaggcgtcg 2340

ccgatgaagg tgctgtcgaa atcgtagacc ccgacgaggc ccatcgtcag gcggcagccc 2400

gtggcggcat tgccgaaatc gagcggcgcc tcaggcgcca ggaggccgcc attgccgacg 2460

ccatcgatga tccaggtgtc gccttcctta cggatgcggg cgcccatcgc ctgcatggcc 2520

ttgcccgtat tgatgacgtc ctcgccttcc agaaggccgg tgatgcgcgt ttcaccgctc 2580

gcgagaccgc cgaacatgaa ggaccggtgg gagatcgact tgtcgccggg aatgcggacg 2640

gttccggaaa ggccagagga tttgcgggcg gttgcgggcc ggctgcttgc accgtgaagc 2700

atgcacgccg tggaaacaga agacatgacc ttaagaggac gaagctcaga gccaattaac 2760

gtcatcccac tcttcttcaa tccccacgac gacgaaatcg gataagctcg tggatgctgc 2820

tgcgtcttca gagaaaccga taagggagat ttgcgttgac tggatttcga gagattggag 2880

ataagagatg ggttctgcac accattgcag attctgctaa cttgcgccat actagtggtt 2940

attgtaaata gtaattgtaa tgttgtttgt tgtttgttgt tgttggtaat tgttgtaaaa 3000

atagtcgacc tgcagaagta acaccaaaca acagggtgag catcgacaaa agaaacagta 3060

ccaagcaaat aaatagcgta tgaaggcagg gctaaaaaaa tccacatata gctgctgcat 3120

atgccatcat ccaagtatat caagatcaaa ataattataa aacatacttg tttattataa 3180

tagataggta ctcaaggtta gagcatatga atagatgctg catatgccat catgtatatg 3240

catcagtaaa acccacatca acatgtatac ctatcctaga tcgatatttc catccatctt 3300

aaactcgtaa ctatgaagat gtatgacaca cacatacagt tccaaaatta ataaatacac 3360

caggtagttt gaaacagtat tctactccga tctagaacga atgaacgacc gcccaaccac 3420

accacatcat cacaaccaag cgaacaaaaa gcatctctgt atatgcatca gtaaaacccg 3480

catcaacatg tatacctatc ctagatcgat atttccatcc atcatcttca attcgtaact 3540

atgaatatgt atggcacaca catacagatc caaaattaat aaatccacca ggtagtttga 3600

aacagaatta attctactcc gatctagaac gaccgcccaa ccagaccaca tcatcacaac 3660

caagacaaaa aaaagcatga aaagatgacc cgacaaacaa gtgcacggca tatattgaaa 3720

taaaggaaaa gggcaaacca aaccctatgc aacgaaacaa aaaaaatcat gaaatcgatc 3780

ccgtctgcgg aacggctaga gccatcccag gattccccaa agagaaacac tggcaagtta 3840

gcaatcagaa cgtgtctgac gtacaggtcg catccgtgta cgaacgctag cagcacggat 3900

ctaacacaaa cacggatcta acacaaacat gaacagaagt agaactaccg ggcccctaacc 3960

atggaccgga acgccgatct agagaaggta gagagggggg gggggggagg acgagcggcg 4020

taccttgaag cggaggtgcc gacgggtgga tttgggggag atctggttgt gtgtgtgtgc 4080

gctccgaaca acacgaggtt ggggaaagag ggtgtggagg gggtgtctat ttattacggc 4140

gggcgaggaa gggaaagcga aggagcggtg ggaaaggaat cccccgtagc tgccggtgcc 4200

gtgagaggag gaggaggccg cctgccgtgc cggctcacgt ctgccgctcc gccacgcaat 4260

ttctggatgc cgacagcgga gcaagtccaa cggtggagcg gaactctcga gaggggtcca 4320

gaggcagcga cagagatgcc gtgccgtctg cttcgcttgg cccgacgcga cgctgctggt 4380

tcgctggttg gtgtccgtta gactcgtcga cggcgtttaa caggctggca ttatctactc 4440

gaaacaagaa aaatgtttcc ttagtttttt taatttctta aagggtattt gtttaatttt 4500

tagtcacttt attttattct attttatatc taaattatta aataaaaaaa ctaaaataga 4560

gttttagttt tcttaattta gaggctaaaa tagaataaaa tagatgtact aaaaaaatta 4620

gtctataaaa accattaacc ctaaacccta aatggatgta ctaataaaat ggatgaagta 4680

ttatataggt gaagctattt gcaaaaaaaa aggagaacac atgcacacta aaaagataaa 4740

actgtagagt cctgttgtca aaatactcaa ttgtccttta gaccatgtct aactgttcat 4800

ttatatgatt ctctaaaaca ctgatattat tgtagtacta tagattat tattcgtaga 4860

gtaaagttta aatatatgta taaagataga taaactgcac ttcaaacaag tgtgacaaaa 4920

aaaatatgtg gtaatttttt ataacttaga catgcaatgc tcattatctc tagagagggg 4980

cacgaccggg tcacgctgca ctgcaggcat gccaactttt gtatacaaag ttgccgatct 5040

agtaacatag atgacaccgc gcgcgataat ttatcctagt ttgcgcgcta tattttgttt 5100

tctatcgcgt cgcgtattaa atgtataatt gcgggactct aatcataaaa acccatctca 5160

taaataacgt catgcattac atgttaatta ttacatgctt aacgtaattc aacagaaatt 5220

atatgataat catcgcaaga ccggcaacag gattcaatct taagaaactt tattgccaaa 5280

tgtttgaacg atcggggaaa ttcgagctct ttaaattttt tttttttttt ttttttttgt 5340

taaatttttt tttttttttt tttttttttt gttaacttga tgtccgaaaa caaaactgaa 5400

agaacacagt aaattacaag cagaacaatg gcttttccaa tgccataata ctcaaagtta 5460

accttactca gcctcgaggg ggggcccggt accgacgtct catcacttaa tgctaacgtc 5520

gtaaaagtga acgattgggc cgccgtacag gttgttgccc tggctcagct cgatgtagaa 5580

gttgtccttc tcgaacttgg tggtgaacat ctcgctcacg tccttggcgc cggacatgta 5640

tctcttctcg aacagcactt ccctgctgtt cctgatcctc acgttagcgt cgccggaaac 5700

ggagaaatag accctgtagg tggagaagct atcgagctga agattttgct tcaggatgcc 5760

gcggccgccc tggtaaaggg tcagggtgtt gccgctgatg ttggtcgagc cggtgctggt 5820

ccagttgttg gtgttgatca gctccggaga gagcagcttc tcagacgggc tgatctcgag 5880

gataatgaag ttgtcacccc aggcctcgtc accgttctga gacttgagga tgaggtacac 5940

gcccttgaga tcggtgcccg tggtgaagcg cttgttgatc gtctggtagt cctccaagtt 6000

attgttggtg tcctcgtagt ggatgtagcc ggtgttctcg tccttcaaat ggatgcttgg 6060

cttgcccttc acggtgtatt ggatcacata ctcagtttta ggcttgagct tatcgccaat 6120

gaattggctg atgccgccgt ccttatgcac gtaaagagcc ttagtaccgt tgacgccgcc 6180

cgtgtggtca acataggcgt tcttgttgtt agccttccag ggctcgaggt tgtcttcctc 6240

aatgctgccg ttctccacga tatttgagat gaagccccgat ggggggacga tcagcttagt 6300

ctccttgttg gacaggtcag tagccagaag gagttcacgg aggtaagact tgcacgtgag 6360

ggtgatcagc ctgctattct cgtcggcctg caggccaaag ccgttgattg gggtcaggaa 6420

ggtctcgctg atcacgccca gaggcatgta aacaccgtcg tcgttggcag agagagtgcg 6480

gtattcagcc tcggagctct cgaccttctt tttattaaga tcgatctcgc cggtggatga 6540

gtcgtagaaa ttggcggtga cctcgtacct cagcgtcttc attttcttgg tgaagtcgat 6600

cttggtgatc acgtactcat ttgggaagac gatattatta gtgtagtaga tttgctcgct 6660

ctggtcgggg cagaggagct tgtccatgtc accgtaaatc acctcggaca agctgtcctt 6720

gtccacctgg tagttctgct tgagcttggc ttcatagacc ttgagcacgg tgatgctatc 6780

gttggaaatc tcaaagccga tcagggcgtg accaggcttg gcctcaacaa tcatcttggc 6840

atcctcatct gaacccttca ccttagcgta gttcgggttg ctgaaggtgt tggacagggt 6900

gggcaggatg ttcacgcgaa actcctcttt ttccttgttc aggtgctcat tcatgatgct 6960

ggtgtagtcg atgtcggcca aacccagcag cttcctgcag gtggtcagag tcaagaaagc 7020

cttggcctgg agggcggtca gcacgatcaa gaagttgtac acgttgccga cctcgctgcc 7080

gctggtcttc acgttttcct tggtgatcag ctctgaggca gtcttgaggg cggatctgcc 7140

gaacaggttg ttgcccacca tcacgtcgtg aaaagtgttc aggtagaact cgaagccgtc 7200

cacgtcattc ttagtcacgc tcttagcaag ctcggtcagc tcggtcagtt catccaggat 7260

gtcggctggg ctgccatcct tcttgacttt ggacgaggtc tcggtagcga aagtcagttc 7320

ctcaaacttt tcgttaacgt acttgatacg ttggtaggcg ggagtgattt cggtcagagt 7380

tgagttaatc agcacgttca cgttgatgat gtccagcttg tcagagattt cttgcaattg 7440

tttggacagg tactcgatct gcaaagagag ggcatagttc tgcttcatca cgtcgctcag 7500

catggaggta atcttgggga gataaaccct cagcatagtg ttgatggcat ccagcttgtt 7560

gttcacgtcg ttcagcacct ggttttgctc gttagcaatc ttgaggatct ccttgctcag 7620

ttccgtgttc agattgccct gagcaatgag gtcgttcagg ctaccgttga cgccgtccag 7680

cttaccgctg atgtcgttaa ggagctgttg attcttcagg atctcatcga gggtaaggtc 7740

accgccggtg tcggtcttga agatcatgtt catgatgtcc ttgatgccgg tggcgaagcc 7800

gtagatgccg ttgaagtaat caatgaaaga tggaagggcg cgggtgctca gcttggtgtt 7860

gttcttgttc atactagtgg ttattgtaaa tagtaattgt aatgttgttt gttgtttgtt 7920

gttgttggta attgttgtaa aaatagtcga cctgcagaag taacaccaaa caacagggtg 7980

agcatcgaca aaagaaacag taccaagcaa ataaatagcg tatgaaggca gggctaaaaa 8040

aatccacata tagctgctgc atatgccatc atccaagtat atcaagatca aaataattat 8100

aaaacatact tgtttattat aatagatagg tactcaaggt tagagcatat gaatagatgc 8160

tgcatatgcc atcatgtata tgcatcagta aaacccacat caacatgtat acctatccta 8220

gatcgatatt tccatccatc ttaaactcgt aactatgaag atgtatgaca cacacataca 8280

gttccaaaat taataaatac accaggtagt ttgaaacagt attctactcc gatctagaac 8340

gaatgaacga ccgcccaacc acaccacatc atcacaacca agcgaacaaa aagcatctct 8400

gtatatgcat cagtaaaacc cgcatcaaca tgtataccta tcctagatcg atatttccat 8460

ccatcatctt caattcgtaa ctatgaatat gtatggcaca cacatacaga tccaaaatta 8520

ataaatccac caggtagttt gaaacagaat taattctact ccgatctaga acgaccgccc 8580

aaccagacca catcatcaca accaagacaa aaaaaagcat gaaaagatga cccgacaaac 8640

aagtgcacgg catatattga aataaaggaa aagggcaaac caaaccctat gcaacgaaac 8700

aaaaaaaatc atgaaatcga tcccgtctgc ggaacggcta gagccatccc aggattcccc 8760

aaagagaaac actggcaagt tagcaatcag aacgtgtctg acgtacaggt cgcatccgtg 8820

tacgaacgct agcagcacgg atctaacaca aacacggatc taacacaaac atgaacagaa 8880

gtagaactac cgggccctaa ccatggaccg gaacgccgat ctagagaagg tagagagggg 8940

gggggggggga ggacgagcgg cgtaccttga agcggaggtg ccgacgggtg gatttggggg 9000

agatctggtt gtgtgtgtgt gcgctccgaa caacacgagg ttggggaaag agggtgtgga 9060

gggggtgtct atttattacg gcgggcgagg aagggaaagc gaaggagcgg tgggaaagga 9120

atcccccgta gctgccggtg ccgtgagagg aggaggaggc cgcctgccgt gccggctcac 9180

gtctgccgct ccgccacgca atttctggat gccgacagcg gagcaagtcc aacggtggag 9240

cggaactctc gagaggggtc cagaggcagc gacagagatg ccgtgccgtc tgcttcgctt 9300

ggcccgacgc gacgctgctg gttcgctggt tggtgtccgt tagactcgtc gacggcgttt 9360

aacaggctgg cattatctac tcgaaacaag aaaaatgttt ccttagtttt tttaatttct 9420

taaagggtat ttgtttaatt tttagtcact ttattttat ctattttata tctaaattat 9480

taaataaaaa aactaaaata gagttttagt tttcttaatt tagaggctaa aatagaataa 9540

aatagatgta ctaaaaaaat tagtctataa aaaccattaa ccctaaaccc taaatggatg 9600

tactaataaa atggatgaag tattatatag gtgaagctat ttgcaaaaaa aaaggagaac 9660

acatgcacac taaaaagata aaactgtaga gtcctgttgt caaaatactc aattgtcctt 9720

tagaccatgt ctaactgttc atttatatga ttctctaaaa cactgatatt attgtagtac 9780

tatagattat attattcgta gagtaaagtt taaatatatg tataaagata gataaactgc 9840

acttcaaaca agtgtgacaa aaaaaatata ttgtggtgta aacaaattga cgcttagaca 9900

acttaataac acattgcgga cgtttttaat gtactgaatt aacgccgaat tgaggtaccc 9960

accatccgcg ggatatcacc actttgtaca agaaagctgg gtccgatcta gtaacataga 10020

tgacaccgcg cgcgataatt tatcctagtt tgcgcgctat attttgtttt ctatcgcgtc 10080

gcgtattaaa tgtataattg cgggactcta atcataaaaa cccatctcat aaataacgtc 10140

atgcattaca tgttaattat tacatgctta acgtaattca acagaaatta tatgataatc 10200

atcgcaagac cggcaacagg attcaatctt aagaaacttt attgccaaat gtttgaacga 10260

tcggggaaat tcgagctctt taaatttttt tttttttttt ttttttgtta aatttttttt 10320

tttttttttt tttttgttaa cttgatgtcc gaaaacaaaa ctgaaagaac acagtaaatt 10380

acaagcagaa caatggcttt tccaatgcca taatactcaa agttaacctt actcagcctc 10440

gagggggggc ccggtacctc atcaggcagc cttcgtatcg gagagttcga tcttcgcgcc 10500

cagcccggcc atcaggtcca tgaactccgg gaagctcgtg gcgatcatcg tggcatcgtc 10560

caccgtgaca gggttttccg acacgaggcc catgacgagg aagctcatgg cgatgcggtg 10620

atcgagatgg gtggcgacgg cggcgcccga ggcgttgccg agccccttgc cgtcagggcg 10680

gccacgcacg acgagcgacg tctcgccctc atcgcaatcc acgccattga gcttgaggcc 10740

attggcgacg gccgagaggc ggtcgctttc cttgacgcgg agttcttcca gaccgttcat 10800

cacggtcgcc ccttccgcga aggcggcggc gacagcgaga atcggatatt cgtcgatcat 10860

cgaaggcgcg cggtcttccg gcaccgtgac gcccttcagc gtggaggagc gaacgcgcag 10920

gtccgccacg tcttcgccgc cggcaaggcg cgggttgatg acttcgatgt cggcgcccat 10980

ttcctgcagc gtcaggatga ggccggtgcg ggtggggttc atcagcacgt tgaggatggt 11040

gacgtcggag cccggaacaa gcagggccgc aaccagcggg aaggccgtcg aggacgggtc 11100

gcccggcacg tcgatgactt ggccggtgag cttgccgcgg ccttccaggc ggatggtgcg 11160

cacgccgtcc gcatccgtct cgacggtaag gttggcgcca aagccctgca gcatcttttc 11220

cgtatgatcg cgcgtcatga tcggctcgat gaccgtcgtg atgccgggcg tgttgaggcc 11280

ggcgagcagc acggcggact tcacctgtgc ggaggccatc ggcacgcggt aggtgatcgg 11340

cgtcggcgtc ttcggcccgc gcaaggtaac gggaagacgg tcaccgtctt ccgatttcac 11400

ctgcacgccc atttcgcgca gcgggttcaa cacgcggccc atcgggcgct ttgtgagcga 11460

ggcgtcgccg atgaaggtgc tgtcgaaatc gtagaccccg acgaggccca tcgtcaggcg 11520

gcagcccgtg gcggcattgc cgaaatcgag cggcgcctca ggcgccagga ggccgccatt 11580

gccgacgcca tcgatgatcc aggtgtcgcc ttccttacgg atgcgggcgc ccatcgcctg 11640

catggccttg cccgtattga tgacgtcctc gccttccaga aggccggtga tgcgcgtttc 11700

accgctcgcg agaccgccga acatgaagga ccggtgggag atcgacttgt cgccgggaat 11760

gcggacggtt ccggaaaggc cagaggattt gcgggcggtt gcgggccggc tgcttgcacc 11820

gtgaagcatg cacgccgtgg aaacagaaga catgacctta agaggacgaa gctcagagcc 11880

aattaacgtc atcccactct tcttcaatcc ccacgacgac gaaatcggat aagctcgtgg 11940

atgctgctgc gtcttcagag aaaccgataa gggagaatttg cgttgactgg atttcgagag 12000

attggagata agagatgggt tctgcacacc attgcagatt ctgctaactt gcgccatact 12060

agtggttatt gtaaatagta attgtaatgt tgtttgttgt ttgttgttgt tggtaattgt 12120

tgtaaaaata gtcgacctgc agaagtaaca ccaaacaaca gggtgagcat cgacaaaaga 12180

aacagtacca agcaaataaa tagcgtatga aggcagggct aaaaaaatcc acatatagct 12240

gctgcatatg ccatcatcca agtatatcaa gatcaaaata attataaaac atacttgttt 12300

attataatag ataggtactc aaggttagag catatgaata gatgctgcat atgccatcat 12360

gtatatgcat cagtaaaacc cacatcaaca tgtataccta tcctagatcg atatttccat 12420

ccatcttaaa ctcgtaacta tgaagatgta tgacacacac atacagttcc aaaattaata 12480

aatacaccag gtagtttgaa acagtattct actccgatct agaacgaatg aacgaccgcc 12540

caaccacacc acatcatcac aaccaagcga acaaaaagca tctctgtata tgcatcagta 12600

aaacccgcat caacatgtat acctatccta gatcgatatt tccatccatc atcttcaatt 12660

cgtaactatg aatatgtatg gcacacacat acagatccaa aattaataaa tccaccaggt 12720

agtttgaaac agaattaatt ctactccgat ctagaacgac cgcccaacca gaccacatca 12780

tcacaaccaa gacaaaaaaa agcatgaaaa gatgacccga caaacaagtg cacggcatat 12840

attgaaataa aggaaaaggg caaaccaaac cctatgcaac gaaacaaaaa aaatcatgaa 12900

atcgatcccg tctgcggaac ggctagagcc atcccaggat tccccaaaga gaaacactgg 12960

caagttagca atcagaacgt gtctgacgta caggtcgcat ccgtgtacga acgctagcag 13020

cacggatcta acacaaacac ggatctaaca caaacatgaa cagaagtaga actaccgggc 13080

cctaaccatg gaccggaacg ccgatctaga gaaggtagag agggggggggg ggggaggacg 13140

agcggcgtac cttgaagcgg aggtgccgac gggtggattt gggggagatc tggttgtgtg 13200

tgtgtgcgct ccgaacaaca cgaggttggg gaaagagggt gtggaggggg tgtctattta 13260

ttacggcggg cgaggaaggg aaagcgaagg agcggtggga aaggaatccc ccgtagctgc 13320

cggtgccgtg agaggagggag gaggccgcct gccgtgccgg ctcacgtctg ccgctccgcc 13380

acgcaatttc tggatgccga cagcggagca agtccaacgg tggagcggaa ctctcgagag 13440

gggtccagag gcagcgacag agatgccgtg ccgtctgctt cgcttggccc gacgcgacgc 13500

tgctggttcg ctggttggtg tccgttagac tcgtcgacgg cgtttaacag gctggcatta 13560

tctactcgaa acaagaaaaa tgtttcctta gtttttttaa tttcttaaag ggtatttgtt 13620

taatttttag tcactttatttattctatt ttatatctaa attattaaat aaaaaaacta 13680

aaatagagtt ttagttttct taatttagag gctaaaatag aataaaatag atgtactaaa 13740

aaaattagtc tataaaaacc attaacccta aaccctaaat ggatgtacta ataaaatgga 13800

tgaagtatta tataggtgaa gctatttgca aaaaaaaagg agaacacatg cacactaaaa 13860

agataaaact gtagagtcct gttgtcaaaa tactcaattg tcctttagac catgtctaac 13920

tgttcattta tatgattctc taaaacactg atattattgt agtactatag attatattat 13980

tcgtagagta aagtttaaat atatgtataa agatagataa actgcacttc aaacaagtgt 14040

gacaaaaaaa atatgtggta attttttata acttagacat gcaatgctca ttatctctag 14100

agaggggcac gaccgggtca cgctgcactg caggcatgcc aacttttgta tacaaagttg 14160

ccgatctagt aacatagatg acaccgcgcg cgataattta tcctagtttg cgcgctatat 14220

tttgttttct atcgcgtcgc gtattaaatg tataattgcg ggactctaat cataaaaacc 14280

catctcataa ataacgtcat gcattacatg ttaattatta catgcttaac gtaattcaac 14340

agaaattata tgataatcat cgcaagaccg gcaacaggat tcaatcttaa gaaactttat 14400

tgccaaatgt ttgaacgatc ggggaaattc gagctcttta aatttttttt tttttttttt 14460

tttttgttaa attttttttt tttttttttt tttttttgtt aacttgatgt ccgaaaacaa 14520

aactgaaaga acacagtaaa ttacaagcag aacaatggct tttccaatgc cataatactc 14580

aaagttaacc ttactcagcc tcgagggggg gcccggtacc gacgtctcat cacttaatgc 14640

taacgtcgta aaagtgaacg attgggccgc cgtacaggtt gttgccctgg ctcagctcga 14700

tgtagaagtt gtccttctcg aacttggtgg tgaacatctc gctcacgtcc ttggcgccgg 14760

acatgtatct cttctcgaac agcacttccc tgctgttcct gatcctcacg ttagcgtcgc 14820

cggaaacgga gaaatagacc ctgtaggtgg agaagctatc gagctgaaga ttttgcttca 14880

ggatgccgcg gccgccctgg taaagggtca gggtgttgcc gctgatgttg gtcgagccgg 14940

tgctggtcca gttgttggtg ttgatcagct ccggagagag cagcttctca gacgggctga 15000

tctcgaggat aatgaagttg tcaccccagg cctcgtcacc gttctgagac ttgaggatga 15060

ggtacacgcc cttgagatcg gtgcccgtgg tgaagcgctt gttgatcgtc tggtagtcct 15120

ccaagttatttgttggtgtcc tcgtagtgga tgtagccggt gttctcgtcc ttcaaatgga 15180

tgcttggctt gcccttcacg gtgtattgga tcacatactc agttttaggc ttgagcttat 15240

cgccaatgaa ttggctgatg ccgccgtcct tatgcacgta aagagcctta gtaccgttga 15300

cgccgcccgt gtggtcaaca taggcgttct tgttgttagc cttccagggc tcgaggttgt 15360

cttcctcaat gctgccgttc tccacgatat ttgagatgaa gcccgatggg gggacgatca 15420

gcttagtctc cttgttggac aggtcagtag ccagaaggag ttcacggagg taagacttgc 15480

acgtgagggt gatcagcctg ctattctcgt cggcctgcag gccaaagccg ttgattgggg 15540

tcaggaaggt ctcgctgatc acgcccagg gcatgtaaac accgtcgtcg ttggcagaga 15600

gagtgcggta ttcagcctcg gagctctcga ccttcttttt attaagatcg atctcgccgg 15660

tggatgagtc gtagaaattg gcggtgacct cgtacctcag cgtcttcatt ttcttggtga 15720

agtcgatctt ggtgatcacg tactcatttg ggaagacgat attattagtg tagtagattt 15780

gctcgctctg gtcggggcag aggagcttgt ccatgtcacc gtaaatcacc tcggacaagc 15840

tgtccttgtc cacctggtag ttctgcttga gcttggcttc atagaccttg agcacggtga 15900

tgctatcgtt ggaaatctca aagccgatca gggcgtgacc aggcttggcc tcaacaatca 15960

tcttggcatc ctcatctgaa cccttcacct tagcgtagtt cgggttgctg aaggtgttgg 16020

acagggtggg caggatgttc acgcgaaact cctctttttc cttgttcagg tgctcattca 16080

tgatgctggt gtagtcgatg tcggccaaac ccagcagctt cctgcaggtg gtcagagtca 16140

agaaagcctt ggcctggagg gcggtcagca cgatcaagaa gttgtacacg ttgccgacct 16200

cgctgccgct ggtcttcacg ttttccttgg tgatcagctc tgaggcagtc ttgagggcgg 16260

atctgccgaa caggttgttg cccaccatca cgtcgtgaaa agtgttcagg tagaactcga 16320

agccgtccac gtcattctta gtcacgctct tagcaagctc ggtcagctcg gtcagttcat 16380

ccaggatgtc ggctgggctg ccatccttct tgactttgga cgaggtctcg gtagcgaaag 16440

tcagttcctc aaacttttcg ttaacgtact tgatacgttg gtaggcggga gtgatttcgg 16500

tcagagttga gttaatcagc acgttcacgt tgatgatgtc cagcttgtca gagatttctt 16560

gcaattgttt ggacaggtac tcgatctgca aagagagggc atagttctgc ttcatcacgt 16620

cgctcagcat ggaggtaatc ttggggagat aaaccctcag catagtgttg atggcatcca 16680

gcttgttgtt cacgtcgttc agcacctggt tttgctcgtt agcaatcttg aggatctcct 16740

tgctcagttc cgtgttcaga ttgccctgag caatgaggtc gttcaggcta ccgttgacgc 16800

cgtccagctt accgctgatg tcgttaagga gctgttgatt cttcaggatc tcatcgaggg 16860

taaggtcacc gccggtgtcg gtcttgaaga tcatgttcat gatgtccttg atgccggtgg 16920

cgaagccgta gatgccgttg aagtaatcaa tgaaagatgg aagggcgcgg gtgctcagct 16980

tggtgttgtt cttgttcata ctagtggtta ttgtaaatag taattgtaat gttgtttgtt 17040

gtttgttgtt gttggtaatt gttgtaaaaa tagtcgacct gcagaagtaa caccaaacaa 17100

cagggtgagc atcgacaaaa gaaacagtac caagcaaata aatagcgtat gaaggcaggg 17160

ctaaaaaaat ccacatatag ctgctgcata tgccatcatc caagtatatc aagatcaaaa 17220

taattataaa acatacttgt ttattataat agataggtac tcaaggttag agcatatgaa 17280

tagatgctgc atatgccatc atgtatatgc atcagtaaaa cccacatcaa catgtatacc 17340

tatcctagat cgatatttcc atccatctta aactcgtaac tatgaagatg tatgacacac 17400

acatacagtt ccaaaattaa taaatacacc aggtagtttg aaacagtatt ctactccgat 17460

ctagaacgaa tgaacgaccg cccaaccaca ccacatcatc acaaccaagc gaacaaaaag 17520

catctctgta tatgcatcag taaaacccgc atcaacatgt atacctatcc tagatcgata 17580

tttccatcca tcatcttcaa ttcgtaacta tgaatatgta tggcacacac atacagatcc 17640

aaaattaata aatccaccag gtagtttgaa acagaattaa ttctactccg atctagaacg 17700

accgcccaac cagaccacat catcacaacc aagacaaaaa aaagcatgaa aagatgaccc 17760

gacaaacaag tgcacggcat atattgaaat aaaggaaaag ggcaaaccaa accctatgca 17820

acgaaacaaa aaaaatcatg aaatcgatcc cgtctgcgga acggctagag ccatcccagg 17880

attccccaaa gagaaacact ggcaagttag caatcagaac gtgtctgacg tacaggtcgc 17940

atccgtgtac gaacgctagc agcacggatc taacacaaac acggatctaa cacaaacatg 18000

aacagaagta gaactaccgg gccctaacca tggaccggaa cgccgatcta gagaaggtag 18060

agaggggggg gggggagga cgagcggcgt accttgaagc ggaggtgccg acgggtggat 18120

ttgggggaga tctggttgtg tgtgtgtgcg ctccgaacaa cacgaggttg gggaaagagg 18180

gtgtggaggg ggtgtctatt tattacggcg ggcgaggaag ggaaagcgaa ggagcggtgg 18240

gaaaggaatc ccccgtagct gccggtgccg tgagaggagg aggaggccgc ctgccgtgcc 18300

ggctcacgtc tgccgctccg ccacgcaatt tctggatgcc gacagcggag caagtccaac 18360

ggtggagcgg aactctcgag aggggtccag aggcagcgac agagatgccg tgccgtctgc 18420

ttcgcttggc ccgacgcgac gctgctggtt cgctggttgg tgtccgttag actcgtcgac 18480

ggcgtttaac aggctggcat tatctactcg aaacaagaaa aatgtttcct tagttttttt 18540

aatttcttaa agggtatttg tttaattttt agtcacttta ttttatattcta ttttatatct 18600

aaattattaa ataaaaaaac taaaatagag ttttagtttt cttaatttag aggctaaaat 18660

agaataaaat agatgtacta aaaaaattag tctataaaaa ccattaaccc taaaccctaa 18720

atggatgtac taataaaatg gatgaagtat tatataggtg aagctatttg caaaaaaaaa 18780

ggagaacaca tgcacactaa aaagataaaa ctgtagagtc ctgttgtcaa aatactcaat 18840

tgtcctttag accatgtcta actgttcatt tatatgattc tctaaaacac tgatattatt 18900

gtagtactat agattatatt attcgtagag taaagtttaa atatatgtat aaagatagat 18960

aaactgcact tcaaacaagt gtgacaaaaa aaatatgtgg taatttttta taacttagac 19020

atgcaatgct cattatctct agagaggggc acgaccgggt cacgctgcac tgcaggcatg 19080

cagcctgcttttttgtacaa acttgtgata tcactagtgc ggccaagctt agcttagctt 19140

gagcttggat cagattgtcg tttcccgcct tcagtttaag tatgctctat aatggggagac 19200

gccctggcat caaggatggc attggcttcc aaaggggaga caatgtcaaa cttaatgccc 19260

ctcctaagaa cttgtctaac tttgttaagg gcaaggctcc catgcctcag gataacgagg 19320

gttacatttt gtaccctgcc ggctatcctg agagcaaaat taggagaact cattctagga 19380

agtctcactc tggccctaat tatgctttta tgtataaggg tgagacatct agctctaggc 19440

aaccaacccg tgccaagttg cctagaaaga aaactcctag tgcatcaaat gaacatagcc 19500

tttcatttaa aacttttgat gcatctttatg tgttgactaa caaatccgac aaagtagttg 19560

ccaaatatgt tgggggcaag cacaaggggt caaagacttg tgtttgggta cccaaagttc 19620

tagtgtctaa tgccaaagga cccaaaacca tttgggtacc taaagtcaag aactaaaatt 19680

gttttgtagg tttatgcatc cgggggctca agttggatca tcgacagcgg gtgcacaaac 19740

cacatgacag g 19751

Claims (10)

1. The nucleic acid molecule flanking the 5' end of the exogenous insertion fragment of transgenic insect-resistant corn VP3C1 is shown at positions 1-756 in SEQ ID NO.1. 2. The nucleic acid molecule flanking the 3' end of the exogenous insertion fragment of transgenic insect-resistant corn VP3C1 is shown at positions 359-932 in SEQ ID NO.2. 3. A PCR reaction detection specific primer designed according to the 5' side nucleic acid molecule of claim 1. 4. The specific primers for PCR reaction detection according to claim 3 are: VP3C1-Left-F1:5'-GGCAAAGGACGGCAAAAGTA-3', VP3C1-NOS-R1: 5'-TGAATCCTGTTGCCGGTCTT-3'. 5. A specific primer for PCR reaction detection designed according to the 3' end flanking nucleic acid molecule of claim 2. 6. The specific primers for PCR reaction detection according to claim 5 are: VP3C1-Ubi-F1: 5’-CCTGTTGTCAAAATACTCAATTGTCC-3’, VP3C1-Right-R1: 5’-CCGTTCATGTGGTTTGTGCA-3’. 7. A PCR reaction detection method for transgenic insect-resistant corn VP3C1, characterized in that the primers in the PCR reaction are the specific primers for PCR reaction detection according to any one of claims 3 to 6. 8. The PCR reaction detection method according to claim 7, characterized in that: the specific primers used for PCR reaction detection are: VP3C1-Left-F1:5'-GGCAAAGGACGGCAAAAGTA-3', VP3C1-NOS-R1: 5'-TGAATCCTGTTGCCGGTCTT-3', The amplified fragment size of transgenic insect-resistant corn VP3C1 is 1067 bp; or VP3C1-Ubi-F1: 5’-CCTGTTGTCAAAATACTCAATTGTCC-3’, VP3C1-Right-R1: 5’-CCGTTCATGTGGTTTGTGCA-3’, The amplified fragment size of transgenic insect-resistant corn VP3C1 was 932 bp. 9. A kit for detecting insect-resistant corn, characterized in that it contains the specific primers for PCR reaction detection according to claim 3 or 4, and/or contains the specific primers for PCR reaction detection according to claim 5 or 6. 10. Use of the flanking nucleic acid molecule according to claim 1 or 2, the specific primer for PCR reaction detection according to any one of claims 3 to 6, and the insect-resistant corn detection kit according to claim 9 in detecting genetically modified corn.