Transgenic soybean event AS-1 with high expression of alpha-amylase as well as detection method and application of transgenic soybean event AS-1

Abstract

The invention discloses a transgenic soybean event AS-1 with high expression of alpha-amylase and a detection method and application of the transgenic soybean event AS-1. The transgenic soybean event AS-1 can express the alpha-amylase at a high level and has very high amylase activity, and the amylase activity reaches up to 62.22 thousand U / g and is far higher than that of commercial enzymes produced through microbial fermentation; and the method has no obvious adverse effect on growth, development and agronomic traits of plants. The transgenic soybean event can be used as a bioreactor for large-scale production of alpha-amylase, and has a remarkable cost advantage and a wide application prospect. The transgenic soybean can be used as an enzyme preparation, and the production cost is lower than that of microbial fermentation.

Description

(I) Technical field

[0001] The present invention relates to a transgenic soybean event expressing alpha-amylase and an identification method thereof, and in particular to a transgenic soybean event AS-1 containing alpha-amylase and flazasulfuron-resistant genes and a nucleic acid sequence and a method thereof for detecting whether a biological sample contains the transgenic soybean event AS-1. (II) Background technology

[0002] Amylase is a type of enzyme that can hydrolyze starch into glucose, maltose and oligosaccharides. It has a wide range of applications, including feed, food, fermentation, papermaking, textile, washing and other industries. According to the different modes of action, amylase can be divided into α-amylase, β-amylase, glucoamylase and debranching enzyme, among which α-amylase is the most commonly used. α-Amylase occupies an important position in the industrial enzyme market, and the market demand is growing. At present, the industrial production of amylase generally adopts microbial fermentation, but this production method is complicated, the production equipment is expensive, the purification process is time-consuming and labor-intensive, and the temperature requirements are strict during transportation and storage. Therefore, it is of great significance to explore an alternative method for producing α-amylase by microbial fermentation and reduce the production cost of α-amylase.

[0003] In recent years, the use of transgenic plants as bioreactors to express recombinant proteins has become very attractive. Compared with microbial fermentation, plant bioreactors have the following advantages: 1) Plants do not contain known human pathogens that may contaminate the final product; 2) Higher plants can synthesize active proteins from eukaryotic organisms; 3) Plant seeds have high protein content, low protease activity and low moisture content, which is conducive to the stability of recombinant proteins. However, the functional properties of recombinant proteins may affect the growth and development of transgenic plants or seed morphology. For example, transgenic corn expressing exogenous xylanase exhibits dwarf plants and shriveled grains (Gray B, Bougri O, Carlson A, et al. Global and grain-specific accumulation of glycoside hydrolase family 10xylanases in transgenic maize (Zea mays). Plant Biotechnology Journal, 2011, 9: 1100-1108).

[0004] Soybeans and corn are both major crops, but corn seeds are rich in starch. If the endosperm overexpresses room-temperature or medium-temperature amylases that are active at room temperature, or amylases with high specific activity, it will lead to the degradation of the starch in the corn seeds themselves, making the seeds shriveled and stunted, and the endosperm liquefied, seriously affecting the germination rate and yield of the seeds. Soybean seeds mainly contain protein and oil, with a starch content of less than 1%, and are an ideal amylase production platform. Patent CN113584075B discloses a method for producing α-amylase using transgenic soybeans, which achieves a high level of expression of α-amylase, and the growth, development and agronomic traits of transgenic soybeans are not significantly adversely affected.

[0005] Transgenic events or transformation events are generated by the random integration of T-DNA into the plant genome. Since the location of T-DNA insertion is random, each transgenic event obtained is unique. It is not easy to obtain an ideal transformation event. On the one hand, the expression of transgenes in plants is affected by the location of their chromosomes. Different locations will result in large differences in the expression level, expression time and spatial pattern of exogenous genes even in different transformation events from the same transformation vector. Secondly, when T-DNA is integrated into the plant genome, it may be inserted into the internal or regulatory region of the functional gene, destroying the expression of the functional gene, thereby affecting the growth, development or reproduction of the plant. On the other hand, T-DNA is often integrated into the plant genome in the form of multiple copies. In order to facilitate breeding, single-copy or single-site T-DNA insertion is desired. When screening transformation events, it is necessary to comprehensively consider the expression level of the exogenous gene, the insertion site of T-DNA and the copy number. Therefore, a large number of transformation events need to be created and screened in order to screen out excellent transformation events that efficiently express exogenous genes, do not affect the agronomic traits of the plant, and meet production needs.

[0006] The information on the integration site of exogenous genes in transformation events is a unique identity feature of transformation events, and is of great value in the hybrid breeding, production application and supervision of transgenic crops. Only by using the information on the integration site of exogenous genes in transformation events can the presence of transformation events in plants be detected with the help of existing polynucleotide detection methods. Conventional polynucleotide and protein detection methods can detect whether there is a target gene or target protein in a plant, but cannot effectively distinguish between different transformation events, especially transformation events produced using the same transformation vector. Therefore, only the detection of inserted genes and flanking sequences can accurately determine whether a target transgenic event exists. (III) Summary of the invention

[0007] The present invention aims to provide a transgenic soybean event AS-1 that highly expresses α-amylase, and a detection method and use thereof. The transgenic soybean event AS-1 simultaneously expresses two proteins, α-amylase and CdP450, has very high amylase activity, is tolerant to flazasulfuron-methyl, has a single-site T-DNA insertion, is genetically stable, and has no obvious adverse effects on agronomic traits. At the same time, the present invention also provides a method for detecting the transgenic soybean event AS-1, and the method can quickly and accurately detect whether a biological sample contains a DNA molecule of the transgenic soybean event AS-1, thereby realizing the traceability of the soybean event AS-1.

[0008] The technical solution adopted by the present invention is:

[0009] In a first aspect, the present invention provides a nucleic acid sequence of a transgenic soybean event AS-1, wherein the nucleic acid sequence comprises SEQ ID NO: 1 or a complementary sequence thereof, and / or SEQ ID NO: 2 or a complementary sequence thereof.

[0010] Furthermore, the nucleic acid sequence comprises one or more of SEQ ID NO: 3-8 or complementary sequences thereof or SEQ ID NO: 20 or complementary sequences thereof.

[0011] Further, the nucleic acid sequence is derived from a plant, seed or cell comprising transgenic soybean event AS-1.

[0012] Further, the transgenic soybean event AS-1 comprises an exogenous insertion sequence, wherein the junction sequence formed by the 5' end of the exogenous insertion sequence and the right flank genomic DNA includes but is not limited to SEQ ID NO: 1, 3, 5, 7, 20 or its complementary sequence, and the junction sequence formed by the 3' end of the exogenous insertion sequence and the left flank genomic DNA includes but is not limited to SEQ ID NO: 2, 4, 6, 8, 20 or its complementary sequence; the right flank region genomic DNA of the exogenous insertion sequence includes but is not limited to SEQ ID NO: 12, and the left flank region genomic DNA includes but is not limited to SEQ ID NO: 13; the genome lengths of the left and right flanking regions are not limited to those shown in SEQ ID NO: 12 and SEQ ID NO: 13.

[0013] Further, the exogenous insertion sequence comprises an α-amylase gene expression cassette and a flazasulfuron-resistant gene expression cassette, wherein the regulatory genetic elements are necessary for expressing α-amylase AmyS and flazasulfuron-resistant CdP450 in soybean plant cells. The α-amylase gene expression cassette encodes α-amylase and expresses AmyS protein; the α-amylase gene expression cassette is composed of a pCG1 promoter, an Arabidopsis 2S albumin signal peptide (At2S2SP), an α-amylase gene AmyS, an AtHSP18.2 terminator, a regulatory sequence TEV 5′UTR and a MAR, wherein the pCG1 promoter is seed-specific, derived from soybean, and can drive the target gene to be expressed at a high level in seeds, the Arabidopsis 2S albumin signal peptide can locate the target protein to the endoplasmic reticulum, the α-amylase gene AmyS is derived from Geobacillus stearothermophilus, the AtHSP18.2 terminator is derived from Arabidopsis, and the regulatory sequences TEV 5′UTR and MAR are elements for enhancing gene expression, both of which are derived from tobacco. The flazasulfuron-resistant gene expression frame encodes a flazasulfuron-resistant cytochrome P450 oxidase and expresses CdP450 protein; the flazasulfuron-resistant CdP450 gene expression frame is composed of a pCaMV35S promoter, a flazasulfuron-resistant gene CdP450 and a CaMV35S terminator, wherein the pCaMV35S promoter is a constitutive promoter derived from cauliflower mosaic virus and can drive the target gene to be expressed in all plant tissues, and the terminator is a CaMV35S terminator derived from cauliflower mosaic virus.

[0014] The nucleotide sequence of the exogenous insertion sequence is shown as 757-15944 in SEQ ID NO:20, and the SEQ ID NO:20 is a nucleic acid sequence formed by connecting SEQ ID NO:9+SEQ ID NO:10+SEQ ID NO:11 end to end, with a length of 16949 nucleotides. Since the sequence is too long, it is expressed in the form of SEQ ID NO:9+SEQ ID NO:10+SEQ ID NO:11. The specific genome and genetic elements contained therein are shown in Table 1, wherein 1-5923bp corresponds to SEQ ID NO:9, 5924-12977bp corresponds to SEQ ID NO:10, and 12978-16949bp corresponds to SEQ ID NO:11.

[0015] Table 1 Genome and genetic elements contained in SEQ ID NO: 20

[0016]

[0017]

[0018] The transgenic soybean event AS-1 of the present invention is a soybean cotyledon node transformation method mediated by Agrobacterium, in which an exogenous T-DNA sequence containing an α-amylase and a flazasulfuron-resistant gene expression frame is introduced into the soybean genome, a soybean transgenic event population is obtained by regenerating soybean cells containing the exogenous genes, and a soybean transformation event that can meet production needs is screened by molecular biology and bioassay methods. Progeny of the transgenic soybean event AS-1 of the present invention is obtained by selfing or hybridization, and soybean events and seeds whose flanking sequences of the inserted sequences in the genome of any progeny contain SEQ ID NO: 12 or SEQ ID NO: 13 should be regarded as one aspect of the present invention.

[0019] In a second aspect, the present invention provides a specific primer pair for detecting transgenic soybean event AS-1, the primer pair comprising a first primer and a second primer different from the first primer, the first primer comprising at least 11 consecutive nucleotides in SEQ ID NO:20 or its complementary sequence, and the second primer comprising at least 11 consecutive nucleotides in SEQ ID NO:20 or its complementary sequence.

[0020] Furthermore, the first primer comprises any 11 or more consecutive polynucleotides in the transgenic insertion sequence region of SEQ ID NO: 20 or its complementary sequence, and the second primer comprises any 11 or more consecutive polynucleotides in the 5' or 3' flanking genome of SEQ ID NO: 20 or its complementary sequence.

[0021] Furthermore, the nucleotide sequence of the first primer is shown as SEQ ID NO:15 or SEQ ID NO:17, and the nucleotide sequence of the second primer is shown as SEQ ID NO:14 or SEQ ID NO:16.

[0022] In a third aspect, the present invention provides a method for detecting the presence of DNA of transgenic soybean event AS-1 in a sample, the method comprising:

[0023] 1) contacting the sample to be detected with the specific primer pair;

[0024] 2) performing a polynucleotide amplification reaction;

[0025] 3) detecting the presence of an amplification product; if the presence of an amplification product is detected, the DNA of the transgenic soybean event AS-1 is present in the test sample; the amplification product comprises SEQ ID NO: 1 or its complementary sequence, and / or SEQ ID NO: 2 or its complementary sequence.

[0026] The SEQ ID NO:1 or its complementary sequence is a 26-nucleotide sequence near the 5'-end integration site of the inserted sequence in the transgenic soybean event AS-1, and the SEQ ID NO:1 or its complementary sequence simultaneously contains the right flank genomic DNA sequence and the 5'-end DNA sequence of the inserted sequence, and the presence of the transgenic soybean event AS-1 can be identified by including the SEQ ID NO:1 or its complementary sequence. The SEQ ID NO:2 or its complementary sequence is a 26-nucleotide sequence near the 3'-end integration site of the inserted sequence in the transgenic soybean event AS-1, and the SEQ ID NO:2 or its complementary sequence simultaneously contains the left flank genomic DNA sequence and the 3'-end DNA sequence of the inserted sequence, and the presence of the transgenic soybean event AS-1 can be identified by including the SEQ ID NO:2 or its complementary sequence.

[0027] Furthermore, the amplification product comprises one or more of SEQ ID NOs: 3-8 or their complementary sequences.

[0028] The SEQ ID NO: 3, 5, 7 or its complementary sequence spans the 5' end DNA sequence of the flanking genomic sequence and the inserted sequence, and the presence of the transgenic soybean event AS-1 can be identified by including the SEQ ID NO: 3, 5, 7 or its complementary sequence.

[0029] The SEQ ID NO: 4, 6, 8 or their complementary sequence spans the 3' end DNA sequence of the flanking genomic sequence and the inserted sequence, and the presence of the transgenic soybean event AS-1 can be identified by including the SEQ ID NO: 4, 6, 8 or their complementary sequence.

[0030] Furthermore, the amplified product comprises SEQ ID NO: 20 or its complementary sequence. The presence of SEQ ID NO: 20 or its complementary sequence can be identified as the presence of transgenic soybean event AS-1.

[0031] The amplification product can be detected by separation and observation through agarose gel electrophoresis, or by sequencing analysis.

[0032] The amplification product can also be detected by combining the primer pair with an oligonucleotide probe, wherein the oligonucleotide probe can be fluorescently labeled (e.g., 6-FAM TM Fluorescent label), the oligonucleotide probe is selected from SEQ ID NO: 1-8 or its complementary sequence or SEQ ID NO: 20 or its complementary sequence.

[0033] In a fourth aspect, the present invention provides a method for producing α-amylase using the transgenic soybean event AS-1, the method comprising: planting soybean seeds containing a specific region nucleic acid sequence, harvesting soybeans containing α-amylase, and crushing the soybeans to use them directly as amylase; the specific region nucleic acid sequence is from the transgenic soybean event AS-1, and the specific region nucleic acid sequence comprises SEQ ID NO: 1 or its complementary sequence, and / or SEQ ID NO: 2 or its complementary sequence.

[0034] In a fifth aspect, the present invention provides a method for cultivating a soybean plant expressing α-amylase containing the transgenic soybean event AS-1, the method comprising: hybridizing a soybean plant containing a specific region nucleic acid sequence with another soybean plant not containing the specific region nucleic acid sequence, producing and harvesting offspring plants, and the α-amylase activity in the offspring plants is significantly increased compared with the soybean plant not containing the specific region nucleic acid sequence. The specific region nucleic acid sequence is from the transgenic soybean event AS-1, and the specific region nucleic acid sequence comprises SEQ ID NO: 1 or its complementary sequence, and / or SEQ ID NO: 2 or its complementary sequence.

[0035] The progeny of the transgenic soybean event AS-1 include plants or seeds or plant parts and seeds; the plant parts include but are not limited to: pollen, ovules, petals, stems, leaves, seeds, pods and meristems.

[0036] In a sixth aspect, the present invention provides a transgenic plant cell produced from a transgenic soybean event AS-1, wherein the transgenic plant cell is obtained by transferring a specific region nucleic acid sequence of the transgenic soybean event AS-1 into the plant genome, and the specific region nucleic acid sequence comprises SEQ ID NO: 1 or its complementary sequence, and / or SEQ ID NO: 2 or its complementary sequence.

[0037] In the seventh aspect, the present invention provides an agricultural product or processed product produced from a transgenic soybean event AS-1, wherein the agricultural product or processed product comprises a specific region nucleic acid sequence, wherein the specific region nucleic acid sequence comprises SEQ ID NO: 1 or its complementary sequence, and / or SEQ ID NO: 2 or its complementary sequence; the processed product includes but is not limited to: soy protein, soybean meal, soybean flour, soybean flakes, soybean hulls, soy cheese, animal feed comprising soybeans, and fuel products produced using soybean plants and soybean plant parts.

[0038] Compared with the prior art, the present invention has the following beneficial effects:

[0039] The present invention provides a transgenic soybean event AS-1, which can express α-amylase at a high level and has very high amylase activity. The amylase activity is as high as 622,000 U / g, which is much higher than the amylase activity of commercial enzymes produced by microbial fermentation; and has no obvious adverse effects on the growth, development and agronomic traits of the plant.

[0040] The transgenic soybean event can be used as a bioreactor to produce α-amylase on a large scale, which has significant cost advantages and broad application prospects. The transgenic soybean can be used as an enzyme preparation, which has lower production costs than microbial fermentation.

[0041] At the same time, the present invention provides a method for detecting the presence of transgenic soybean event AS-1 DNA in a sample, which is of great significance for achieving the traceability and supervision of soybean event AS-1. (IV) Description of the drawings

[0042] Figure 1 Schematic diagram of the transformation vector.

[0043] Figure 2 Schematic diagram of exogenous inserted genes and soybean genome structure.

[0044] Figure 3 This is the internal reference gene control diagram for the specific detection of transgenic soybean event AS-1. M, protein marker; 1, blank control; 2, W28; 3-6, seeds, leaves, pods, and stems of AS-1 soybean; 7, CAL16; 8, rice genome; 9, corn genome.

[0045] Figure 4 This is the electrophoresis diagram of the right flank sequence specificity detection of the transgenic soybean event AS-1. M, protein marker; 1, blank control; 2, W28; 3-6, seeds, leaves, pods, and stems of AS-1 soybean; 7, CAL16; 8, rice genome; 9, corn genome.

[0046] Figure 5 This is the electrophoresis diagram of the specific detection of the left flank sequence of the transgenic soybean event AS-1. M, protein marker; 1, blank control; 2, W28; 3-6, seeds, leaves, pods, and stems of AS-1 soybean; 7, CAL16; 8, rice genome; 9, corn genome. (V) Specific implementation methods

[0047] The present invention is further described below in conjunction with specific embodiments, but the protection scope of the present invention is not limited thereto:

[0048] Example 1. Obtaining soybean transformation events

[0049] 1. Construction of soybean transformation vector

[0050] The soybean transformation vector of the present invention is constructed based on pCambia1300 (GenBank: AF234296.1). The vector map is shown in Figure 1 . The T-DNA of the transformation vector contains an α-amylase gene expression cassette and a flazasulfuron-resistant gene expression cassette. Among them, the α-amylase gene expression cassette encodes the α-amylase AmyS protein, which is driven by the pCG1 promoter and the terminator is the AtHSP18.2 terminator; the flazasulfuron-resistant gene expression cassette encodes a flazasulfuron-resistant cytochrome P450 oxidase, expresses the CdP450 protein, is driven by the pCaMV35S promoter and the terminator is the CaMV35S terminator. The nucleotide fragment shown in 966-8844 of SEQ ID NO:20 was artificially synthesized and connected to the multiple cloning site of pCambia1300 by seamless cloning to obtain a soybean transformation vector. The components and positions of the above-mentioned artificially synthesized nucleotide fragments can be obtained from Table 1.

[0051] 2. Transformation of soybean transformation vector into Agrobacterium

[0052] The transformation plasmid obtained in step 1 was introduced into the Agrobacterium EHA105 strain by electroporation to obtain Agrobacterium containing the soybean transformation vector.

[0053] 3. Agrobacterium-mediated genetic transformation of soybean

[0054] The soybean transformation process mainly refers to the Agrobacterium-mediated half-seed transformation method of Paz (Paz MM, Martinez JC, Kalvig AB, et al. (2006), Improved cotyledonary node method using an alternative explant derived from mature seed for efficient Agrobacterium-mediated soybean transformation. Plant Cell Reports, 25 (3): 206-213). The details are as follows:

[0055] (1) Sterilization of soybean seeds: Select soybean seeds of good quality (variety: Wandou 28), place them in a desiccator, and treat them with chlorine gas produced by the reaction of 100 mL of 30% sodium hypochlorite aqueous solution and 8 mL of pure hydrochloric acid for 16 hours.

[0056] (2) Seed germination: Sterilized soybean seeds were soaked in sterile distilled water for 16 hours to obtain germinated soybean seeds.

[0057] (3) Preparation of Agrobacterium culture liquid: Agrobacterium containing the transformation vector was streaked on YEP solid medium containing 50 mg / L Kan (kanamycin) and 25 mg / L rifampicin and cultured at 28°C for 2 days. The colonies on the YEP culture plate were scraped and evenly suspended in the infection medium, and the concentration was adjusted to OD650 = 0.5-0.6.

[0058] (3) Explant preparation and infection: Germinated soybean seeds were cut longitudinally along the hilum into two halves, the seed coat of the halves with the hypocotyl and embryo was removed and the halves were quickly placed in the prepared Agrobacterium solution and infected at room temperature for 30 min. The halves were taken out and placed in a sterile culture dish lined with filter paper, sealed and cultured at 22°C in the dark for 3 days.

[0059] (4) Bud induction culture: The explants after co-cultivation were transferred to a bud induction medium containing 0.1 mg / mL flazasulfuron-methyl and cultured at 26°C and 16 h light / 8 h dark for 3 weeks.

[0060] (5) Bud elongation and screening: After induction, the cotyledons of the explants were cut off and the part with clustered buds was transferred to a bud elongation medium supplemented with 0.04 mg / L pyrasulfuron-methyl and cultured at 26°C and 16 h light / 8 h dark for 21 days.

[0061] (6) Rooting and transplanting: After elongation, the clustered shoots were transferred to a rooting medium containing 0.025 mg / L pyrasulfuron-methyl for rooting. When the roots grew out, the medium attached to the roots was washed off and the T0 seedlings were transplanted to soil for cultivation.

[0062] Example 2: Screening of transgenic strains expressing high α-amylase

[0063] (1) Spraying herbicides

[0064] 110 T0 transgenic lines were transplanted into the soil. After 2-3 weeks of transplantation, 25% flazasulfuron water dispersible granules (purchased from Zhengzhou Lane Ping'an Biotechnology Co., Ltd.) were sprayed. According to the instructions, the spraying concentration was 100 mg / L. After 7 days, the leaves of some transgenic lines turned yellow and died, and some lines grew the same as the non-transgenic control Wandou 28 sprayed with water. During the growth period, the surviving transgenic lines were observed and recorded, and the transgenic lines with abnormal growth were excluded. The mature seeds of the T0 plants were harvested for α-amylase activity determination and seed propagation. Two weeks after sowing, the T1 generation plants were sprayed with sulfamethoxam at the same concentration as the T0 plants. One week after spraying, the segregation ratio of surviving plants and dead plants in each line was counted. The chi-square test results showed that the segregation ratio of surviving plants and dead plants in the T1 generation plants of AS1, AS23, AS41, AS53 and AS79 was consistent with 3:1, indicating that the T-DNA in these transgenic lines may be integrated into the soybean genome as a single copy or a single insertion site.

[0065] (2) Determination of α-amylase activity in soybean seeds

[0066] The activity of α-amylase was determined by the DNS method (Miller GL (1959). Using of dinitrosalicylicacid reagent for determination of reducing sugar. Analytical Chemistry, 31: 426-489). The preparation of 3,5-dinitrosalicylic acid (DNS) solution was as follows: 185 g potassium tartrate was dissolved in 500 mL of water, heated to 45°C to dissolve, 6.3 g DNS and 262 mL 2M NaOH solution, 5 g crystalline phenol, 5 g sodium sulfite were added, stirred to dissolve, cooled and diluted to 1000 mL with water.

[0067] Preparation of maltose standard solution and preparation of standard curve: Prepare 1 mg / mL maltose standard solution with water, add different reagents according to Table 2, mix well and boil in boiling water for 5 minutes, measure the absorbance at a wavelength of 540 nm, and draw a standard curve with maltose content as the horizontal axis and absorbance as the vertical axis.

[0068] Table 2 Preparation of maltose standard curve

[0069]

[0070] Substrate preparation: 1 g of soluble starch was dissolved in 5 mL of sodium acetate buffer (20 mM sodium acetate, solvent is water, pH 5.4). Another 95 mL of sodium acetate buffer was heated to boiling and slowly poured into the previously dissolved soluble starch solution and stirred until transparent.

[0071] Preparation of enzyme solution: Weigh the seeds of AS soybean lines identified as positive, add 1 ml of sodium acetate-sodium chloride buffer (20 mM sodium acetate, 250 mM sodium chloride, solvent is water, pH 5.4) for every 20 mg, and grind to obtain enzyme solution.

[0072] Determination of enzyme activity: Dilute the enzyme solution with sodium acetate-sodium chloride buffer (20mM sodium acetate, 250mM sodium chloride, solvent is water, pH 5.4), take 50μL of the diluted enzyme solution and add it to 450μL of substrate, react at 65℃ for 1 hour, add 1mL DNS solution to terminate the reaction, boil in boiling water for 5min and measure OD540. Definition of enzyme activity: The amount of enzyme required to produce 1μmol of reducing sugar per minute at 65℃ is one enzyme activity unit, expressed as U. Calculate the enzyme activity based on the drawn maltose standard curve.

[0073] The harvested mature T0 generation seeds were taken for enzyme activity determination, and 3 seeds were determined for each strain. At the same time, the non-transgenic soybean Wandou 28 (W28) was used as a negative control, and the purchased commercial enzymes C1 and C2 were used as positive controls. The determination results of some transgenic strains are shown in Table 3: Among them, the amylase activities of the commercial enzymes C1 and C2 were 344,000 and 58,000 U / g, respectively, and the amylase activity of AS-1 was the highest. The average enzyme activity of the three seeds reached 622,000 U / g, which was much higher than the activity of the commercial enzymes and had a high prospect for industrial application; the amylase activities of other transformation events were between 22,000 and 210,000 U / g, which was significantly lower than that of the transformation event AS-1, and the industrial application prospects were far less than those of AS-1; the amylase activity in the non-transgenic control W28 seeds was only 357 U / g (average value).

[0074] Table 3 Amylase activity of T0 soybean seeds

[0075]

[0076]

[0077] Example 3: Investigation of agronomic traits of transgenic soybean line AS-1

[0078] This example provides field agronomic trait survey data of the transgenic soybean line AS-1. The specific process is: AS-1 transgenic soybeans were planted in the field, and non-transgenic control Wandou 28 was planted as a control. The field experiment adopted a completely randomized block design with 4 replications. Each plot area was 10m 2, about 40 plants. Representative agronomic traits were evaluated, including emergence rate (%), plant height (cm), number of branches, number of pods per plant (pieces), number of grains per plant (grains), grain weight per plant (g) and 100-grain weight (g). Finally, variance analysis was performed on the quantifiable agronomic traits, and the results are shown in Table 4. This example demonstrates that the transgenic soybean line AS-1 provides similar agronomic traits to the untransformed Wandou 28 soybean plants in the field.

[0079] Table 4 Main agronomic traits of transgenic soybean AS-1 and non-transgenic soybean

[0080] strain AS-1 Wandou 28 Seedling rate (%) 98.5 98.0 Plant height (cm) 61.7±3.9 60.3±4.1 Number of branches 2.7±0.5 2.3±0.5 Number of pods per plant 31.7±4.5 33.0±5.1 Number of grains per plant (grains) 50.3±4.1 51.7±3.7 Single plant grain weight (g) 10.6±0.7 10.9±0.7 100-grain weight (g) 21.7±1.3 21.1±1.0

[0081] Example 4. Analysis of T-DNA insertion site in soybean transformation event AS-1

[0082] (1) Extraction of soybean genome

[0083] The genomic DNA of the soybean transformation line AS-1 screened in Example 2 was extracted using the CTAB (cetyltrimethylammonium bromide) method.

[0084] 100 mg of young T0 leaves of transgenic soybean line AS-1 were ground into powder in liquid nitrogen and then added with 0.8 mL of CTAB buffer (20 g / L CTAB, 1.4 M NaCl, 100 mM Tris-HCl, 20 mM EDTA, water as solvent, pH 7.4) preheated in a 65°C water bath. 8.0), after fully mixing, water bath in a 65°C water bath for 60 minutes; add an equal volume of chloroform, mix by inversion, centrifuge at 12000rpm (revolutions per minute) for 10 minutes, and draw the supernatant into a new centrifuge tube; add 0.7 times the volume of isopropanol, gently shake the centrifuge tube, centrifuge at 12000rpm for 1 minute, and collect DNA to the bottom of the tube; discard the supernatant, add 1mL of 75% ethanol aqueous solution, wash the precipitate, centrifuge at 12000rpm for 1 minute, repeat the washing once, and blow dry in a clean bench; dissolve the DNA precipitate in an appropriate amount of TE buffer (10mM Tris-HCl, 1mM EDTA, solvent is water, pH 8.0), determine the concentration of DNA by Nanodrop, and save it for later use.

[0085] (2) Analysis of flanking DNA sequences

[0086] The hiTAIL-PCR (High-Efficiency Thermal asymmetric interlaced PCR) method reported by Liu et al. (Liu YG, Chen Y (2007). High-efficiency thermal asymmetric interlaced PCR for amplification of unknown flanking sequences. Biotechniques 43 (5): 649-656) was used to determine the regional sequences on both sides of the exogenous T-DNA insertion point of the excellent transformation event AS-1 screened in Example 2. The primer sequences are shown in Table 5, the PCR reaction conditions are shown in Table 6, and the PCR reaction system is shown in Table 7.

[0087] Table 5 TAIL-PCR primer sequences

[0088]

[0089] In Table 5, V represents G, A, C, N represents A, T, C, G, and B represents G, T, C.

[0090] Table 6 TAIL-PCR reaction conditions

[0091]

[0092] Table 7 PCR reaction system

[0093]

[0094]

[0095] The PCR product recovery kit of Axygen was used to recover the second or third round of PCR amplification products with strong specificity, connected to the PMD20-T cloning vector (TaKaRa, Code: D107A), transformed into Escherichia coli, and the obtained positive clones were sequenced. The obtained sequence information was compared and analyzed with the soybean online database (http: / / www.soybase.org) to retrieve similar soybean genome sequences. After comparison and verification, the flanking sequence of the AS-1 insertion sequence of the transformation event was obtained, wherein the left flank sequence is shown in SEQ ID NO: 13, and the right flank sequence is shown in SEQ ID NO: 12.

[0096] (3) AS-1 junction sequence information

[0097] The junction sequence is a unique sequence formed when the exogenous T-DNA is inserted into the plant genome, which is composed of the plant genomic DNA and the 5' end DNA of the exogenous insertion sequence connected together or the 3' end DNA of the exogenous insertion sequence and the plant genomic DNA connected together. The junction sequence is unique and can be used as an identity feature to identify a certain transgenic event. The nucleotide length of the junction sequence is not particularly specified. In the present invention, the nucleotide sequences shown in SEQ ID NO:1 and SEQ ID NO:2 or their complementary sequences can be used as the junction sequence of the transgenic event AS-1. Longer junction sequences can be selected from SEQ ID NO:3-8 or their complementary sequences or SEQ ID NO:20 or its complementary sequence. The genome and genetic elements contained in SEQ ID NO:20 are shown in Table 1.

[0098] Example 5: Specific detection of transgenic soybean event AS-1

[0099] In this embodiment, a pair of PCR primers are designed to generate an amplification product after reacting with a template, and the amplification product is further detected to identify the presence of transgenic soybean event AS-1 DNA. The amplification product that can be identified as the presence of transgenic soybean event AS-1 DNA is a nucleotide sequence containing one or more of SEQ ID NO: 1-8 or its complementary sequence or SEQ ID NO: 20 or its complementary sequence.

[0100] The primer pairs used for the amplification reaction are designed based on the inserted sequence and its flanking sequence. In order to obtain an amplified product containing the nucleotide sequence of SEQ ID NO:1, SEQ ID NO:3 or SEQ ID NO:5, the forward primer F1 (SEQ ID NO:14) is designed based on the right flank region in SEQ ID NO:7, and the reverse primer R1 (SEQ ID NO:15) is designed based on the inserted sequence region in SEQ ID NO:7. The forward primer F1 (SEQ ID NO:14) is identical to the 502-524 nucleic acid sequence corresponding to SEQ ID NO:20, and the reverse primer R1 (SEQ ID NO:15) is identical to the 785-809 nucleic acid sequence corresponding to SEQ ID NO:20. Primer information is shown in Table 8.

[0101] In this embodiment, in order to obtain an amplification product containing the nucleotide sequence of SEQ ID NO:2, SEQ ID NO:4 or SEQ ID NO:6, a forward primer F2 (SEQ ID NO:16) was designed based on the left wing region in SEQ ID NO:8, and a reverse primer R2 (SEQ ID NO:17) was designed based on the insertion sequence region in SEQ ID NO:8. The forward primer F2 (SEQ ID NO:16) is identical to the 16216-16240 nucleic acid sequence corresponding to SEQ ID NO:20, and the reverse primer R2 (SEQ ID NO:17) is identical to the 15876-15899 nucleic acid sequence corresponding to SEQ ID NO:20. Primer information is shown in Table 8.

[0102] Table 8 Primer information

[0103]

[0104] The seeds, leaves, pods and stems of the transgenic soybean event AS-1 in Example 1 were taken respectively, and the genomic DNA was extracted by the CTAB method as a template, and the control without template addition, the genome of non-transgenic soybean Wandou 28 (W28), the genome of transgenic soybean CAL16, the genome of conventional rice and the genome of conventional corn were used as controls, and PCR amplification was performed according to the reaction system in Table 9 under the action of primer pairs F1 / R1, F2 / R2 and Lec-F / Lec-R, and the amplified products were detected by agarose gel electrophoresis. The PCR reaction program was: denaturation at 95°C for 3min, denaturation at 95°C for 15s, annealing at 58°C for 30s, extension at 72°C for 30s, 32 cycles in total, and finally extension at 72°C for 3min.

[0105] Table 9 PCR reaction system

[0106]

[0107] In this experiment, the endogenous gene Lectin of soybean was used as the reference gene, and the primer pair Lec-F / Lec-R was used to amplify it. The results showed that the samples containing soybean genome were able to detect a band of about 118bp, which was consistent with the expected size, while the target band could not be detected in other samples ( Figure 3 ), indicating that the quality of the soybean genome used for testing meets the requirements. M, protein marker; 1, blank control; 2, W28; 3-6, seeds, leaves, pods, and stems of AS-1 soybean; 7, CAL16; 8, rice genome; 9, corn genome.

[0108] The results of agarose gel electrophoresis of the amplified products using primer pair F1 / R1 showed that only the AS-1 sample could detect a band of about 308 bp, and the band size was consistent with expectations, while the target bands could not be detected in other samples ( Figure 4 ), indicating that the primer pair provided by the present invention can specifically detect the presence of AS-1. M, protein marker; 1, blank control; 2, W28; 3-6, seeds, leaves, pods, and stems of AS-1 soybean; 7, CAL16; 8, rice genome; 9, corn genome.

[0109] The results of agarose gel electrophoresis of the amplified products using primer pair F2 / R2 showed that only the AS-1 sample could detect a band of 365 bp, and the band size was consistent with expectations, while the target bands could not be detected in other samples ( Figure 5 ), indicating that the primer pair provided by the present invention can specifically detect the presence of AS-1. M, protein marker; 1, blank control; 2, W28; 3-6, seeds, leaves, pods, and stems of AS-1 soybean; 7, CAL16; 8, rice genome; 9, corn genome.

[0110] In addition to F1 (SEQ ID NO: 14), R1 (SEQ ID NO: 15), F2 (SEQ ID NO: 16), R2 (SEQ ID NO: 17), it should be apparent to those skilled in the art that other primers can be designed to amplify sequences within SEQ ID NO: 20 that are unique to the presence of DNA derived from transgenic soybean event AS-1 in a sample and are used to detect the presence of DNA derived from transgenic soybean event AS-1 in a sample; other primer sequences can be selected from SEQ ID NO: 7, SEQ ID NO: 8 or SEQ ID NO: 20 by those skilled in the art of DNA amplification methods. It is within the scope of the present invention to use these DNA primer sequences with modifications to the methods of this embodiment. The primer sequences of the present invention that can obtain an amplicon from a sample containing AS-1 include at least one DNA primer sequence derived from SEQ ID NO: 7, SEQ ID NO: 8 or SEQ ID NO: 20.

[0111] Example 6: Hybridization to produce offspring containing transgenic soybean event AS-1

[0112] To produce soybean plants or plant parts thereof comprising expression of alpha-amylase and herbicide tolerance traits, soybean plants containing transgenic soybean event AS-1 can be crossed with soybean plants containing potentially any other soybean event or combination thereof and the phenotypes evaluated to determine the resulting characteristics of the progeny plants.

[0113] Specific steps of hybridization:

[0114] (1) Flower selection: Select flowers from the middle to upper nodes (6th to 12th) of healthy, pest-free, undamaged, non-GMO soybean plants, with petals about to emerge from the calyx and the color of the petals visible;

[0115] (2) Emasculation: Use the index finger and thumb of your left hand to gently pinch the pedicel and the base of the flower. Use tweezers in your right hand to tear off most of the calyx downward or diagonally downward to expose the closed corolla. At this time, clamp about 1 / 3 of the upper part of the corolla diagonally downward from the standard petal to the keel petal (at an angle of about 45 degrees to the pedicel) (because the stigma bends toward the standard petal, this can avoid pinching the stigma), and pull it up slightly diagonally toward the standard petal;

[0116] (3) Flower pollination: Tear off the sepals at the keel of the male parent flower of the transgenic soybean event AS-1, separate the corolla from between the two keels, and expose the yellow anthers (which look fluffy on the surface); then, remove the pollen mass from the middle of the filament, gently pinch off the male flower with your left hand, aim the anther at the stigma, and gently rub it once or twice; then, carefully insert the pollen mass upside down on the style, so that it can continue to scatter pollen and protect the exposed stigma.

[0117] The properties conferred by the cross to the offspring plants produced by the plant breeding described herein may extend beyond the expression alpha-amylase and herbicide tolerance of event AS-1, including but not limited to aboveground pest control, herbicide tolerance, nematicidal properties, drought resistance, virus resistance, antifungal control, bacterial resistance, male sterility, cold tolerance, salt tolerance, increased yield, enhanced oil composition, increased oil content, enhanced nutrient use efficiency, or altered amino acid content. Examples of transgenic events with improved agronomic traits are well known in the art.

Claims

1. A nucleic acid sequence of a transgenic soybean event AS-1, characterized in that: The nucleotide sequence comprises SEQ ID NO: 1 or a complementary sequence thereof, and / or SEQ ID NO: 2 or a complementary sequence thereof.

2. The nucleic acid sequence according to claim 1, characterized in that The nucleic acid sequence comprises one or more of SEQ ID NOs: 3-8 or their complementary sequences or SEQ ID NO: 20 or its complementary sequence.

3. The nucleic acid sequence according to any one of claims 1 to 2, characterized in that The nucleic acid sequence is derived from a plant, seed or cell comprising a transgenic soybean event AS-1.

4. A specific primer pair for detecting the transgenic soybean event AS-1 according to claim 1, characterized in that: The primer pair comprises a first primer and a second primer different from the first primer, the first primer comprising at least 11 consecutive nucleotides in SEQ ID NO: 20 or its complementary sequence, and the second primer comprising at least 11 consecutive nucleotides in SEQ ID NO: 20 or its complementary sequence.

5. The specific primer pair according to claim 4, characterized in that: The nucleotide sequence of the first primer is shown as SEQ ID NO: 15 or SEQ ID NO: 17, and the nucleotide sequence of the second primer is shown as SEQ ID NO: 14 or SEQ ID NO:

16.

6. A method for detecting the presence of DNA of the transgenic soybean event AS-1 of claim 1 in a sample, characterized in that: The method comprises: 1) contacting a sample to be detected with the specific primer pair according to claim 4; 2) performing a polynucleotide amplification reaction; 3) detecting the presence of an amplification product; if the presence of an amplification product is detected, the DNA of the transgenic soybean event AS-1 is present in the test sample; the amplification product comprises SEQ ID NO: 1 or its complementary sequence, and / or SEQ ID NO: 2 or its complementary sequence.

7. A method for producing α-amylase using the transgenic soybean event AS-1 of claim 1, characterized in that: The method comprises: planting soybean seeds containing a specific region nucleic acid sequence, harvesting soybeans containing α-amylase, and crushing the soybeans to be directly used as amylase; the specific region nucleic acid sequence is from a transgenic soybean event AS-1, and the specific region nucleic acid sequence comprises SEQ ID NO: 1 or a complementary sequence thereof, and / or SEQ ID NO: 2 or a complementary sequence thereof.

8. A method for cultivating soybean plants expressing α-amylase containing the transgenic soybean event AS-1 of claim 1, characterized in that: The method comprises: hybridizing a soybean plant containing a specific region nucleic acid sequence with another soybean plant not containing the specific region nucleic acid sequence, generating and harvesting offspring plants, wherein the α-amylase activity in the offspring plants is significantly increased compared with the soybean plant not containing the specific region nucleic acid sequence; the specific region nucleic acid sequence is from a transgenic soybean event AS-1, and the specific region nucleic acid sequence comprises SEQ ID NO: 1 or a complementary sequence thereof, and / or SEQ ID NO: 2 or a complementary sequence thereof.

9. A transgenic plant cell produced from the transgenic soybean event AS-1 of claim 1, characterized in that: The transgenic plant cell is obtained by transferring the specific region nucleic acid sequence of the transgenic soybean event AS-1 into the plant genome, and the specific region nucleic acid sequence comprises SEQ ID NO: 1 or its complementary sequence, and / or SEQ ID NO: 2 or its complementary sequence.

10. An agricultural product or processed product produced from the transgenic soybean event AS-1 of claim 1, characterized in that: The agricultural products or processed products contain a specific region nucleic acid sequence, and the specific region nucleic acid sequence contains SEQ ID NO:1 or its complementary sequence, and / or SEQ ID NO:2 or its complementary sequence; the processed products include but are not limited to: soy protein, soybean meal, soybean flour, soybean flakes, soy hulls, soy cheese, animal feed containing soybeans, and fuel products produced using soybean plants and soybean plant parts.

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