Transgenic soybean event HLAF-3 with high expression of human lactoferrin as well as detection method and application of transgenic soybean event HLAF-3

Abstract

The invention discloses a transgenic soybean event HLAF-3 highly expressing human lactoferrin and a detection method and application thereof, the transgenic soybean event HLAF-3 simultaneously expresses two proteins of human lactoferrin and CP4EPSPS, has high lactoferrin content, is stable in heredity and has no obvious adverse effect on agronomic traits; the gene cp4epsps for expressing the CP4EPSPS protein is only used as a selection marker; the invention also provides a method for detecting the transgenic soybean event HLAF-3. The method can be used for rapidly and accurately detecting whether a biological sample contains the DNA molecule of the transgenic soybean event HLAF-3 or not.

Description

(I) Technical field

[0001] The present invention relates to a transgenic soybean event expressing human lactoferrin and an identification method thereof, and in particular to a transgenic soybean event HLAF-3 containing human lactoferrin and a nucleic acid sequence and a method thereof for detecting whether a biological sample contains the transgenic soybean event HLAF-3. (II) Background technology

[0002] Lactoferrin is a natural non-heme iron-binding glycoprotein first discovered in milk in 1939. Lactoferrin is mainly synthesized and secreted by mammalian mucosal epithelial cells and is widely present in breast milk, as well as in internal and external secretions such as tears, saliva, plasma, pancreatic juice, and bile. The content of lactoferrin in breast milk varies at different stages of lactation. The content of lactoferrin in colostrum is relatively high. For example, the content of lactoferrin in human colostrum is 1.5-10.1 mg / mL, while the content of lactoferrin in normal milk is only 2.0-3.3 mg / mL (Wang B, Timilsena Y, Blanch E, et al. Lactoferrin: Structure, Function, Denaturation and Digestion. Critical Reviews in Food Science & Nutrition, 2017(2): 00-00). In addition, the content of lactoferrin varies greatly between species. For example, the lactoferrin content in bovine colostrum (0.28–1.36 mg / mL) is lower than that in human colostrum, and the lactoferrin content in bovine milk is even lower, at only 0.03-0.49 mg / mL (Krolitzki E, Schwaminger S, Pagel M, et al. Current practices with commercial scale bovine lactoferrin production and alternative approaches, International Dairy Journal, 2022 (126) 105263). There is also a small amount of lactoferrin in the milk of animals such as goats, camels, pigs, horses, rabbits, and dogs, but the main sources of lactoferrin are still human milk and cow milk.

[0003] Lactoferrin has the functions of regulating body iron metabolism, enhancing antibacterial and antiviral ability, regulating immunity, and anti-oxidation, so it is widely used in the fields of biomedicine, food health care, feed industry, etc., and the demand is large. Human lactoferrin is an ideal baby food supplement, but there is little industrially produced human lactoferrin available. At present, commercially available lactoferrin is mainly separated from milk or sweet whey, and it is necessary to use ion exchange chromatography, salting out, ultrafiltration and a combination of methods. These separation and purification processes are costly and require a large amount of raw materials. Therefore, it is of great significance to explore a method for low-cost production of lactoferrin, especially human lactoferrin.

[0004] In recent years, the use of transgenic plants as bioreactors to express recombinant proteins has become very attractive. Plant bioreactors have the following advantages: 1) Higher plants can synthesize active proteins from eukaryotic organisms; 2) Plant seeds have high protein content, low protease activity and low moisture content, which is conducive to the stability of recombinant proteins; 3) It is easy to expand the scale of production, low planting cost and environmental sustainability. Soybean is one of the main crops and is currently also used as a bioreactor. Compared with other crops, soybean has the following advantages: First, it is a self-pollinating crop with genetic stability; second, the rhizobia of soybean have nitrogen fixation, which can reduce the demand for chemical fertilizers; third, soybean is rich in protein, about 40%, and has a high synthesis potential.

[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. 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, and may even affect the agronomic traits of the plant. 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 plants, 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 HLAF-3 highly expressing human lactoferrin and a detection method and application thereof. The transgenic soybean event HLAF-3 simultaneously expresses two proteins, human lactoferrin and CP4 EPSPS, has a very high lactoferrin content, is genetically stable and has no obvious adverse effects on agronomic traits; the gene cp4 epsps expressing the CP4 EPSPS protein is only used as a screening marker; the present invention also provides a method for detecting the transgenic soybean event HLAF-3, and the method can quickly and accurately detect whether a biological sample contains a DNA molecule of the transgenic soybean event HLAF-3.

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

[0009] In a first aspect, the present invention provides a transgenic soybean event HLAF-3 that highly expresses human lactoferrin, wherein the transgenic soybean event HLAF-3 comprises an exogenous insertion sequence, wherein the exogenous insertion sequence has a nucleotide sequence shown in SEQ ID NO: 10 as a left wing region and a nucleotide sequence shown in SEQ ID NO: 11 as a right wing region.

[0010] Furthermore, the exogenous insertion sequence comprises two human lactoferrin gene expression cassettes and one glyphosate-resistant gene expression cassette; the human lactoferrin gene expression cassette encodes human lactoferrin HLF protein, wherein the HLF protein in one expression cassette is driven by a pGM2S-1 promoter and a terminator is an AtHSP18.2 terminator, and the HLF protein in another expression cassette is driven by a pGY5 promoter and a terminator is an NOS terminator, and the promoter and the HLF protein are both connected with an At2S2 signal peptide and a TEV 5'UTR; the glyphosate-resistant gene expression cassette encodes a glyphosate-resistant CP4 EPSPS protein, which is driven by a pCaMV35S promoter and a terminator is a CaMV35S terminator, and the promoter and the CP4 EPSPS protein are connected with a CTP transduction peptide and a TEV 5'UTR; and the two human lactoferrin gene expression cassettes and one glyphosate-resistant gene expression cassette are provided with MAR sequences mutually.

[0011] Furthermore, the nucleotide sequence of the exogenous insertion sequence is shown as 1001-14834 in SEQ ID NO:9.

[0012] In a second aspect, the present invention provides a nucleic acid sequence for detecting transgenic soybean event HLAF-3, wherein the nucleic acid sequence comprises SEQ ID NO: 1 or its complementary sequence, and / or SEQ ID NO: 2 or its complementary sequence.

[0013] The SEQ ID NO:1 or its complementary sequence is a 50-nucleotide sequence near the 5'-end integration site of the insertion sequence of the transgenic soybean event HLAF-3. The SEQ ID NO:1 or its complementary sequence spans the flanking genomic sequence and the 5'-end DNA sequence of the insertion sequence. The presence of the transgenic soybean event HLAF-3 can be identified by containing the SEQ ID NO:1 or its complementary sequence.

[0014] The SEQ ID NO: 2 or its complementary sequence is a 50-nucleotide sequence near the 3'-end integration site of the insertion sequence of the transgenic soybean event HLAF-3. The SEQ ID NO: 2 or its complementary sequence spans the flanking genomic sequence and the 3'-end DNA sequence of the insertion sequence. The presence of the transgenic soybean event HLAF-3 can be identified by containing the SEQ ID NO: 2 or its complementary sequence.

[0015] Furthermore, the nucleic acid sequence comprises one or more of SEQ ID NOs: 3-8 or their complementary sequences.

[0016] The SEQ ID NO: 3, 5, 7 or their complementary sequences span the 5' end DNA sequence of the flanking genomic sequence and the inserted sequence, and the presence of the transgenic soybean event HLAF-3 can be identified by including the SEQ ID NO: 3, 5, 7 or their complementary sequences.

[0017] 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 HLAF-3 can be identified by including the SEQ ID NO: 4, 6, 8 or their complementary sequence.

[0018] Furthermore, the nucleic acid sequence comprises SEQ ID NO: 9 or its complementary sequence. SEQ ID NO: 9 includes an insertion sequence and left and right flanking regions of the insertion sequence, and has a length of 15834 nucleotides. The genome and genetic elements specifically included are shown in Table 1. The presence of SEQ ID NO: 9 or its complementary sequence can be identified as the presence of transgenic soybean event HLAF-3.

[0019] The nucleic acid sequence is derived from a plant, seed or cell comprising a transgenic soybean event HLAF-3.

[0020] The present invention also provides a recombinant vector or recombinant cell containing the transgenic soybean event HLAF-3, which contains the exogenous insertion sequence (1001-14834 in SEQ ID NO: 9).

[0021] The transgenic soybean event HLAF-3 of the present invention is a soybean cotyledon node transformation method mediated by Agrobacterium, in which an exogenous T-DNA sequence containing human lactoferrin and a glyphosate-resistant gene expression frame is introduced into the soybean genome, a soybean transgenic population is obtained by regenerating soybean cells containing the exogenous gene, and a soybean transformation event that can meet production needs is screened by molecular biology and bioassay methods. The offspring of the transgenic soybean event HLAF-3 of the present invention are obtained by selfing or hybridization, and the soybean events and seeds in which the flanking sequence of the inserted sequence in the genome of any offspring is SEQ ID NO: 10 or SEQ ID NO: 11 should be regarded as one aspect of the present invention.

[0022] In a third aspect, the present invention provides a specific primer pair for detecting transgenic soybean event HLAF-3, 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:9 or its complementary sequence, and the second primer comprising at least 11 consecutive nucleotides in SEQ ID NO:9 or its complementary sequence.

[0023] Furthermore, the first primer comprises any 11 or more consecutive polynucleotides in the transgenic insertion sequence region of SEQ ID NO: 9 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: 9 or its complementary sequence.

[0024] Furthermore, the nucleotide sequence of the first primer is shown as SEQ ID NO:13 or SEQ ID NO:15, and the nucleotide sequence of the second primer is shown as SEQ ID NO:12 or SEQ ID NO:14.

[0025] In a fourth aspect, the present invention provides a method for detecting the presence of DNA of a transgenic soybean event HLAF-3 in a sample, the method comprising:

[0026] 1) contacting the sample to be detected with the specific primer pair;

[0027] 2) performing a polynucleotide amplification reaction;

[0028] 3) detecting the presence of an amplification product; if the presence of the amplification product is detected, DNA of the transgenic soybean event HLAF-3 is present in the sample to be tested; the amplification product comprises SEQ ID NO: 1 or its complementary sequence, and / or SEQ ID NO: 2 or its complementary sequence.

[0029] Furthermore, the amplified product comprises one of SEQ ID NO: 3-8 or a complementary sequence thereof.

[0030] Furthermore, the amplification product comprises SEQ ID NO: 9 or its complementary sequence.

[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 The oligonucleotide probe is selected from SEQ ID NO: 1-9 or its complementary sequence.

[0033] In a fifth aspect, the present invention provides a method for producing human lactoferrin using the transgenic soybean event HLAF-3, the method comprising: planting soybean seeds containing a specific region nucleotide sequence, harvesting soybeans that highly express human lactoferrin, and separating and extracting human lactoferrin; the specific region nucleotide sequence is from the transgenic soybean event HLAF-3, and the specific region nucleotide sequence comprises SEQ ID NO: 1 or its complementary sequence, and / or SEQ ID NO: 2 or its complementary sequence.

[0034] In a sixth aspect, the present invention provides a method for cultivating soybean plants expressing human lactoferrin containing the transgenic soybean event HLAF-3, the method comprising: hybridizing a soybean plant containing a specific region nucleotide sequence with another soybean, generating and harvesting offspring plants, and harvesting plants having significantly increased human lactoferrin content in the offspring plants compared to soybeans not containing the specific region nucleotide sequence. The specific region nucleotide sequence is from the transgenic soybean event HLAF-3, and the specific region nucleotide 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 HLAF-3 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 the seventh aspect, the present invention provides a transgenic plant cell produced from the transgenic soybean event HLAF-3, wherein the transgenic plant cell is obtained by transferring the specific region nucleic acid sequence of the transgenic soybean event HLAF-3 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 eighth aspect, the present invention provides an agricultural product or processed product produced from the transgenic soybean event HLAF-3, wherein the agricultural product or processed product comprises a specific region nucleotide sequence, wherein the specific region nucleotide 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 containing soybeans, and fuel products produced using soybean plants and soybean plant parts.

[0038] Compared with the prior art, the beneficial effects of the present invention are mainly reflected in:

[0039] The present invention provides a transgenic soybean event HLAF-3, which can express human lactoferrin at a high level and has a very high lactoferrin content of up to 6.12 mg / g, which is much higher than the lactoferrin content in bovine colostrum and also higher than the lactoferrin content in human milk; 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 mass produce human lactoferrin, filling the gap in the market of human lactoferrin in infant food supplements. The use of the transgenic soybean to separate and purify lactoferrin has the advantages of low planting cost and easy mass production, and has significant cost advantages and broad application prospects.

[0041] At the same time, the present invention provides a method for detecting the presence of transgenic soybean event HLAF-3 DNA in a sample, which is of great significance for achieving the traceability and supervision of the soybean event HLAF-3. (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 electrophoresis diagram of the specific detection of the sequence on the right flank of the transgenic soybean event HLAF-3. M, protein marker; 1, blank control; 2, TL1; 3-6, seeds, leaves, pods, and stems of HLAF-3 soybean; 7, CAL16; 8, rice genome; 9, corn genome.

[0045] Figure 4 This is the electrophoresis diagram of the sequence-specific detection of the left flank of the transgenic soybean event HLAF-3. M, protein marker; 1, blank control; 2, TL1; 3-6, seeds, leaves, pods, and stems of HLAF-3 soybean; 7, CAL16; 8, rice genome; 9, corn genome. (V) Specific implementation methods

[0046] The present invention is further described below in conjunction with specific embodiments, but the protection scope of the present invention is not limited thereto:

[0047] Example 1. Obtaining soybean transformation events

[0048] 1. Obtaining soybean transformation vector

[0049] The soybean transformation vector of the present invention is constructed based on pCambia1300 (GenBank: ARB-2B34296.1). The vector map is as follows: Figure 1 As shown. The T-DNA of the transformation vector contains two lactoferrin gene expression cassettes and one glyphosate-resistant gene expression cassette. The human lactoferrin gene expression cassette encodes the human lactoferrin HLF protein, in which the HLF protein in one expression cassette is driven by the pGM2S-1 promoter and the terminator is the AtHSP18.2 terminator, and the HLF protein in the other expression cassette is driven by the pGY5 promoter and the terminator is the NOS terminator; the glyphosate-resistant gene expression cassette encodes the glyphosate-resistant CP4 EPSPS protein, which is driven by the pCaMV35S promoter and the terminator is the CaMV35S terminator. The specific components and positions of the T-DNA are shown in Table 1. The nucleotide fragment shown in 1076-14603 of SEQ ID NO:9 is artificially synthesized and connected to the multiple cloning site of pCambia1300 by seamless cloning to obtain the soybean transformation vector.

[0050] Table 1. Genome and genetic elements contained in SEQ ID NO:9

[0051]

[0052]

[0053] 2. Transformation of soybean transformation vector into Agrobacterium

[0054] The transformation vector obtained in step 1 was introduced into the Agrobacterium EHA105 strain by electroporation to obtain Agrobacterium containing the soybean transformation vector.

[0055] 3. Agrobacterium-mediated genetic transformation of soybean

[0056] 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:

[0057] (1) Sterilization of soybean seeds: Select soybean seeds of good quality (variety: Tianlong No. 1), put 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.

[0058] (2) Seed germination: Sterilized soybean seeds were soaked in sterile distilled water for 16 hours to obtain germinated soybean seeds.

[0059] (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.

[0060] (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.

[0061] (4) Bud induction culture: The explants after co-cultivation were transferred to a bud induction medium supplemented with 25 mg / L glyphosate and cultured at 26°C, 16 h light / 8 h dark for 3 weeks.

[0062] (5) Shoot elongation culture: After induction, the cotyledons of the explants were cut off and the part with clustered shoots was transferred to a shoot elongation medium supplemented with 5 mg / L glyphosate and cultured at 26°C and 16 h light / 8 h dark for 21 days.

[0063] (6) Rooting and transplanting: The elongated buds were transferred to a rooting medium containing 5 mg / L glyphosate 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.

[0064] Example 2. Screening of Transgenic Lines with High Yield of Lactoferrin

[0065] (1) Spraying Herbicides

[0066] After the T0 generation plants transplanted into the soil survived for 2 - 3 weeks, they were sprayed with glyphosate (Roundup, produced by Sinochem Crop Protection Co., Ltd. as an agent, aqueous solution, with an active ingredient of 30%), and the application dose was 200 mL / acre, and the water consumption per acre was 40 L. After 7 days of spraying, those with yellowing and withering leaves were negative, and those with the same growth as the clear water control were positive plants. The positive plants were retained and numbered for subsequent identification and detection. The screened positive plants were numbered respectively as: HLAF - 3, HLF - 8, HLF - 11, HLF - 17, HLF - 22, HLF - 24, HLF - 29, HLAF - 37.

[0067] (2) Determination of Human Lactoferrin Content in Soybean Seeds

[0068] T0 generation seeds of HLAF - 3, HLF - 8, HLF - 11, HLF - 17, HLF - 22, HLF - 24, HLF - 29, and HLAF - 37 lines were respectively taken for the determination of human lactoferrin content. Three seeds of each line were measured, and at the same time, non - transgenic soybean Tianlong 1 (TL1) was used as a control. The determination method was enzyme - linked immunosorbent assay (ELISA). The human lactoferrin ELISA kit was purchased from Shanghai Sangon Biotech Co., Ltd. (product number: D711315), and the operation steps were carried out according to the instructions.

[0069] The determination results are shown in Table 2: Among them, the lactoferrin content of HLAF - 3 was the highest, and the average human lactoferrin content of the three seeds reached 6.12 mg / g, far higher than the lactoferrin content in bovine colostrum (0.28 - 1.36 mg / mL), and also higher than the lactoferrin content in human normal milk (2.0 - 3.3 mg / mL), indicating that HLAF - 3 transgenic soybeans have broad application prospects and can be used for the separation and purification of human lactoferrin to fill the gap in the market of human lactoferrin in infant food supplements; the human lactoferrin content of other transgenic soybean lines was between 0.91 - 4.43 mg / g, much lower than that of the transgenic line HLAF - 3, and the industrial application prospects were not as good as those of HLAF - 3; the human lactoferrin content in the non - transgenic control TL1 seeds was below the detection limit.

[0070] Table 2. Human Lactoferrin Expression in T0 Generation Soybean Seeds

[0071] strain Human lactoferrin content (mg / g) HLAF-3 6.12±1.05 HLF-8 2.68±0.52 HLF-11 1.86±0.37 HLF-17 4.43±0.80 HLF-22 3.19±0.67 HLF-24 2.85±0.49 HLF-29 3.80±0.80 HLAF-37 0.91±0.20 TL1 <LOD

[0072] Note: The data in the table are mean ± standard deviation; <LOD refers to below the detection limit.

[0073] Example 3: Investigation of agronomic traits of transgenic soybean line HLAF-3

[0074] This example provides field agronomic trait survey data of the transgenic soybean line HLAF-3. The specific process is: HLAF-3 transgenic soybeans were planted in the field, and non-transgenic control Tianlong No. 1 was planted as a control. The field experiment adopted a completely randomized block design with 4 replications. Each plot had an area of ​​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 3. This example demonstrates that the transgenic soybean line HLAF-3 provides similar agronomic traits to the untransformed Tianlong No. 1 soybean plant in the field.

[0075] Table 3 Main agronomic traits of transgenic soybean HLAF-3 and non-transgenic soybean

[0076] strain HLAF-3 Tianlong No. 1 Seedling rate (%) 97.4 98.2 Plant height(cm) 50.1±3.7 49.5±3.5 Number of branches 2.4±0.3 2.6±0.3 Number of pods per plant 23.9±3.7 23.7±3.4 Number of grains per plant (grains) 39.9±3.2 39.5±2.9 Single plant grain weight (g) 7.6±0.6 7.5±0.5 100-grain weight (g) 18.4±0.9 18.9±1.0

[0077] Example 4: Analysis of insertion sites of soybean transformation event HLAF-3

[0078] (1) Extraction of soybean genome

[0079] The genomic DNA of the transgenic soybean line HLAF-3 screened in Example 2 was extracted using the CTAB (cetyltrimethylammonium bromide) method.

[0080] 100 mg of young T0 leaves of transgenic soybean line HLAF-3 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.

[0081] (2) Analysis of flanking DNA sequences

[0082] 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 HLAF-3 screened in Example 2. The primer sequences are shown in Table 4, the PCR reaction conditions are shown in Table 5, and the PCR reaction system is shown in Table 6.

[0083] Table 4 TAIL-PCR primer sequences

[0084]

[0085] In Table 4, V represents G, A, C, N represents A, T, C, G, and B represents G, T, C.

[0086] Table 5 TAIL-PCR reaction conditions

[0087]

[0088] Table 6 PCR reaction system

[0089]

[0090]

[0091] Axygen's PCR product recovery kit was used to recover the second or third round of PCR amplification products with strong specificity, and the recovered products were sequenced. The obtained sequence information was compared and analyzed with the soybean online database (https: / / venyao.xyz / soybeangdb / ) to retrieve similar soybean genome sequences. After comparison and verification, the flanking sequence of the HLAF-3 insertion sequence of the transformation event was obtained, wherein the left flank sequence is shown in SEQ ID NO: 10, and the right flank sequence is shown in SEQ ID NO: 11.

[0092] (3) HLAF-3 junction sequence information

[0093] The junction sequence is a unique sequence formed when the exogenous T-DNA is inserted into the plant genome. It is composed of the 5' or 3' DNA of the exogenous insertion sequence and the plant genomic DNA. The junction sequence is unique and can be used as an identity feature to identify a certain transgenic event. There is no special regulation on the nucleotide length of the junction sequence. 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 HLAF-3. Longer junction sequences can be selected from SEQ ID NO:3-9 or their complementary sequences. The genome and genetic elements contained in SEQ ID NO:9 are shown in Table 1, and the schematic diagram is shown in Figure 2 .

[0094] Example 5: Specific detection of transgenic soybean event HLAF-3

[0095] In this embodiment, a pair of PCR primers are designed to generate amplification products after reacting with the template, and the amplification products are further detected to identify the presence of transgenic soybean event HLAF-3 DNA. The amplification products that can be identified as the presence of transgenic soybean event HLAF-3 DNA are nucleotide sequences containing one or more of SEQ ID NOs: 1-9 or their complementary sequences.

[0096] The primer pairs used for the amplification reaction are designed based on the insertion sequence and its flanking sequence. In order to obtain an amplification product containing the nucleotide sequence of SEQ ID NO:1, SEQ ID NO:3 or SEQ ID NO:5, the forward primer LB-F (SEQ ID NO:12) is designed based on the left flank sequence in SEQ ID NO:9, and the reverse primer LB-SP3 (SEQ ID NO:13) is designed based on the insertion sequence in SEQ ID NO:9. The forward primer LB-F (SEQ ID NO:12) corresponds to nucleotides 893-917 of SEQ ID NO:9, and the reverse primer LB-SP3 (SEQ ID NO:13) corresponds to nucleotides 1091-1118 of SEQ ID NO:9. The primer information is shown in Table 7.

[0097] In this embodiment, in order to obtain an amplification product containing the nucleotide sequence of SEQ ID NO: 2 or SEQ ID NO: 4, a forward primer RB-F (SEQ ID NO: 14) was designed based on the right wing sequence in SEQ ID NO: 9, and a reverse primer RB-2b (SEQ ID NO: 15) was designed based on the insertion sequence in SEQ ID NO: 9. The forward primer RB-F (SEQ ID NO: 14) corresponds to nucleotides 14892-14915 of SEQ ID NO: 9, and the reverse primer RB-2b (SEQ ID NO: 15) corresponds to nucleotides 14713-14737 of SEQ ID NO: 9. The primer information is shown in Table 7.

[0098] Table 7 Primer information

[0099]

[0100] The seeds, leaves, pods and stems of the transgenic soybean strain HLAF-3 screened in Example 2 were taken respectively, and the genomic DNA was extracted by the CTAB method as a template, and the blank control was used without adding template, and the genome of non-transgenic soybean Tianlong No. 1 (TL1), the genome of transgenic soybean CAL16, the genome of conventional rice and the genome of conventional corn were used as controls. PCR amplification was performed according to the reaction system in Table 8 under the action of primer pairs LB-F / LB-SP3 and RB-F / RB-2b, 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, a total of 32 cycles, and finally extension at 72°C for 3min.

[0101] Table 8 PCR reaction system

[0102]

[0103] In this experiment, the agarose gel electrophoresis results of the amplified products using primer pair LB-F / LB-SP3 showed that only the HLAF-3 sample could detect a band of about 226 bp in size, and the band size was consistent with expectations, while the target bands could not be detected in other samples ( Figure 3 ), indicating that the primer pair provided by the present invention can specifically detect the presence of soybean transformation event HLAF-3. M, protein marker; 1, blank control; 2, TL1; 3-6, seeds, leaves, pods, and stems of HLAF-3 soybean; 7, CAL16; 8, rice genome; 9, corn genome.

[0104] The results of agarose gel electrophoresis of the products amplified using primer pair RB-F / RB-2b showed that only the HLAF-3 sample could detect a 203 bp band, which was consistent with the expected size, while the other samples could not detect the target band ( Figure 4 ), indicating that the primer pair provided by the present invention can specifically detect the presence of soybean transformation event HLAF-3. M, protein marker; 1, blank control; 2, TL1; 3-6, seeds, leaves, pods, and stems of HLAF-3 soybean; 7, CAL16; 8, rice genome; 9, corn genome.

[0105] In addition to primers LB-F (SEQ ID NO: 12), LB-SP3 (SEQ ID NO: 13), RB-F (SEQ ID NO: 14), RB-2b (SEQ ID NO: 15), it is well known to those skilled in the art that other primers can be designed to detect the presence of DNA derived from soybean transformation event HLAF-3 in a sample; other primer sequences can be selected from SEQ ID NO: 7, SEQ ID NO: 8 or SEQ ID NO: 9. It is within the scope of the present invention to use these DNA primer sequences with modifications to the method of this example.

[0106] Example 6. Hybridization to produce progeny containing soybean transformation event HLAF-3

[0107] To produce soybean plants or plant parts thereof containing expression of human lactoferrin, soybean plants containing soybean transformation event HLAF-3 can be crossed with soybean plants containing potentially any other soybean event or combination thereof and the phenotype evaluated to determine the resulting characteristics of the progeny plants.

[0108] Specific steps of hybridization:

[0109] (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;

[0110] (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;

[0111] (3) Flower pollination: Tear off the sepals at the keel of the male parent flower of the transgenic soybean event HLAF-3, 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.

[0112] The properties conferred by the cross to the progeny plants produced by the plant breeding described herein may extend beyond the human lactoferrin expressing properties of event HLAF-3, including but not limited to aboveground pest control, herbicide tolerance, nematicidal properties, drought resistance, viral 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 transgenic soybean event HLAF-3 that highly expresses human lactoferrin, characterized in that: The transgenic soybean event HLAF-3 comprises an exogenous insertion sequence, wherein the exogenous insertion sequence has a nucleotide sequence shown in SEQ ID NO: 10 as a left flank region and a nucleotide sequence shown in SEQ ID NO: 11 as a right flank region.

2. The transgenic soybean event HLAF-3 according to claim 1, characterized in that: The exogenous insertion sequence comprises two human lactoferrin gene expression cassettes and one glyphosate-resistant gene expression cassette.

3. A nucleic acid sequence for detecting the transgenic soybean event HLAF-3 according to claim 1, characterized in that: 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.

4. The nucleic acid sequence according to claim 3, characterized in that The nucleic acid sequence comprises one or more of SEQ ID NOs: 3-8 or their complementary sequences.

5. The nucleic acid sequence according to claim 3, characterized in that The nucleotide sequence comprises SEQ ID NO: 9 or its complementary sequence.

6. A specific primer pair for detecting the transgenic soybean event HLAF-3 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 comprises at least 11 consecutive nucleotides in SEQ ID NO: 9 or its complementary sequence, and the second primer comprises at least 11 consecutive nucleotides in SEQ ID NO: 9 or its complementary sequence.

7. The specific primer pair according to claim 6, characterized in that: The nucleotide sequence of the first primer is shown as SEQ ID NO:13 or SEQ ID NO:15, and the nucleotide sequence of the second primer is shown as SEQ ID NO:12 or SEQ ID NO:

14.

8. A method for detecting the presence of DNA of the transgenic soybean event HLAF-3 of claim 1 in a sample, characterized in that: The method comprises: 1) contacting a sample to be tested with the specific primer pair described in any one of claims 6-7; 2) performing a polynucleotide amplification reaction; 3) detecting the presence of an amplification product; if the presence of an amplification product is detected, DNA of the transgenic soybean event HLAF-3 is present in the test sample, and the amplification product comprises SEQ ID NO: 1 or its complementary sequence, and / or SEQ ID NO: 2 or its complementary sequence.

9. A method for producing human lactoferrin using the transgenic soybean event HLAF-3 of claim 1, characterized in that: The method comprises: planting soybean seeds containing a specific region nucleotide sequence, harvesting soybeans that highly express human lactoferrin, and separating and extracting human lactoferrin; the specific region nucleotide sequence is from a transgenic soybean event HLAF-3, and the specific region nucleotide sequence comprises SEQ ID NO: 1 or a complementary sequence thereof, and / or SEQ ID NO: 2 or a complementary sequence thereof.

10. A method for cultivating soybean plants expressing human lactoferrin containing the transgenic soybean event HLAF-3 of claim 1, characterized in that: The method comprises: hybridizing a soybean plant containing a specific region nucleotide sequence with another soybean to produce and harvest offspring plants, and harvesting plants having significantly increased human lactoferrin content in the offspring plants compared with soybeans not containing the specific region nucleotide sequence; the specific region nucleotide sequence is from a transgenic soybean event HLAF-3, and the specific region nucleotide sequence comprises SEQ ID NO: 1 or a complementary sequence thereof, and / or SEQ ID NO: 2 or a complementary sequence thereof.

11. A transgenic plant cell produced from the transgenic soybean event HLAF-3 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 HLAF-3 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.

12. An agricultural product or processed product produced from the transgenic soybean event HLAF-3 of claim 1, characterized in that: The agricultural products or processed products contain a specific region nucleotide sequence, and the specific region nucleotide 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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