WO2016086884A1 - 抗草甘膦转基因大豆及其制备方法与应用 - Google Patents

抗草甘膦转基因大豆及其制备方法与应用 Download PDF

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WO2016086884A1
WO2016086884A1 PCT/CN2015/096326 CN2015096326W WO2016086884A1 WO 2016086884 A1 WO2016086884 A1 WO 2016086884A1 CN 2015096326 W CN2015096326 W CN 2015096326W WO 2016086884 A1 WO2016086884 A1 WO 2016086884A1
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soybean
transgenic
fragment
transgenic soybean
dna fragment
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French (fr)
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邱丽娟
郭兵福
郭勇
张丽娟
金龙国
洪慧龙
化宿南
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中国农业科学院作物科学研究所
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Publication of WO2016086884A1 publication Critical patent/WO2016086884A1/zh
Priority to ZA2016/08507A priority patent/ZA201608507B/en

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids

Definitions

  • the present invention relates to the field of plant genetic engineering technology or breeding, and in particular, the present invention relates to genetically modified soybeans and their use, and more particularly to the glyphosate resistant genetically modified soybean and a preparation method and application thereof.
  • Soybean (Glycine max (L.). Merr) originated in China and is an important oil crop and cash crop. It is also the main source of edible vegetable oil and plant protein. Weeds are an important part of the agro-ecosystem. Weeds and crops compete for light, water, nutrients and other resources to reduce crop yields. Therefore, effective control of soybean field weeds is one of the key factors for stable yield and high yield of soybean.
  • Traditional manual weeding, including manual weeding and simple agricultural tools, is time-consuming and labor-intensive, and has low efficacy and cannot be controlled in large areas in time.
  • the use of herbicides has greatly improved the control efficiency of farmland weeds.
  • the traditional herbicides such as chlorsulfuron-methyl and metsulfuron-methyl have serious residues in the soil and pollute the environment, and the use range is small.
  • Glyphosate is a systemic, broad-spectrum, herbicide with broad-spectrum, high-efficiency, low-toxicity, low-residue, especially for humans and animals. It is the most widely used and sold worldwide.
  • One of the most abundant herbicides, the mechanism of action is to catalyze the metabolic pathway of shikimic acid in plants and microorganisms by irreversibly binding to EPSPS synthase (5-enolpyruvylshikimate-3-phosphate synthase) in plants.
  • EPSPS synthase 5-enolpyruvylshikimate-3-phosphate synthase
  • the shikimate synthesis pathway leads to disturbances in the metabolic process, interferes with protein synthesis, prevents the formation of secondary products, and ultimately causes the plants to die. Because of its extinction, glyphosate also damages soybeans while killing weeds.
  • breeding glyphosate-tolerant genetically modified crops is one of the important ways to protect crops from glyphosate and improve the efficiency of integrated weed control.
  • the existing research results and the history of commercialization of GM crops prove that the herbicide glyphosate tolerance is an effective target trait for the control and management of soybean weeds.
  • EPSPS genes that increase glyphosate tolerance in plants
  • EPSPS genes are usually derived from microorganisms and have glyphosate resistance, thus in grasses. Their catalytic activity was maintained in the case of glyphosate (PCT/CN03/00651).
  • Most of the current globally commercialized glyphosate-resistant GM crops are designed for EPSPS.
  • N-acetyltransferase In plant tissues, N-acetyltransferase (GAT) is able to effectively degrade glyphosate by N-acetylation, thereby eliminating herbicide activity, and acetylated glyphosate is not EPSPS.
  • the substrate is effectively acted upon to confer tolerance to the plant to glyphosate (ZL 2005 1 00866626.X).
  • the use of N-acetylation to grow transgenic crops allows glyphosate to be applied throughout the plant's growth cycle, independent of the growth and development stages.
  • the method provided by the present invention is to insert an exogenous DNA fragment into the 7th, 980th, 5th, 7th, and 980th, 541th position of chromosome 17 of the target soybean genome, and replace the 13 bp base sequence between the 7th, 980th, 5th, 980th, and 980th positions of the 17th chromosome to obtain the transgenic soybean. ;
  • the transgenic soybean has higher glyphosate resistance than the target soybean
  • the exogenous DNA fragment is a DNA molecule comprising a 5-enolpyruvylshikimate-3-phosphate synthase gene and an N-acetyltransferase gene.
  • the 5-enolpyruvylshikimate-3-phosphate synthase gene is G2-aroA
  • the N-acetyltransferase gene is GAT;
  • the exogenous DNA fragment is SEQ ID NO: 10 or SEQ ID NO: 1 from nucleotides 6189 to 10927 at the 5' end.
  • the exogenous DNA fragment is 0 to 5 in the upstream direction of the transgenic soybean, and the length of the nucleotide of chromosome 17 of the target soybean genome extends from the nucleotides 7,980,527 to the upstream direction thereof.
  • the upstream flanking fragment is specifically a nucleotide represented by SEQ ID NO: 8 in the sequence listing;
  • the exogenous DNA fragment in the downstream flanking fragment of the transgenic soybean is any one of 0 to 5 Kb in length from the nucleotide number 7,980,541 of the chromosome 17 of the target soybean genome.
  • the downstream flanking fragment is specifically a nucleotide represented by SEQ ID NO: 9 in the Sequence Listing.
  • the upstream flanking fragment is a fragment of the transgenic soybean genome immediately adjacent to the 5' end of the exogenous DNA fragment;
  • the downstream flanking fragment is a fragment of the transgenic soybean genome immediately adjacent to the 3' end of the exogenous DNA fragment;
  • the exogenous DNA fragment is introduced into the soybean of interest by a recombinant vector containing the exogenous DNA fragment;
  • nucleotide sequence of the recombinant vector is specifically Sequence 1 in the Sequence Listing.
  • the target soybean is Zhonghuang No. 10.
  • the transgenic soybean is a homozygous strain.
  • the above transgenic plant is soybean ZH10-6 CGMCC No.11108 (also known as T2 transgenic soybean ZH10-6 homozygous strain), which was deposited with the General Microbiology Center of China Microbial Culture Collection Management Committee on December 1, 2015 (referred to as CGMCC, Address: No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, China Institute of Microbiology, Chinese Academy of Sciences, 100101), the deposit number is CGMCCNo.11108, and the classification is named Glycine max.
  • Another object of the present invention is to provide a method for detecting or assisting in detecting whether a plant sample is derived from the transgenic soybean or its progeny prepared by the above method or for detecting or assisting in detecting whether the preparation contains the transgenic soybean prepared by the above method or its progeny.
  • the method provided by the present invention comprises the steps of: detecting whether the DNA fragment A is contained in the genomic DNA of the plant sample or in the preparation,
  • the DNA fragment A is as follows 1) or 2);
  • the plant sample is or can be the transgenic soybean or its progeny; or the preparation contains or can be selected to contain the transgenic soybean or its progeny;
  • the plant sample is not or the candidate is not the transgenic soybean or its progeny; or the preparation is free or candidate free of the transgenic soybean or its progeny.
  • the method is as follows 1) or 2) or 3):
  • the primer pair 1 is capable of amplifying the 5' end of the foreign DNA fragment and the immediately adjacent thereto a primer pair of DNA molecule A consisting of part or all of the upstream flanking sequence; the corresponding amplification product of interest is the DNA molecule A;
  • the primer pair 2 is a primer pair capable of amplifying a DNA molecule B comprising a part or all of the downstream flanking sequence of the exogenous DNA fragment and the downstream flanking sequence thereof; the corresponding amplification product of interest is DNA molecule B;
  • the primer pair 1 consists of a single-stranded DNA molecule represented by SEQ ID NO: 11 in the Sequence Listing and a single-stranded DNA molecule represented by SEQ ID NO: 12 in the Sequence Listing;
  • the primer-specific corresponding fragment size of the primer pair 1 is 810 bp, and the nucleotide sequence thereof is specifically the sequence 15;
  • the primer pair 2 is composed of a single-stranded DNA molecule represented by SEQ ID NO: 13 in the Sequence Listing and a single-stranded DNA molecule represented by SEQ ID NO: 14 in the Sequence Listing;
  • the primer-specific corresponding fragment size of the primer pair 2 is 1627 bp, and the nucleotide sequence thereof is specifically the sequence 16;
  • nucleotide sequence of the probe is sequence 6.
  • the DNA molecule is a 810 bp fragment of interest, and the nucleotide sequence thereof is sequence 15;
  • the DNA molecule B is a 1627 bp fragment of interest, and its nucleotide sequence is sequence 16.
  • the progeny of the transgenic soybean is a transgenic material derived from the transgenic soybean as a parent, including a derived progeny obtained by mutagenesis of the transgenic soybean or hybridized with other soybeans or the mutagenized or hybrid progeny Derived descendants.
  • a third object of the present invention is to provide a kit for detecting or assisting in detecting whether a plant sample is derived from the above-described method, or a progeny thereof, or a kit for detecting or assisting the detection of whether the preparation contains the transgenic soybean or the progeny thereof prepared by the above method. .
  • the kit provided by the invention comprises 1) the exogenous DNA fragment, 2) the primer pair 1, 3) the primer pair 2 or 4) the probe.
  • the above kit may also describe the instructions of the above method.
  • transgenic soybean prepared by the above method in breeding and/or production processing.
  • Figure 1 shows the PCR analysis of the transgenic soybean T0 generation resistant to 1.5L / ha glyphosate spray identification material.
  • Figure 2 is a copy number analysis of exogenous T-DNA of transgenic soybean ZH10-6.
  • Figure 3 shows the glyphosate resistance identification of T1 plants of transgenic soybean ZH10-6.
  • Figure 4 is a PCR amplification diagram of T1 plants of transgenic soybean ZH10-6.
  • Figure 5 shows the resistance of the transgenic soybean ZH10-6 homozygous resistant strain to 12 L/ha glyphosate isopropylamine salt.
  • Figure 6 shows the PCR molecular detection of the transgenic soybean ZH10-6 homozygous strain.
  • Figure 7 is a schematic diagram showing the insertion site and integration of exogenous T-DNA of transgenic soybean ZH10-6.
  • Figure 8 is a schematic diagram showing the position of the GM soybean ZH10-6 verification primer.
  • Figure 9 is a qualitative PCR amplification diagram of the progeny plants of the transgenic soybean ZH10-6.
  • Figure 10 is a qualitative PCR amplification diagram of the derivative plants formed by the transgenic soybean ZH10-6 in breeding.
  • Figure 11 is a qualitative PCR amplification diagram of different tissues of the transgenic plants formed by the transgenic soybean ZH10-6 in breeding.
  • the soybean variety is Zhonghuang 10 (Zhonghuang10, its serial number: ZDD23873, which is recorded in the following literature: ⁇ China Soybean Variety>>, edited by Qiu Lijuan and Wang Yuming, China Agricultural Press, 2007, the public can be cropped from the Chinese Academy of Agricultural Sciences.
  • the scientific research institute obtained the cotyledonary node of Zhonghuang No. 10 as a conversion material;
  • Agrobacterium tumefaciens strain is Ag10, which is reported in the following literature: Cheng Wei. Preliminary study on ectopic expression of AtMGT4 gene in rice (D). Hunan Normal University, 2012; public available from the Chinese Academy of Agricultural Sciences, Crop Science Institute obtained.
  • the soybean tissue culture medium was mainly MS and B5 medium, and sterilized at 121 ° C for 15-20 min.
  • Germination medium B5 + 20g / L sucrose + 8g / L agar powder, pH 5.8;
  • Co-culture medium 1/10 B5+30g/L sucrose +3.9g/L 2-(N-morpholine)ethanesulfonic acid (MES)+1.67mg/L 6-BA+39mg/L acetosyringone+0.25mg /L gibberellic acid (GA3) +1mmol / L dithiothreitol +1mmol / L sodium thiosulfate + 1mmol / L cysteine + 5g / L agar Powder, pH 5.4;
  • MES 2-(N-morpholine)ethanesulfonic acid
  • GA3 gibberellic acid
  • Co-culture solution 1/10 B5+30g/L sucrose +3.9g/L 2-(N-morpholine)ethanesulfonic acid (MES)+1.67mg/L 6-BA+39mg/L acetosyringone+0.25mg /L gibberellin (GA3);
  • MES 2-(N-morpholine)ethanesulfonic acid
  • GA3 gibberellin
  • Cluster bud induction medium B5+30g/L sucrose +8g/L agar powder+0.6 g/L 2-(N-morpholine)ethanesulfonic acid (MES)+1.67mg/L 6-BA+150mg/L thiophene Sporemycin + 400mg / L carbenicillin + 15mg / L glyphosate, pH 5.7;
  • MES 2-(N-morpholine)ethanesulfonic acid
  • Bud elongation medium MS+B5 organic+30g/L sucrose+8g/L agar powder+0.6g/L 2-(N-morpholine)ethanesulfonic acid (MES)+50mg/L aspartic acid+50mg /L glutamine +0.3mg / L ⁇ -3-acetic acid (IAA) +0.5mg / L gibberellin (GA3) + 150mg / L thiaphipin + 400mg / L carbenicillin + 0.1mg / L Zein (Ze) + 5 mg / L glyphosate, pH 5.7;
  • Rooting medium MS+B5 organic +30g/L sucrose+8g/L agar powder+0.6g/L 2-(N-morpholine)ethanesulfonic acid (MES) 50mg/L aspartic acid+50mg/L valley Aminoamide, pH 5.7;
  • MES 2-(N-morpholine)ethanesulfonic acid
  • Agrobacterium cultures were cultured with YEP and LB media.
  • Acetyl syringone, MS and B5 dry powder medium and acetosyringone are products of sigma, 2-(N-morpholine)ethanesulfonic acid (MES), streptomycin, carbenicillin, agar powder, zeatin, day Aspartic acid, glutamine, gibberellin (GA3) and 6-benzylaminoadenine (6-BA) are products of Biodee, and sucrose is a domestic reagent.
  • pBI121 Wang Huaxin, Cao Jiashu, Xiang Yu et al. Construction of pBI121 expression vector and rapid identification of transformed plants. Journal of Zhejiang University (Agriculture and Life Sciences), 2008, 34(2): 137-142; Obtained from the Crop Science Institute of the Chinese Academy of Agricultural Sciences) and pCAMBIA2300 (Gong Yuanyong, Feng Yongkun, Ni Wanchao et al. Construction and validation of plant expression vector pCAMBIA2300-35S-GUS-CaMVterm.
  • the p35S-2300-rbcS-G2-EPSPS (aroA) was excised from the plant selection marker kan with XhoI monozyme, and a 10.4 kb intermediate vector vector backbone was collected.
  • the gene has a single restriction site XhoI upstream and downstream, and the optimized GAT gene and the intermediate vector vector skeleton obtained in the above (3) are added to the recombinant vector pKT-rGE; Sequence 1.
  • the promoters Rbcs and the glyphosate resistance gene Rbcs-EPSPS (G2-aroA) from the 5' end are the promoter-enhanced 35S and the 5' end of the 8204.
  • nucleotides are the peptide Rbcs and the glyphosate resistance gene Rbcs-EPSPS (G2-aroA), and the 5' end nucleotides 7926-8196 are the glyphosate resistance gene EPSPS (G2-aroA)
  • the NOS at the 5' end of the terminator, the nucleotides 6903-7673 at the 5' end are the glyphosate-degrading gene N-acetyltransferase gene-enhanced 35S, and the nucleotides 6456-6896 at the 5' end are the glyphosate-degrading gene N-
  • the acetyltransferase gene GAT and the nucleotides 6248-6455 at the 5' end are the glyphosate-degrading gene N-acetyltransferase gene terminator CaMV 35S polyA.
  • the recombinant vector pKT-rGE obtained in the above 1 was introduced into Agrobacterium tumefaciens Ag10 to obtain recombinant Agrobacterium Ag10/pKT-rGE.
  • Recombinant Agrobacterium Ag10/pKT-rGE was transformed into cotyledonary explants of Huang 10 in in vitro cultured soybean (Glycine max), and Agrobacterium infected explants for 3 days in a screening environment containing glyphosate.
  • Transgenic plants are induced by the organ regeneration pathway, and the bud induction stage is used.
  • Glyphosate (Sigma) at a concentration of 15 mg/L was used as a screening agent for 3-6 weeks; in the elongation bud induction phase, glyphosate (sigma) at a concentration of 5 mg/L was used as a screening agent to induce 4-8 weeks.
  • the medium was changed every 2 weeks to regenerate the transformed cells.
  • a concentration of 150 mg/L of thiamycin and 400 mg/in the induction and elongation medium were respectively added.
  • Carbendazimycin at a concentration of L when the elongation buds are elongated to 4-6 cm, is transferred to a rooting medium to induce rooting, and a regenerated soybean plant is obtained.
  • the regenerated plants are transplanted into the soil and cultured in a greenhouse or incubator.
  • the light conditions are 16h light and 8h darkness.
  • the glyphosate isopropylamine salt (Roundup) is sprayed separately.
  • an aqueous herbicide solution was applied at a dose of 1.5 L of glyphosate isopropylamine per hectare using a graduated sprayer.
  • the response of each plant to glyphosate treatment was investigated, and 5 of the transformants showed strong tolerance to 1.5 L/ha of glyphosate isopropylamine salt (Roundup) spray treatment.
  • Sex (given the number ZH10-1, 2, 3, 5, 6), of which the number ZH10-6 is the most tolerant, and the growth of the control plant (Zhonghuang No. 10) is strong, the growth vigor is not affected, and the leaves are not found.
  • Symptoms of yellow chlorosis, regenerative plants numbered ZH10-1, 2, 3, 5, 6 are T0 generation transgenic soybeans, and glyphosate resistance of T0 generation transgenic soybeans is higher than that of wild type soybean Zhonghuang No. 10.
  • PCR Polymerase chain reaction
  • the 20 ⁇ l PCR reaction system contained 2 ⁇ l of 10 ⁇ EX-Taq buffer, 2 ⁇ l of 2 mM dNTPs, and 0.5 ⁇ l of 10 ⁇ M gene-specific upstream and downstream primers, EX-Taq. 0.2 ⁇ l of enzyme, hydrated to 20 ⁇ l; PCR reaction procedure was 94 ° C, 4 min (1 cycle); 94 ° C, 30 s (denaturation), 60 ° C, 30 s (annealing), 72 ° C, 45 s (extension) 35 cycles; 72 ° C ( Final extension) 10 min (1 cycle).
  • Amplification of the primer pair of EPSPS can positively amplify the 743 bp product:
  • the primer pair that amplifies the GAT gene can positively amplify the 338 bp product:
  • M 100 bp DNA Marker
  • 1 wild type Zhonghuang No. 10 negative control
  • 2 sterile water control
  • 3 plasmid pKT-rGE positive control
  • 4, 5, 6, and 7. 8 were transgenic soybean ZH10-1, 2, 3, 5, 6 respectively; the results showed that the transgenic soybeans numbered ZH10-1, 2, 3, 5, 6 were PCR positive, numbered ZH10-1, 2, 3
  • the 5,6 GM soybeans were transferred to the foreign genes EPSPS and GAT.
  • the transgenic soybeans having glyphosate resistance are propagated by conventional cultivation and breeding methods, and the transgenic soybean seeds are harvested.
  • T0 generation transgenic soybean ZH10-6 seeds were collected and seeded to obtain T1 generation transgenic soybean ZH10-6.
  • Each plant was coated with 1 ⁇ l of glyphosate isopropylamine salt (Roundup) in the seedling stage (the period when the cotyledon unearthed leaves were not fully developed). 0.3 mg ae ⁇ l -1 glyphosate), and the toxic reaction was investigated after 2 weeks.
  • the phenotype of non-resistant plants is leaf chlorosis, leaf curling, shrinkage, apical meristem necrosis, until the whole plant dies; resistant plants phenotype is strong growth trend, leaves are not chlorotic, no curl, no shrinkage .
  • T1 generation transgenic soybean ZH10-6 identification are shown in Figure 3, 1: T1 single plant that is not resistant; 2: untreated T1 individual plant: 3: resistant T1 single plant, indicating T1 single of transgenic soybean ZH10-6
  • the resistance of 1 ⁇ l of glyphosate isopropylamine salt (Roundup) stock solution/strain was isolated, 3 strains were treated, the survival was resistant, the death was not resistant, and 2 resistant plants were found.
  • 1 plant was not resistant; compared with the untreated T1 transgenic soybean ZH10-6, the plant height and growth trend of the resistant T1 plants were not inhibited, and the leaves did not chlorotic, so the resistant T1 generation was identified by smearing.
  • glyphosate resistant T1 generation transgenic soybean ZH10-6 for glyphosate resistant T1 generation transgenic soybean ZH10-6.
  • the two homozygous lines of the above T2 generation transgenic soybean ZH10-6 have higher resistance to glyphosate than Zhonghuang No. 10; it has better breeding prospects and utilization value.
  • Glyphosate-tolerant transgenic soybean ZH10-6 obtained a homozygous strain, and its resistance was stably inherited in the offspring.
  • the probe for Southern blotting was to select a 338 bp vector DNA sequence design probe from the T-DNA region of the vector (sequence 6), and refer to the instruction manual of the PCR method DIG labeling kit produced by Beijing Meilaibo Medical Technology Co., Ltd. High-six-labeled probe.
  • the recombinant plasmid pKT-rGE was used as a positive control, and the genomic DNA of Zhonghuang 10 was a negative control.
  • the genomic DNA of the glyphosate-tolerant T1 transgenic soybean ZH10-6 obtained in the above Example 2 was extracted, and 50-70 ⁇ g of genomic DNA was digested in a 200 ⁇ l digestion system at 37 ° C with 5 units of restriction enzymes.
  • the digested DNA was precipitated and redissolved in 25 ⁇ l of sterile water, 6 ⁇ l of 6 ⁇ Loading Buffer per sample, digested DNA, positive control, negative control, standard molecular size marker (Digoxigenin) -labeled (Roche) and ⁇ Hind III marker), DNA was separated on a 0.8-1.0% agarose gel electrophoresis at 45V. The DNA was observed with ethidium bromide and included on a fluorescent scale for imaging recording.
  • the DNA was then transferred to a Hybond nylon membrane by referring to the instructions of the What Down Schleicher & Schuell Corporation's Rapid Downward transfer system, which hybridized with the probe and displayed by hybridization (Beijing Meilaibo Medical Technology Co., Ltd.).
  • Copy number determination can be performed by analyzing genomic DNA adjacent to the left and right border regions, and digesting genomic DNA with restriction enzymes DraI, HindIII, and XbaI.
  • the genomic DNA of T1 transgenic soybean ZH10-6 in Example 2 was extracted.
  • a 20 ul PCR reaction system about 50 ng of genomic DNA was used as template DNA, and the EPSPS (G2-aroA) primer was amplified using Example 1.
  • PCR amplification was performed on the amplified and amplified GAT gene primer pairs, respectively.
  • the genomic DNA of the T2 generation transgenic soybean ZH10-6 homozygous strain obtained in Example 2 was extracted, and about 50 ng of genomic DNA was used as template DNA in the 20 ul PCR reaction system, and the EPSPS was amplified using Example 1.
  • the (G2-aroA) primer pair and the amplified GAT gene primer pair were subjected to PCR amplification, respectively.
  • T2 transgenic soybean ZH10-6 homozygous strains were different plants; 20: plasmid pKT-rGE was a positive control; 21, 22: sterile water control; 23, 24: wild Type Zhonghuang No. 10 negative control; M: 100bp DNA marker, indicating that all the plants of T2 transgenic soybean ZH10-6 homozygous strain were PCR positive, molecular identification was homozygous insertion, and resistant to glyphosate Sexual correspondence.
  • the above-mentioned T2 generation transgenic soybean ZH10-6 homozygous was sequenced and analyzed, and by comparison analysis, it was found that in the T2 generation transgenic soybean ZH10-6 homozygous strain, T -
  • the DNA was inserted into the soybean 17 chromosome physical position at 7,980, 527-7, 980, 541, and the soybean soybean endogenous gene coding region was not inserted.
  • the T-DNA integration replaced the 13 bp base sequence on the genome, and the replaced sequence was 5' CAAATGCAAAAAT 3' (SEQ ID NO: 7), which did not disrupt the soy endogenous gene coding region.
  • the binding region of the genomic DNA of the foreign T-DNA and the insertion site was PCR-amplified to verify the insertion position of the foreign T-DNA. The result further confirmed the correctness of the T-DNA insertion site, and the T2 generation transgenic soybean ZH10-6 was pure.
  • the results of T-DNA insertion of the mutant line are shown in Figure 7.
  • the upstream flanking sequence of the 5' end of the exogenous T-DNA inserted in the T2 transgenic soybean ZH10-6 homozygous strain is sequence 8, exogenous T-DNA.
  • the 3' end downstream flanking sequence is sequence 9.
  • the complete insertion of the T2 transgenic soybean ZH10-6 was obtained.
  • the exogenous T-DNA sequence is sequence 10
  • the exogenously inserted exogenous DNA molecule is 4739 bp.
  • the transgenic soybean exogenous DNA molecule integration does not contain the vector backbone sequence.
  • the T2 transgenic soybean ZH10-6 homozygous strain was a glyphosate-resistant plant, which was analyzed by flanking sequences and insertion sites, which was sequence 10 or sequence 1 in the sequence listing from the 5' end of the sequence 6189- 10927 nucleotides were inserted between the 7th, 980th, 527th, and 980th, 541th chromosomes of the No. 17 soybean genome, and the 13th bp base sequence of the 7th, 980th, 528th, 980th, and 540th chromosomes of the 17th chromosome was replaced, and the transgenic soybean was obtained.
  • sequence 8 The nucleotide sequence of the upstream flanking fragment of nucleotides 7,980,527 upstream and adjacent to nucleotides 7,980,527 is sequence 8, and the nucleotide sequence of the downstream flanking fragment downstream of the 7,980,541 nucleotide and immediately adjacent to nucleotides 7,980,541 is sequence 9; Sequence 10 is a DNA molecule comprising a 5-enolpyruvylshikimate-3-phosphate synthase gene and an N-acetyltransferase gene.
  • the insertion position of the foreign DNA molecule and the flanking sequences on both sides can be used to identify whether or not the target transgenic soybean (T2 generation transgenic soybean ZH10-6 homozygous strain).
  • T2 generation genetically modified soybean ZH10-6 homozygous strain (named ZH10-6) was deposited on December 1, 2015 at the General Microbiology Center of China Microbial Culture Collection Management Committee (CGMCC, Address: Beichenxi, Chaoyang District, Beijing) No. 3, No. 1 Hospital, Institute of Microbiology, Chinese Academy of Sciences, Zip Code 100101), the accession number is CGMCC No.11108, and the classification is named Glycine max.
  • transgenic soybean to be tested is a T2 transgenic soybean ZH10-6 homozygous strain
  • a specific primer is developed according to the position of the exogenous DNA molecule inserted in Example 4 and its upstream and downstream flanking gene sequences.
  • a qualitative PCR identification method for glyphosate-tolerant transgenic soybean ZH10-6 and its self-crossing or hybrid progeny was established.
  • Primers ZH10P-1 and GAT-1 were designed based on the upstream flanking sequence of the 5' end soybean genomic DNA of the inserted T-DNA (sequence 8) and the inserted TAT DNA (sequence 10). 1); Primers G2EP-2 and ZH10P-2 were designed based on the EPSPS gene fragment inserted in the exogenous T-DNA (SEQ ID NO: 10) and the downstream flanking sequence of the 3' end soybean genomic DNA of the inserted exogenous T-DNA (SEQ ID NO: 9). (primer pair 2) (Fig. 8).
  • Primer ZH10P-1 5'TAATAGTAGAATGGGACTGGTGGAT 3' (sequence 11)
  • Primer GAT-1 5'GCGGACTTGCTTTGGTGTAAT 3' (SEQ ID NO: 12).
  • Primer G2EP-2 5'CCCGAATCATCAGGCAAACA 3' (sequence 13)
  • Primer ZH10P-2 5'AACACATCATAGTATTCTAAAACGCTT 3' (sequence 14).
  • the genomic DNA of the sample to be tested is extracted as a template, and PCR amplification is performed using primer pair 1 and primer pair 2 to detect PCR amplification products:
  • the transgenic soybean to be detected is the T2 generation transgenic soybean ZH10-6 homozygous strain obtained in Example 4;
  • primer pair 1 is not amplified, a 810 bp target fragment is obtained and/or primer pair 2 is not amplified.
  • the transgenic soybean to be tested was not the T2 generation transgenic soybean ZH10-6 homozygous strain obtained in Example 4.
  • transgenic soybean ZH10-6 The experiment indicated that the PCR amplification of the flanking sequence of transgenic soybean ZH10-6 can specifically detect the molecular characteristics of soybean transgenic soybean ZH10-6, and can be used to identify the above-mentioned transgenic soybean and its progeny, cells, seeds and vegetative organs.
  • Heihe 43 (Heihe 43, its serial number: ZDD24325), is recorded in the following literature: "New Early-maturing and High-yield Soybean Variety Heihe 43", Jia Hongchang et al., Heilongjiang Agricultural Sciences, 2007, 05: 124-125);
  • Heihe 38 (Heihe38, its serial number: ZDD24320), is recorded in the following literature: "Selection and Genetic Composition of Soybean Variety Heihe No. 38", Wu Ji'an, Journal of Plant Genetic Resources, 2007, 3: 313-316);
  • Keshan No. 1 (Keshan 1, Guo Judou 2009002), recorded in the following literature: "Using Space Mutagenesis Technology to Breed New Soybean Variety Keshan No.1", Zhang Yong et al., Journal of Black Agricultural, 2013, 27 (9) ): 1241-1246);
  • Fen ⁇ 22 (Kenfen 22, its serial number is ZDD24348), is recorded in the following literature: “Selection and Cultivation Techniques of New Soybean Variety Jifeng 22", Yang Danxia et al., Soybean Science and Technology, 2010, 01: 64-65) .
  • the soybean varieties Hehe 43 , Heihe 38, Keshan No. 1, Qifeng 16, Yufeng 20 and Qifeng 22, which are mainly cultivated in China, are the female parent, and the T2 generation transgenic soybean ZH10-6 homozygous strain (anti-grass Phosphine, hereinafter referred to as ZH10-6), is a male parent.
  • the seeds of F1 are prepared by traditional hybrid breeding techniques, and the seeds of F1 are harvested. In the first to third three-leaf stage, 200 ml/mu of grass is sprayed.
  • Glyphosate harvesting 200ml/mu glyphosate sprayed seeds of hybrid F1 plant, planting F2; spraying 800ml/mu of glyphosate isopropylamine salt solution on different hybrid combinations of F2 materials (Roundup, Monsanto, Co) .
  • the results of the identification of glyphosate tolerance in the F2 population are shown in Table 1, indicating that the resistance is genetically stable in the hybrid progeny and is consistent with a 3:1 separation ratio, further indicating that the exogenous gene has only one insertion in the ZH10-6 genome.
  • the site is consistent with the results of Southern detection as a single copy.
  • the electrophoresis results of the PCR product of the resistant F2 plant are shown in Figure 10, A: amplification result of primer pair 1; B: amplification result of primer pair 2; 1:200 bp DNA Marker; 2: sterile water control; 4: non-transgenic soybean Heihe 43 and Heihe 38; 5-25: randomly selected resistant hybrid F2 plants; results showed that water and non-transgenic plants had no amplified bands, and resistant F2 generation genomic DNA amplification
  • the target bands of 810 bp (Fig. 10A) and 1626 bp (Fig. 10B) were obtained, and the nucleotide sequences of the 810 bp fragment and the 1626 bp fragment were sequence 15 and sequence 16, respectively.
  • the plant single plant that was positive by PCR was the hybrid progeny of the T2 transgenic soybean ZH10-6 homozygous strain.
  • FIG. 11A A: amplification results of primer pair 1; B: amplification results of primer pair 2; 1:200 bp DNA Marker; 2: sterile water control; 3-7: roots, stems, leaves, flowers and seeds of resistant F2; 8-12: non-transgenic Because of the roots, stems, leaves, flowers and seeds of the plant Heihe 43; it can be seen that there is no amplified band in water and non-transgenic plants, and the DNA of the resistant F2 roots, stems, leaves, flowers and seeds is amplified by 810 bp. (Fig. 11A) and the target band of 1626 bp (Fig. 11B), and the nucleotide sequences of the 810 bp fragment and the 1626 bp fragment are sequence 15 and sequence 16, respectively.
  • the plant is derived from the T2 transgenic soybean ZH10-6 homozygous strain and the derived hybrid progeny can be detected from any plant tissue such as roots, stems, leaves, flowers, seeds, and the like.
  • the experiment of the present invention proves that the present invention adopts the insertion of an exogenous DNA fragment between positions 7980527 and 7980541 of soybean chromosome 17, and obtains a transgenic soybean containing an exogenous DNA fragment; the exogenous DNA fragment includes the glyphosate resistance gene G2-aroA. And glyphosate degrading gene N-acetyltransferase gene GAT gene; transgenic soybean has higher resistance to glyphosate than wild-type soybean without transgenic.
  • the transgenic soybean is tolerant to high doses of glyphosate, which can be further modified by mutagenesis or by crossing with elite soybean lines to optimize other agronomic traits such as yield, quality, and the like.
  • the exogenous DNA fragment soybean co-expressed the glyphosate-resistant EPSPS (G2-aroA) and the glyphosate-degrading gene N-acetyltransferase gene (GAT) at the seedling stage (the cotyledon was unearthed and the true leaves were not unfolded).
  • the strain is tolerant to 1-1.5 ul of glyphosate isopropylamine salt solution, and the field is tolerant to about 3 to 12 liters of glyphosate isopropylamine salt per hectare.
  • the total standard measure of good weed control varies between 3-6 liters per hectare depending on weed pressure.

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Abstract

提供了抗草甘膦转基因大豆及其制备方法与应用。该方法将外源DNA片段插入目的大豆基因组第17号染色体的第7,980,527-7,980,541位间，得到转基因大豆。该转基因大豆对高剂量草甘膦有耐受性。进一步提供了检测方法，可以鉴定插入的T-DNA和植物基因组DNA的结合区域并鉴定植物组织或制品中是否含有所述转基因大豆成分。

Description

抗草甘膦转基因大豆及其制备方法与应用 技术领域
本发明涉及植物基因工程技术或育种领域，具体地，本发明涉及转基因大豆及其应用，更具体地，本发明涉及抗草甘膦转基因大豆及其制备方法与应用。
背景技术
大豆(Glycine max(L.).Merr)起源于中国，是重要的油料作物和经济作物，也是食用植物油与植物蛋白质的主要来源。杂草是农业生态系统中的重要组成部分，杂草与作物竞争光、水、养分等资源，使作物减产。因此，大豆田间杂草的有效防除是大豆稳产高产的关键因素之一。传统的人工除草包括手工拔草和使用简单农具除草等，耗时耗力，功效低且不能大面积及时防除。除草剂的使用大大提高了农田杂草的防治效率，传统除草剂氯嘧磺隆、甲磺隆等在土壤中残留严重、污染环境，使用范围较小。
草甘膦(Glyphosate)为内吸传导型、广谱灭生型除草剂，具有杀草谱广、高效、低毒、低残留等优点，尤其对人畜毒害小，是全球应用范围最广、销售量最大的除草剂之一，其作用机制是通过不可逆地与植物体内EPSPS合酶(5-烯醇丙酮酸莽草酸-3-磷酸合成酶)结合催化植物和微生物体内莽草酸的代谢途径，扰乱莽草酸合成途径导致代谢过程紊乱，干扰蛋白质合成，阻止次生产物的形成，最终使植株死亡。草甘膦因其灭生性，在杀灭杂草的同时也损伤大豆，因此培育抗草甘膦转基因作物是保护作物不受草甘膦伤害，提高杂草综合防治效率的重要途径之一。已有研究结果和转基因作物商业化发展历史证明除草剂草甘膦耐受性是应用于大豆杂草防除、管理的有效目标性状。
为赋予农作物草甘膦的耐受性，研究者集中于向植物体内导入能增加草甘膦耐受性的基因，如EPSPS，EPSPS基因通常来自于微生物，具有草甘膦抗性，因而在草甘膦作用的情况下保持了他们的催化活性(PCT/CN03/00651)。当前全球商业化种植的草甘膦抗性转基因作物绝大多数为针对EPSPS所设计。
在植物组织中N-乙酰转移酶(GAT)能够通过N-乙酰化作用使得草甘膦被有效的降解，从而失去除草剂活性，且乙酰化的草甘膦不是EPSPS的 有效作用底物，从而赋予植物对草甘膦的耐受性(ZL 2005 1 0086626.X)。利用N-乙酰化的手段培育转基因作物可以使得草甘膦在植物整个生长周期都可以应用，不受生长发育阶段的限制。
发明公开
本发明的一个目的是提供一种培育转基因大豆的方法。
本发明提供的方法，为将外源DNA片段插入目的大豆基因组第17号染色体的第7,980,527-7,980,541位间，替换掉第17号染色体的第7,980,527-7,980,541位间13bp的碱基序列，得到转基因大豆；
所述转基因大豆的草甘膦抗性高于所述目的大豆；
所述外源DNA片段为含有5-烯醇丙酮酸莽草酸-3-磷酸合成酶基因和N-乙酰转移酶基因的DNA分子。
上述方法中，所述5-烯醇丙酮酸莽草酸-3-磷酸合成酶基因为G2-aroA；
所述N-乙酰转移酶基因为GAT；
所述外源DNA片段为序列表中序列10或序列1自5’末端第6189-10927位核苷酸。上述方法中，所述外源DNA片段在所述转基因大豆的上游侧翼片段为所述目的大豆基因组第17号染色体的自第7,980,527位核苷酸起向其上游方向延伸得到的长度为0至5 Kb的任意一个DNA片段；
所述上游侧翼片段具体为序列表中序列8所示的核苷酸；
所述外源DNA片段在所述转基因大豆的下游侧翼片段为所述目的大豆基因组第17号染色体的自第7,980,541位核苷酸起向其下游方向延伸得到的长度为0至5 Kb的任意一个DNA片段；
所述下游侧翼片段具体为序列表中序列9所示的核苷酸。
所述上游侧翼片段为所述转基因大豆基因组中紧邻所述外源DNA片段5’末端的片段；
所述下游侧翼片段为所述转基因大豆基因组中紧邻所述外源DNA片段3’末端的片段；
上述方法中，所述外源DNA片段通过含有所述外源DNA片段的重组载体导入所述目的大豆；
所述重组载体的核苷酸序列具体为序列表中序列1。
上述方法中，所述目的大豆为中黄10号。
所述转基因大豆为纯合株系。
由上述方法制备的转基因植物。
上述转基因植物为大豆ZH10-6 CGMCC No.11108(又名T2代转基因大豆ZH10-6纯合株系)，已于2015年12月1日保藏于中国微生物菌种保藏管理委员会普通微生物中心(简称CGMCC，地址：北京市朝阳区北辰西路1号院3号，中国科学院微生物研究所，邮编100101)，保藏号为CGMCCNo.11108，分类命名为大豆Glycine max。
本发明另一个目的是提供用于检测或辅助检测植物样品是否来源于上述方法制备的转基因大豆或其后代或用于检测或辅助检测制品是否含有上述方法制备的转基因大豆或其后代的方法。
本发明提供的方法，包括如下步骤：检测所述植物样品的基因组DNA中或所述制品中是否含有DNA片段A，
所述DNA片段A为如下1)或2)；
1)由权利要求3中的所述外源DNA片段在所述转基因大豆的上游侧翼片段、权利要求3中的所述外源DNA片段和权利要求3中的所述外源DNA片段在所述转基因大豆的下游侧翼片段组成；
2)与1)所示的DNA片段A同源性大于95％的DNA片段；
若含有所述DNA片段A，则所述植物样品为或候选为所述转基因大豆或其后代；或所述制品含有或候选含有所述转基因大豆或其后代；
若不含有所述DNA片段A，则所述植物样品不为或候选不为所述转基因大豆或其后代；或所述制品不含或候选不含所述转基因大豆或其后代。
上述方法中，
所述方法为如下1)或2)或3)：
1)直接测序植物样品的基因组DNA，判断所述测序植物样品是否含有所述DNA片段A；
2)用引物对1或引物对2进行PCR扩增，若有目的扩增产物，则所述植物样品或制品中含有所述DNA片段A；
所述引物对1为能够扩增由所述外源DNA片段5’端和紧邻其的所述 上游侧翼序列部分或全部片段组成的DNA分子甲的引物对；其对应的目的扩增产物为所述DNA分子甲；
所述引物对2为能够扩增含有所述外源DNA片段3’端和紧邻其的所述下游侧翼序列的部分或全部组成的DNA分子乙的引物对；其对应的目的扩增产物为所述DNA分子乙；
3)用能特异结合所述DNA分子甲或其DNA分子乙的探针对所述待测植物样品或制品的DNA进行Southern杂交，若能杂交得到杂交片段，则所述植物样品来源于或制品中含有所述转基因大豆或其后代。
上述方法中，所述引物对1由序列表中序列11所示的单链DNA分子和序列表中序列12所示的单链DNA分子组成；
所述引物对1对应的目的特异片段大小为810bp，其核苷酸序列具体为序列15；
所述引物对2由序列表中序列13所示的单链DNA分子和序列表中序列14所示的单链DNA分子组成；
所述引物对2对应的目的特异片段大小为1627bp，其核苷酸序列具体为序列16；
3)中，所述探针的核苷酸序列为序列6。
所述DNA分子甲为810bp目的片段，其核苷酸序列为序列15；
所述DNA分子乙为1627bp目的片段，其核苷酸序列为序列16。
上述方法中，所述转基因大豆的后代为以所述转基因大豆为亲本衍生的转基因材料，包括用所述转基因大豆诱变或与其他大豆杂交得到的衍生后代或所述诱变或杂交后代再进行衍生得到的后代。
本发明第三个目的是提供用于检测或辅助检测植物样品是否来源于上述方法制备的转基因大豆或其后代或用于检测或辅助检测制品是否含有上述方法制备的转基因大豆或其后代的试剂盒。
本发明提供的试剂盒，包括1)所述外源DNA片段，2)所述引物对1，3)所述引物对2或4)所述探针。
上述试剂盒还可以记载上述方法的说明书。
上述方法制备的转基因大豆在育种和/或生产加工中的应用。
附图说明
图1为转基因大豆T0代耐1.5L/公顷草甘膦喷施鉴定材料PCR分析。
图2为转基因大豆ZH10-6外源T-DNA的拷贝数分析。
图3为转基因大豆ZH10-6的T1植株的草甘膦抗性鉴定。
图4为转基因大豆ZH10-6的T1植株的PCR扩增图。
图5为转基因大豆ZH10-6纯合抗性株系在12L/公顷草甘膦异丙胺盐处理下抗性。
图6为转基因大豆ZH10-6纯合株系的PCR分子检测。
图7为转基因大豆ZH10-6外源T-DNA插入位点及整合方式示意图。
图8为转基因大豆ZH10-6验证引物所在位置示意图。
图9为所述转基因大豆ZH10-6后代植株定性PCR扩增图。
图10为所述转基因大豆ZH10-6在育种中所形成衍生植株的定性PCR扩增图。
图11为所述转基因大豆ZH10-6在育种中所形成衍生植株不同组织的定性PCR扩增图。
实施发明的最佳方式
下述实施例中所使用的实验方法如无特殊说明，均为常规方法。
下述实施例中所用的材料、试剂等，如无特殊说明，均可从商业途径得到。
大豆品种为中黄10号(Zhonghuang10，其统编号：ZDD23873，记载在如下文献中：<<中国大豆品种志>>，邱丽娟、王曙明主编，中国农业出版社，2007，公众可从中国农业科学院作物科学研究所获得，取中黄10号的子叶节作为转化材料；
根癌农杆菌(Agrobacterium tumefaciens)菌株为Ag10，记载在如下文献中：程伟.AtMGT4基因在水稻中异位表达的初步研究(D).湖南师范大学，2012；公众可从中国农业科学院作物科学研究所获得。
大豆组织培养基以MS和B5培养基为主,121℃灭菌15-20min。
萌发培养基：B5+20g/L蔗糖+8g/L琼脂粉，pH5.8；
共培养基：1/10 B5+30g/L蔗糖+3.9g/L 2-(N-吗啡啉)乙磺酸(MES)+1.67mg/L 6-BA+39mg/L乙酰丁香酮+0.25mg/L赤霉素(GA3)+1mmol/L二硫苏糖醇+1mmol/L硫代硫酸钠+1mmol/L半胱氨酸+5g/L琼脂 粉，pH5.4；
共培养液：1/10 B5+30g/L蔗糖+3.9g/L 2-(N-吗啡啉)乙磺酸(MES)+1.67mg/L 6-BA+39mg/L乙酰丁香酮+0.25mg/L赤霉素(GA3)；
丛生芽诱导培养基：B5+30g/L蔗糖+8g/L琼脂粉+0.6 g/L 2-(N-吗啡啉)乙磺酸(MES)+1.67mg/L 6-BA+150mg/L噻孢霉素+400mg/L羧苄青霉素+15mg/L glyphosate，pH5.7；
芽伸长培养基：MS+B5有机+30g/L蔗糖+8g/L琼脂粉+0.6 g/L 2-(N-吗啡啉)乙磺酸(MES)+50mg/L天冬氨酸+50mg/L谷氨酰胺+0.3mg/L吲哚-3-乙酸(IAA)+0.5mg/L赤霉素(GA3)+150mg/L噻孢霉素+400mg/L羧苄青霉素+0.1mg/L玉米素(Ze)+5mg/L glyphosate，pH5.7；
生根培养基：MS+B5有机+30g/L蔗糖+8g/L琼脂粉+0.6 g/L 2-(N-吗啡啉)乙磺酸(MES)50mg/L天冬氨酸+50mg/L谷氨酰胺,pH5.7；
农杆菌培养用YEP和LB培养基。
乙酰丁香酮、MS和B5干粉培养基和乙酰丁香酮为sigma公司产品，2-(N-吗啡啉)乙磺酸(MES)、噻孢霉素、羧苄青霉素、琼脂粉、玉米素、天冬氨酸、谷氨酰胺、赤霉素素(GA3)和6-苄氨基腺嘌呤(6-BA)为Biodee公司产品，蔗糖为国产试剂。
以下实施例进一步描述了得到本发明和随后的结果时所用的材料和方法，它们通过举例说明提供，并且它们的叙述不应认为是要求权利和本发明的限制。
实施例1、转基因大豆的遗传转化
1、重组载体的获得
(1)根据A.thaliana-rbcS加G2-EPSPS(G2-aroA)基因优化后合成序列，重新设计PCR引物，上游带有酶切位点是XbaI，下游带有酶切位点SacI，PCR扩增rbcS-G2-EPSPS(aroA)片段(序列1自5’末端第8204-9784位核苷酸)，连接到T-A载体上，获得质粒prbcS-G2-EPSPS(aroA)，酶切及测序验证。
(2)pBI121(王华新，曹家树，向珣等.pBI121表达载体构建及其转化植株的快速鉴定.浙江大学学报(农业与生命科学版)，2008，34(2)：137-142；公众可从中国农业科学院作物科学研究所获得)和pCAMBIA2300 (巩元勇,冯永坤,倪万潮等.植物表达载体pCAMBIA2300-35S-GUS-CaMVterm的构建及验证.中国生物工程杂志，2013，33(3):86-91；公众可从中国农业科学院作物科学研究所获得)用HindⅢ/EcoRⅠ双酶切，将pBI121带有p35S-GUS-Nos的3.0kb片段连入pCAMBIA2300，形成中间载体p35S-2300-GUS；
(3)p35S-2300-GUS载体及prbcS-G2-EPSPS(aroA)用XbaI/SacⅠ酶切后，9.8kb的p35S-2300-GUS载体骨架与1.5kb rbcS-G2-EPSPS(aroA)片段连接，得到中间载体p35S-2300-rbcS-G2-EPSPS(aroA)，该载体为将prbcS-G2-EPSPS(aroA)中的rbcS-G2-EPSPS(aroA)片段替换(2)得到的p35S-2301-GUS相应酶切位点中的GUS片段形成的。将p35S-2300-rbcS-G2-EPSPS(aroA)用XhoI单酶切除掉植物筛选标记kan，收集10.4kb中间载体载体骨架。(4)根据优化后GAT基因合成序列，基因上下游带有单酶切位点XhoI，连接优化后GAT基因和上述(3)得到的中间载体载体骨架到重组载体pKT-rGE；其全序列为序列1。
序列表中序列1中，自5’末端第9789-10360位核苷酸为导肽Rbcs和草甘膦抗性基因Rbcs-EPSPS(G2-aroA)的启动子增强型35S、5’末端第8204-9784位核苷酸为导肽Rbcs和草甘膦抗性基因Rbcs-EPSPS(G2-aroA)、5’末端第7926-8196位核苷酸为草甘膦抗性基因EPSPS(G2-aroA)终止子NOS、5’末端第6903-7673位核苷酸为草甘膦降解基因N-乙酰转移酶基因增强型35S、5’末端第6456-6896位核苷酸为草甘膦降解基因N-乙酰转移酶基因GAT、5’末端第6248-6455位核苷酸为草甘膦降解基因N-乙酰转移酶基因终止子CaMV 35S polyA。
2、转基因大豆的获得
1)、重组根瘤农杆菌的获得
将上述1获得的重组载体pKT-rGE导入根癌农杆菌Ag10中，得到重组农杆菌Ag10/pKT-rGE。
2)再生大豆植株的获得
重组农杆菌Ag10/pKT-rGE转化离体培养的大豆(Glycine max)中黄10号的子叶节外植体，农杆菌侵染外植体后共培养3天，在含有草甘膦的筛选环境下经器官再生途径诱导转基因植株，丛生芽诱导阶段，使用 15mg/L浓度的草甘膦(Sigma公司)作为筛选剂，诱导3-6周；伸长芽诱导阶段，使用5mg/L浓度的草甘膦(sigma公司)作为筛选剂，诱导4-8周；诱导和伸长阶段，每2周更换一次培养基，使转化细胞再生，为抑制根癌农杆菌的快速生长，在诱导和伸长培养基中分别添加150mg/L浓度的噻孢霉素和400mg/L浓度的羧苄青霉素，待伸长芽伸长至4-6cm时，转入生根培养基中诱导生根，获得再生大豆植株。
3)转基因大豆的鉴定
(1)草甘膦筛选
将再生植株移栽至土壤中在温室或培养箱继续培养，光照条件是16h光照和8h黑暗，在第一至第三复叶期，用草甘膦异丙胺盐(Roundup)喷施处理各独立转化体，使用刻度喷雾器，以每公顷1.5L的草甘膦异丙胺盐(Roundup)剂量施用除草剂水溶液。草甘膦施用2周后，调查每个植株对草甘膦处理的反应，其中5个转化子对1.5L/公顷的草甘膦异丙胺盐(Roundup)喷施处理表现出较强的耐受性(给予编号为ZH10-1，2，3，5，6)，其中编号ZH10-6耐受性最高，较对照植株(中黄10号)生长旺盛，生长活力不受影响，叶片未发现变黄褪绿的症状，编号为ZH10-1，2，3，5，6的再生植株为T0代转基因大豆，T0代转基因大豆的草甘膦抗性高于野生型大豆中黄10号。
(2)PCR筛选
聚合酶链式反应(PCR)用于鉴定EPSPS(G2-aroA)和GAT基因的存在，从编号ZH10-1，2，3，5，6的T0代转基因大豆植株上收集20-50mg新鲜幼嫩的叶片置于2mL离心管中用于提取植物基因组DNA，DNA提取方法参照Murray and Thompson(1980)介绍的方法。用Takara公司生产的EX-Taq PCR试剂盒，参照说明书进行PCR反应，20μl PCR反应体系含10×EX-Taq buffer 2μl,2mM dNTPs 2μl,10μM的基因特异性上下游引物各0.5μl，EX-Taq酶0.2μl，补水至20μl；PCR反应程序为94℃,4min(1循环)；94℃,30s(变性),60℃,30s(退火),72℃,45s(延伸)35循环；72℃(终延伸)10min(1循环)。
扩增EPSPS(G2-aroA)的引物对，能够扩增出743bp产物的为阳性：
5’-ACCAGGAGCCTTGTACCTTGAG-3’(序列2)和
5’-ATCGGGTTCGATCAGGTAATC-3’(序列3)
扩增GAT基因的引物对，能够扩增出338bp产物的为阳性：
5’-CTCAGACCAAACCAGCCGATAG-3’(序列4)和
5’-GTGTCGAATACCTCTCCCTGCTC3’(序列5)
PCR鉴定结果如图1所示，M为100bp DNA Marker；1为野生型中黄10号阴性对照；2为无菌水对照；3为质粒pKT-rGE阳性对照；4、5、6、7、8分别为转基因大豆ZH10-1，2，3，5，6；结果表明，编号为ZH10-1，2，3，5，6的转基因大豆均呈PCR阳性，编号为ZH10-1，2，3，5，6的转基因大豆均转入了外源基因EPSPS和GAT。用传统的栽培和育种办法繁殖具有草甘膦抗性的转基因大豆，收获该转基因大豆种子。
实施例2、转基因大豆的草甘膦抗性分析
对实施例1中所得到的T0代转基因大豆ZH10-6进行连续多代的草甘膦抗性分析发现，其比非转基因对照中黄10号对草甘膦具有显著的抗性，具体如下：
收集T0代转基因大豆ZH10-6种子，播种，得到T1代转基因大豆ZH10-6，其在苗期(子叶出土真叶未完全展开时期)每株涂抹1μl草甘膦异丙胺盐(Roundup)原液(0.3mg a.e.μl-1 glyphosate)，2周后调查药害反应。
不抗植株表型为叶片失水褪绿，叶卷曲、皱缩，顶端分生组织坏死，直至整株死亡；抗性植株表型为生长趋势旺盛、叶片不褪绿、不卷曲、不皱缩。
T1代转基因大豆ZH10-6鉴定结果如图3所示，1：不抗的T1单株；2：未处理T1单株：3：抗性T1单株,表明，转基因大豆ZH10-6的T1单株在苗期对1μl草甘膦异丙胺盐(Roundup)原液/株处理的耐药性存在分离，处理3株，存活为具有抗性，死亡则不具有抗性，具有抗性植株2株，不抗植株1株；与未处理的T1代转基因大豆ZH10-6相比，抗性T1代单株植株株高、生长趋势不受抑制，叶片不褪绿，因此涂抹鉴定抗性T1代单株为抗草甘膦T1代转基因大豆ZH10-6。
在上述涂抹剂量下，中黄10号不具备任何抗性，2周后植株全部枯死，死亡。利用传统的育种方法，收集抗草甘膦T1代转基因大豆ZH10-6单株 的种子，播种，得到T2代转基因大豆ZH10-6。将T2代转基因大豆的不同株系分别进行3L/公顷和6L/公顷的草甘膦异丙胺盐(Roundup)喷施鉴定，从中筛选出2个抗性不分离T2代转基因ZH10-6的株系为T2代转基因大豆ZH10-6纯合株系；
对2个T2代转基因大豆ZH10-6纯合株系、转CP4-EPSPS耐草甘膦转基因材料Ag4501(王秀荣、廖红、何勇等.不同大豆种质材料蛋白质和脂肪含量分析.华南农业大学学报，2006，27(3)：9-11)和野生型对照中黄10号进行12升/公顷草甘膦异丙胺盐(Roundup)喷施处理。
结果如图5所示，1：T2代转基因大豆ZH10-6纯合株系未处理；2：T2代转基因大豆ZH10-6纯合株系喷施12L/公顷草甘膦异丙胺盐(Roundup)；3：转CP4-EPSPS抗草甘膦转基因材料Ag4501喷施12L/公顷草甘膦异丙胺盐(Roundup)；4：转CP4-EPSPS抗草甘膦转基因材料Ag4501未处理；5：T2代转基因大豆ZH10-6纯合株系喷施12L/公顷草甘膦异丙胺盐(Roundup)新叶不变黄、不褪绿；6、转CP4-EPSPS抗草甘膦转基因材料Ag4501喷施12L/公顷草甘膦异丙胺盐(Roundup)新叶变黄褪绿；表明，较未喷施对照，上述2个纯合株系无植株枯死死亡，上述2个纯合株系生长不受抑制，叶片不褪绿、不皱缩。且在高浓度12升/公顷草甘膦异丙胺盐(Roundup)喷施处理条件下，上述2个T2代转基因大豆ZH10-6纯合株系较转CP4-EPSPS耐草甘膦转基因材料对照，新叶未发现黄化、褪绿的现象，对高浓度的草甘膦喷施鉴定均具有较强的耐受性。
在上述喷施剂量下，中黄10号不具备任何抗性，2周后植株全部枯死，死亡。
上述T2代转基因大豆ZH10-6的2个纯合株系的对草甘膦的抗性高于中黄10号；具有更好的育种前景和利用价值。
抗草甘膦转基因大豆ZH10-6获得纯合株系，抗性能在后代中稳定遗传。
实施例3、转基因大豆中外源DNA拷贝数及分子整合稳定性分析
1、整合的外源T-DNA的拷贝数
通过对在所用转化载体的左和右边界序列之外延伸的限制性片段的Southern blotting分析测定抗草甘膦T1代转基因大豆ZH10-6的基因组 内整合的外源DNA片段的拷贝数。
Southern blotting分析的探针为，从载体T-DNA区域选择338bp的载体DNA序列设计探针(序列6)，参照北京美莱博医学科技有限公司生产的PCR法DIG标记试剂盒使用说明书，制备地高辛标记的探针。
参照北京美莱博医学科技有限公司生产的地高辛杂交试剂盒使用说明书，所用重组质粒pKT-rGE为阳性对照，中黄10号的基因组DNA为阴性对照。提取上述实施例2获得的抗草甘膦T1代转基因大豆ZH10-6的基因组DNA，用5个单位的限制性内切酶在200μl的酶切体系中于37℃中消化50-70μg基因组DNA 5-10h，使消化的DNA沉淀并重新溶解于25μl的无菌水中，每样品加6μl 6×Loading Buffer，消化的DNA、阳性对照、阴性对照、标准的分子大小标记(DNA molecular weight marker III,Digoxigenin-labeled(Roche)和λHind III marker)，45V电压下在0.8-1.0％的琼脂糖凝胶电泳上分离DNA。用溴化乙锭观察DNA，并包括荧光标尺进行摄像记录。然后再参照Whatman Schleicher&Schuell公司的Rapid Downward transfer system的使用说明书，将DNA转移至Hybond尼龙膜上，所述与探针杂交，用化学显示法显示杂交结果(北京美莱博医学科技有限公司)。
拷贝数测定可以通过对临近左右边界区域的基因组DNA的分析进行，用限制性内切酶DraI、HindIII、XbaI消化基因组DNA。
不同酶切后的Southern blotting杂交结果如图2所示，1:DIGMarker、2:质粒pKT-rGE阳性对照、3：ZH10-6-HindIII(HindIII酶切抗草甘膦T1代转基因大豆ZH10-6基因组)、4:ZH10-6-XbaI(XbaI酶切抗草甘膦T1代转基因大豆ZH10-6基因组)、5:ZH10-6-DraI(DraI酶切抗草甘膦T1代转基因大豆ZH10-6基因组)、6:中黄10号阴性对照；结果表明，整合的外源DNA为单拷贝插入。
2、T1代转基因大豆ZH10-6的PCR分析
提取实施例2中T1代转基因大豆ZH10-6单株基因组DNA，在20ul的PCR反应体系中，约50ng的基因组DNA用作模板DNA，且使用实施例1中扩增EPSPS(G2-aroA)引物对和扩增GAT基因引物对分别进行PCR扩增。
结果如图4所示，1：100bp Marker；2、3：无菌水对照；4：中黄 10号阴性对照；5：不抗草甘膦T1代转基因大豆ZH10-6；6、7：抗草甘膦T1代转基因大豆ZH10-6；8：pKT-rGE质粒阳性对照，可以看出，不抗草甘膦T1代转基因大豆ZH10-6没有目的产物，抗草甘膦T1代转基因大豆ZH10-6有目的产物。
3、纯合抗性株系的PCR分析
提取实施例2中所得到的T2代转基因大豆ZH10-6纯合株系的基因组DNA，在20ul的PCR反应体系中，约50ng的基因组DNA用作模板DNA，且使用实施例1中扩增EPSPS(G2-aroA)引物对和扩增GAT基因引物对分别进行PCR扩增。
结果如图6所示，1-19：T2代转基因大豆ZH10-6纯合株系不同单株；20：质粒pKT-rGE为阳性对照；21、22：无菌水对照；23、24：野生型中黄10号阴性对照；M：100bp DNA marker，表明，T2代转基因大豆ZH10-6纯合株系的所有单株均呈PCR阳性，分子鉴定均为纯合插入，且与草甘膦抗性完全对应。
实施例4、转基因大豆中外源DNA分子插入位置确定
用改良的CTAB法提取实施例2中所得到的T2代转基因大豆ZH10-6纯合株系基因组DNA，以已经公布的大豆基因组(http://phytozome.jgi.doe.gov/pz/portal.html#！info？alias＝Org_Gmax；2010)为参照，对上述T2代转基因大豆ZH10-6纯合进行测序分析，通过比对分析，发现在T2代转基因大豆ZH10-6纯合株系中，T-DNA插入到大豆17号染色体物理位置为7,980,527-7,980,541位，未插入已知大豆内源基因编码区。T-DNA整合替换了基因组上13bp碱基序列，被替换序列为5’CAAATGCAAAAAT 3’(序列7)，该被替换序列未破坏大豆内源基因编码区。
对外源T-DNA与插入位置的基因组DNA的结合区域进行PCR扩增来验证外源T-DNA插入位置，结果进一步证实了T-DNA插入位点的正确性，T2代转基因大豆ZH10-6纯合株系T-DNA插入结果如图7所示，T2代转基因大豆ZH10-6纯合株系中插入的外源T-DNA的5’端上游侧翼序列为序列8，外源T-DNA的3’端下游侧翼序列为序列9。
进一步的，通过测序分析，获得插入T2代转基因大豆ZH10-6的完整 外源T-DNA序列为序列10，整合外源插入外源DNA分子为4739bp，该转基因大豆外源DNA分子整合不含载体骨架序列。
上述结果表明，T2代转基因大豆ZH10-6纯合株系为草甘膦抗性植物，经侧翼序列和插入位点分析，其为将序列表中序列10或序列1自5’末端第6189-10927位核苷酸插入中黄10号大豆基因组第17号染色体的第7,980,527-7,980,541位间，替换掉第17号染色体的第7,980,528-7,980,540位间13bp的碱基序列，得到转基因大豆，且自第7,980,527位上游且紧邻第7,980,527位核苷酸的上游侧翼片段的核苷酸序列为序列8，自第7,980,541位下游且紧邻第7,980,541位核苷酸的下游侧翼片段的核苷酸序列为序列9；序列10为含有5-烯醇丙酮酸莽草酸-3-磷酸合成酶基因和N-乙酰转移酶基因的DNA分子。
可以看出，外源DNA分子的插入位置及两侧的侧翼序列可以用来鉴定是否为或源自目标转基因大豆(T2代转基因大豆ZH10-6纯合株系)。
T2代转基因大豆ZH10-6纯合株系(命名为ZH10-6)已于2015年12月1日保藏于中国微生物菌种保藏管理委员会普通微生物中心(简称CGMCC，地址：北京市朝阳区北辰西路1号院3号，中国科学院微生物研究所，邮编100101)，保藏号为CGMCC No.11108，分类命名为大豆Glycine max。
实施例5、检测转基因大豆的方法
为了更快速方便的检测待测转基因大豆是否为T2代转基因大豆ZH10-6纯合株系，根据实施例4中的外源DNA分子插入的位置及其上下游侧翼基因序列，开发特异性引物，建立抗草甘膦转基因大豆ZH10-6及其自交或杂交后代的定性PCR鉴定方法。
依据插入的外源T-DNA的5’端大豆基因组DNA上游侧翼序列(序列8)和插入的外源T-DNA(序列10)中GAT基因片段设计引物ZH10P-1和GAT-1(引物对1)；依据插入的外源T-DNA(序列10)中EPSPS基因片段和插入的外源T-DNA的3’端大豆基因组DNA下游侧翼序列(序列9)设计引物G2EP-2和ZH10P-2(引物对2)(图8)。
序列如下：
引物对1：
引物ZH10P-1：5'TAATAGTAGAATGGGACTGGTGGAT 3'(序列11)
引物GAT-1：5'GCGGACTTGCTTTGGTGTAAT 3'(序列12)。
引物对2：
引物G2EP-2：5'CCCGAATCATCAGGCAAACA 3'(序列13)
引物ZH10P-2：5'AACACATCATAGTATTCTAAAACGCTT 3'(序列14)。
大豆基因组DNA提取和PCR反应的参照实施例1中的方法，用上述引物对1进行PCR扩增，水和非转基因植株无扩增条带，T2代转基因大豆ZH10-6纯合株系叶片、种子、茎和花的基因组DNA扩增出目标条带，结果如图9A所示，M，DL 2K Plus DNA Marker；1，水；2，非转基因大豆中黄10；3，T2代转基因大豆ZH10-6纯合株系叶片；4，T2代转基因大豆ZH10-6纯合株系种子；5，T2代转基因大豆ZH10-6纯合株系花；6，T2代转基因大豆ZH10-6纯合株系茎，表明，T2代转基因大豆ZH10-6纯合株系叶片、种子和茎基因组DNA扩增均得到特异性的810bp目的片段，该目的片段具体的核苷酸序列如序列15所示，而非转基因大豆中黄10没有目的片段。
大豆基因组DNA提取和PCR反应的参照实施例1中的方法，用上述引物对2进行PCR扩增，水和非转基因植株无扩增条带，T2代转基因大豆ZH10-6纯合株系叶片、种子、花和茎的基因组DNA扩增，结果如图9B所示，M，DL2K Plus DNA Marker；1，水；2，非转基因大豆中黄10；3，T2代转基因大豆ZH10-6纯合株系叶片；4，T2代转基因大豆ZH10-6纯合株系种子；5，T2代转基因大豆ZH10-6纯合株系花；6，T2代转基因大豆ZH10-6纯合株系茎，表明，T2代转基因大豆ZH10-6纯合株系叶片、花、种子和茎基因组DNA扩增均得到特异性1627bp目的片段，且该目的片段具体的核苷酸序列如序列16所示，而非转基因大豆中黄10没有目的片段。
从上述可以看出，提取待测样品的基因组DNA作为模板，用引物对1和引物对2进行PCR扩增，检测PCR扩增产物：
若引物对1扩增得到810bp目的片段，引物对2扩增得到1627bp目的片段，则待检测转基因大豆为实施例4获得的T2代转基因大豆ZH10-6纯合株系；
若引物对1没有扩增得到810bp目的片段和/或引物对2没有扩增得 到1627bp目的片段，则待测转基因大豆不为实施例4获得的T2代转基因大豆ZH10-6纯合株系。
试验说明利用转基因大豆ZH10-6的侧翼序列进行PCR扩增，可以特异性的检测大豆转基因大豆ZH10-6的分子特征，用于鉴定上述转基因大豆及其后代、细胞、种子、营养器官等。
实施例6、检测转基因大豆方法的应用
黑河43(Heihe 43,其统编号：ZDD24325)，记载在如下文献中：《早熟高产优质大豆新品种黑河43》，贾鸿昌等，黑龙江农业科学，2007,05:124-125)；
黑河38(Heihe38,其统编号：ZDD24320),记载在如下文献中：《黑河38号大豆品种的选育和遗传组成》，吴纪安，植物遗传资源学报，2007,3:313-316)；
克山1号(Keshan 1,国审豆2009002)，记载在如下文献中：《利用空间诱变技术选育大豆新品种克山1号》，张勇等，黑农学报，2013，27(9)：1241-1246)；
垦丰16(Kenfeng 16,其统编号：ZDD24344),记载在如下文献中：《大豆品种垦丰16的选育及体会分析》，杨丹霞等，大豆科技，2010,3:65-66)；
垦丰20(Kenfen 20，黑垦审豆2008004)，记载在如下文献中：《不同大豆品种豆浆口感的研究》，姜玉久等，大豆科技，2015,04:11-13)；
垦丰22(Kenfen 22，其统编号为ZDD24348)，记载在如下文献中：《大豆新品种垦丰22号的选育及栽培技术》，杨丹霞等，大豆科技，2010，01:64-65)。
以中国主栽的大豆品种黑河43、黑河38、克山1号、垦丰16、垦丰20、垦丰22等为母本，以T2代转基因大豆ZH10-6纯合株系(抗草甘膦，以下称为ZH10-6)为父本，利用传统的杂交育种技术制备F1的种子，收获种植F1代种子，在第一至第三个三出复叶期，喷施200ml/亩的草甘膦，收获抗200ml/亩草甘膦喷施杂交F1单株的种子，种植F2；对不同杂交组合的F2材料喷施800ml/亩的草甘膦异丙胺盐溶液(Roundup,Monsanto,Co)。
F2群体对草甘膦耐受性鉴定结果见表1，表明抗性在杂交后代中遗传稳定且符合3:1的分离比例，进一步的说明了外源基因在ZH10-6的基因组上只有一个插入位点，与Southern检测为单拷贝的结果相吻合。
表1 ZH10-6衍生的F2分离群体对草甘膦耐受性的抗感分析
Figure PCTCN2015096326-appb-000001
χ0.052＝3.841， χ0.052 符合3:1； P＞0.05符合3∶1
随机提取不同杂交组合F2抗性单株的植株DNA及其根、茎、叶、花、种子的DNA，以上述DNA为模板，用实施例5中的引物对1和引物对2进行PCR扩增，以非转基因大豆黑河43和黑河38为阴性对照；扩增结果如图10和图11所示。
抗性F2单株PCR产物电泳结果如图10所示，A：引物对1的扩增结果；B：引物对2的扩增结果；1:200 bp DNA Marker；2:无菌水对照；3、4：非转基因大豆黑河43和黑河38；5-25：随机选择的抗性杂交F2单株；结果表明，水和非转基因植株无扩增条带，抗性F2代单株基因组DNA扩增出810bp(图10A)和1626bp(图10B)的目标条带，且810bp片段和1626bp片段的核苷酸序列分别为序列15和序列16。
因此，PCR检测为阳性的植物单株为T2代转基因大豆ZH10-6纯合株系的杂交后代。
抗性F2的单株的根、茎、叶、花、种子PCR产物电泳结果如图11所示，A：引物对1的扩增结果；B：引物对2的扩增结果；1:200 bp DNA Marker；2:无菌水对照；3-7：抗性F2的根、茎、叶、花和种子；8-12：非转基 因植株黑河43的根、茎、叶、花和种子；可以看出，水和非转基因植株无扩增条带，抗性F2代根、茎、叶、花、种子的DNA均扩增出810bp(图11A)和1626bp(图11B)的目标条带，且810bp片段和1626bp片段的核苷酸序列分别为序列15和序列16。
因此，从根、茎、叶、花、种子等任何植物组织中都可以检测该植物是否来源于T2代转基因大豆ZH10-6纯合株系及其衍生的杂交后代。
工业应用
本发明的实验证明，本发明采用在大豆17号染色体的7980527至7980541位置间插入外源DNA片段，得到含有外源DNA片段的转基因大豆；外源DNA片段包括草甘膦抗性基因G2-aroA和草甘膦降解基因N-乙酰转移酶基因GAT基因；转基因大豆对草甘膦抗性高于未转基因的野生型大豆。该转基因大豆对高剂量草甘膦有耐受性，其可进一步通过诱变或与优良大豆株系杂交来改良以优化其他农艺性状如产量、品质等。该转外源DNA片段大豆共表达抗草甘膦基因EPSPS(G2-aroA)和草甘膦降解基因N-乙酰转移酶基因(GAT)，在苗期(子叶出土且真叶未展开时期)单株对1-1.5ul的草甘膦异丙胺盐(Roundup)原液处理具有耐受性，田间对每公顷约3至12升的草甘膦异丙胺盐(Roundup)处理具有耐受性。获得良好杂草控制的总标准计量根据杂草压力在每公顷3-6升之间变动。本发明中的转基因大豆在这些浓度处理时甚至更高浓度(12升/公顷)处理时，除草剂处理对植物活力和叶持绿等性状不产生任何可测的影响。

Claims (14)

  1. 一种培育转基因大豆的方法，为将外源DNA片段插入目的大豆基因组第17号染色体的第7,980,527-7,980,541位间，替换掉第17号染色体的第7,980,527-7,980,541位间13bp的碱基序列，得到转基因大豆；
    所述转基因大豆的草甘膦抗性高于所述目的大豆；
    所述外源DNA片段为含有5-烯醇丙酮酸莽草酸-3-磷酸合成酶基因和N-乙酰转移酶基因的DNA分子。
  2. 根据权利要求1所述的方法，其特征在于：
    所述5-烯醇丙酮酸莽草酸-3-磷酸合成酶基因为G2-aroA；
    所述N-乙酰转移酶基因为GAT；
    所述外源DNA片段为序列表中序列10或序列1自5’末端第6189-10927位核苷酸。
  3. 根据权利要求1或2所述的方法，其特征在于：所述外源DNA片段在所述转基因大豆的上游侧翼片段为所述目的大豆基因组第17号染色体的自第7,980,527位核苷酸起向其上游方向延伸得到的长度为0至5Kb的任意一个DNA片段；
    所述外源DNA片段在所述转基因大豆的下游侧翼片段为所述目的大豆基因组第17号染色体的自第7,980,541位核苷酸起向其下游方向延伸得到的长度为0至5Kb的任意一个DNA片段。
  4. 根据权利要求3所述方法，其特征在于：
    所述上游侧翼片段为序列表中序列8所示的核苷酸；
    所述下游侧翼片段为序列表中序列9所示的核苷酸。
  5. 根据权利要求4所述方法，其特征在于：
    所述外源DNA片段通过含有所述外源DNA片段的重组载体导入所述目的大豆；
    所述重组载体的核苷酸序列为序列表中序列1；
    所述目的大豆为中黄10号。
  6. 由权利要求1-5中任一所述方法制备的转基因植物。
  7. 根据权利要求6所述的转基因植物，其特征在于：所述转基因植物为大豆ZH10-6CGMCC No.11108。
  8. 用于检测或辅助检测植物样品是否来源于权利要求6或7所述的转基因大豆或权利要求或其后代或用于检测或辅助检测制品是否含有权利要求6或7所述的转基因大豆或其后代的方法，包括如下步骤：检测所述植物样品的基因组DNA中或所述制品中是否含有DNA片段A，
    所述DNA片段A为如下1)或2)；
    1)由权利要求3中的所述外源DNA片段在所述转基因大豆的上游侧翼片段、权利要求3中的所述外源DNA片段和权利要求3中的所述外源DNA片段在所述转基因大豆的下游侧翼片段组成；
    2)与1)所示的DNA片段A同源性大于95％的DNA片段；
    若含有所述DNA片段A，则所述植物样品为或候选为所述转基因大豆或其后代；或所述制品含有或候选含有所述转基因大豆或其后代；
    若不含有所述DNA片段A，则所述植物样品不为或候选不为所述转基因大豆或其后代；或所述制品不含或候选不含所述转基因大豆或其后代。
  9. 根据权利要求8所述的方法，其特征在于：
    所述方法为如下1)或2)或3)：
    1)直接测序植物样品的基因组DNA，判断所述测序植物样品含有所述DNA片段A；
    2)用引物对1或引物对2进行PCR扩增，若有目的扩增产物，则所述测序植物样品或制品含有所述DNA片段A；
    所述引物对1为能够扩增由所述外源DNA片段5’端和紧邻其的所述上游侧翼序列部分或全部片段组成的DNA分子甲的引物对；其对应的目的扩增产物为所述DNA分子甲；
    所述引物对2为能够扩增含有所述外源DNA片段3’端和紧邻其的所述下游侧翼序列的部分或全部组成的DNA分子乙的引物对；其对应的目的扩增产物为所述DNA分子乙；
    3)用能特异结合所述DNA分子甲或能特异结合所述DNA分子乙的探针对所述待测植物样品或制品的DNA进行Southern杂交，若能杂交得到杂交片段，则所述植物样品来源于所述转基因大豆或其后代，所述制品含有所述转基因大豆或其后代。
  10. 根据权利要求9所述的方法，其特征在于：
    2)中，所述引物对1由序列表中序列11所示的单链DNA分子和序列表中序列12所示的单链DNA分子组成；
    所述引物对2由序列表中序列13所示的单链DNA分子和序列表中序列14所示的单链DNA分子组成；
    3)中，所述探针的核苷酸序列为序列6。
  11. 根据权利要求8-10中任一所述的方法，其特征在于：所述转基因大豆的后代为以所述转基因大豆为亲本衍生的转基因材料，包括用所述转基因大豆诱变或与其他大豆杂交得到的衍生后代或所述诱变或杂交后代再进行衍生得到的后代。
  12. 用于检测或辅助检测植物样品是否来源于权利要求6或7所述的转基因大豆或其后代或用于检测或辅助检测制品是否含有权利要求6或7所述的转基因大豆或其后代的试剂盒，其包括：1)权利要求7中所述外源DNA片段、2)权利要求7中所述引物对1、3)权利要求7中所述引物对2或4)权利要求7中所述探针。
  13. 根据权利要求12所述的试剂盒，其特性在于：所述试剂盒还包括记载权利要求1-5中任一所述方法或权利要求8-10中任一所述方法的说明书。
  14. 权利要求6或7所述的转基因植物在育种和/或生产加工中的应用。
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CN110229843A (zh) * 2019-04-04 2019-09-13 中国农业科学院棉花研究所 陆地棉转化事件19pfa1-135-17及其特异性鉴定方法
CN110229843B (zh) * 2019-04-04 2023-02-28 中国农业科学院棉花研究所 陆地棉转化事件19pfa1-135-17及其特异性鉴定方法
CN112852867A (zh) * 2021-04-08 2021-05-28 中国农业科学院生物技术研究所 草甘膦抗性基因gr79和gat的表达载体及应用

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