CN116200529B - Nucleic acid sequence of corn transformation event LG11 and detection method thereof - Google Patents
Nucleic acid sequence of corn transformation event LG11 and detection method thereof Download PDFInfo
- Publication number
- CN116200529B CN116200529B CN202310139736.6A CN202310139736A CN116200529B CN 116200529 B CN116200529 B CN 116200529B CN 202310139736 A CN202310139736 A CN 202310139736A CN 116200529 B CN116200529 B CN 116200529B
- Authority
- CN
- China
- Prior art keywords
- seq
- dna
- sequence
- corn
- event
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
- 240000008042 Zea mays Species 0.000 title claims abstract description 182
- 235000002017 Zea mays subsp mays Nutrition 0.000 title claims abstract description 165
- 235000005824 Zea mays ssp. parviglumis Nutrition 0.000 title claims abstract description 111
- 235000005822 corn Nutrition 0.000 title claims abstract description 111
- 150000007523 nucleic acids Chemical group 0.000 title claims abstract description 75
- 230000009466 transformation Effects 0.000 title abstract description 62
- 108091028043 Nucleic acid sequence Proteins 0.000 title abstract description 59
- 238000001514 detection method Methods 0.000 title abstract description 29
- 230000009261 transgenic effect Effects 0.000 claims abstract description 105
- 230000000295 complement effect Effects 0.000 claims abstract description 46
- 102000039446 nucleic acids Human genes 0.000 claims abstract description 39
- 108020004707 nucleic acids Proteins 0.000 claims abstract description 39
- 230000002441 reversible effect Effects 0.000 claims abstract description 35
- 238000000034 method Methods 0.000 claims description 74
- 235000016383 Zea mays subsp huehuetenangensis Nutrition 0.000 claims description 54
- 235000009973 maize Nutrition 0.000 claims description 54
- 238000003780 insertion Methods 0.000 claims description 46
- 230000037431 insertion Effects 0.000 claims description 46
- 239000002773 nucleotide Substances 0.000 claims description 36
- 125000003729 nucleotide group Chemical group 0.000 claims description 34
- 239000012634 fragment Substances 0.000 claims description 28
- 238000002493 microarray Methods 0.000 claims description 7
- 238000012360 testing method Methods 0.000 claims description 2
- 108020004414 DNA Proteins 0.000 abstract description 108
- 239000004009 herbicide Substances 0.000 abstract description 61
- 230000002363 herbicidal effect Effects 0.000 abstract description 57
- 241000238631 Hexapoda Species 0.000 abstract description 55
- IAJOBQBIJHVGMQ-UHFFFAOYSA-N 2-amino-4-[hydroxy(methyl)phosphoryl]butanoic acid Chemical compound CP(O)(=O)CCC(N)C(O)=O IAJOBQBIJHVGMQ-UHFFFAOYSA-N 0.000 abstract description 40
- 239000005561 Glufosinate Substances 0.000 abstract description 38
- 102000053602 DNA Human genes 0.000 abstract description 16
- 230000009418 agronomic effect Effects 0.000 abstract description 13
- 239000012472 biological sample Substances 0.000 abstract description 6
- 241000196324 Embryophyta Species 0.000 description 101
- 108090000623 proteins and genes Proteins 0.000 description 66
- 239000000523 sample Substances 0.000 description 59
- 239000013615 primer Substances 0.000 description 56
- 238000009396 hybridization Methods 0.000 description 36
- 230000014509 gene expression Effects 0.000 description 33
- 238000003199 nucleic acid amplification method Methods 0.000 description 29
- 241000607479 Yersinia pestis Species 0.000 description 27
- 230000003321 amplification Effects 0.000 description 27
- 238000003752 polymerase chain reaction Methods 0.000 description 27
- 108700019146 Transgenes Proteins 0.000 description 25
- 210000004027 cell Anatomy 0.000 description 21
- 241000701489 Cauliflower mosaic virus Species 0.000 description 18
- 108091093088 Amplicon Proteins 0.000 description 16
- 239000000463 material Substances 0.000 description 16
- 102000004169 proteins and genes Human genes 0.000 description 15
- 238000006243 chemical reaction Methods 0.000 description 14
- 238000003776 cleavage reaction Methods 0.000 description 13
- 230000007017 scission Effects 0.000 description 13
- 241000589158 Agrobacterium Species 0.000 description 12
- 241001057636 Dracaena deremensis Species 0.000 description 12
- 241000346285 Ostrinia furnacalis Species 0.000 description 12
- 239000013612 plasmid Substances 0.000 description 11
- 108020001019 DNA Primers Proteins 0.000 description 10
- 230000004544 DNA amplification Effects 0.000 description 10
- 239000003155 DNA primer Substances 0.000 description 10
- 241000256251 Spodoptera frugiperda Species 0.000 description 10
- 230000002068 genetic effect Effects 0.000 description 10
- 239000013598 vector Substances 0.000 description 10
- 102000040430 polynucleotide Human genes 0.000 description 9
- 108091033319 polynucleotide Proteins 0.000 description 9
- 239000002157 polynucleotide Substances 0.000 description 9
- 210000001519 tissue Anatomy 0.000 description 9
- 108010058731 nopaline synthase Proteins 0.000 description 8
- 108091008146 restriction endonucleases Proteins 0.000 description 8
- 229920000742 Cotton Polymers 0.000 description 7
- 108091034117 Oligonucleotide Proteins 0.000 description 7
- 108010076504 Protein Sorting Signals Proteins 0.000 description 7
- 238000002105 Southern blotting Methods 0.000 description 7
- 238000009395 breeding Methods 0.000 description 7
- 230000006378 damage Effects 0.000 description 7
- 238000002866 fluorescence resonance energy transfer Methods 0.000 description 7
- 238000011835 investigation Methods 0.000 description 7
- 239000000203 mixture Substances 0.000 description 7
- 108010082527 phosphinothricin N-acetyltransferase Proteins 0.000 description 7
- 239000002987 primer (paints) Substances 0.000 description 7
- 230000001568 sexual effect Effects 0.000 description 7
- QGZKDVFQNNGYKY-UHFFFAOYSA-N Ammonia Chemical compound N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 description 6
- 238000012408 PCR amplification Methods 0.000 description 6
- 231100000674 Phytotoxicity Toxicity 0.000 description 6
- 238000004458 analytical method Methods 0.000 description 6
- 230000027455 binding Effects 0.000 description 6
- 230000001488 breeding effect Effects 0.000 description 6
- 238000012216 screening Methods 0.000 description 6
- 238000012163 sequencing technique Methods 0.000 description 6
- 230000000694 effects Effects 0.000 description 5
- 239000003623 enhancer Substances 0.000 description 5
- 239000013604 expression vector Substances 0.000 description 5
- 235000013305 food Nutrition 0.000 description 5
- 230000012010 growth Effects 0.000 description 5
- 239000003550 marker Substances 0.000 description 5
- 239000013642 negative control Substances 0.000 description 5
- 230000008488 polyadenylation Effects 0.000 description 5
- 239000013641 positive control Substances 0.000 description 5
- 230000008569 process Effects 0.000 description 5
- 230000000306 recurrent effect Effects 0.000 description 5
- 238000005507 spraying Methods 0.000 description 5
- 241000758794 Asarum Species 0.000 description 4
- 102000016928 DNA-directed DNA polymerase Human genes 0.000 description 4
- 108010014303 DNA-directed DNA polymerase Proteins 0.000 description 4
- 108091029865 Exogenous DNA Proteins 0.000 description 4
- 108020004511 Recombinant DNA Proteins 0.000 description 4
- 101150103518 bar gene Proteins 0.000 description 4
- 235000013339 cereals Nutrition 0.000 description 4
- 230000008859 change Effects 0.000 description 4
- 230000000749 insecticidal effect Effects 0.000 description 4
- 230000001404 mediated effect Effects 0.000 description 4
- 230000001105 regulatory effect Effects 0.000 description 4
- 239000011540 sensing material Substances 0.000 description 4
- 241000894007 species Species 0.000 description 4
- 238000011144 upstream manufacturing Methods 0.000 description 4
- 238000005406 washing Methods 0.000 description 4
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 3
- 241000589155 Agrobacterium tumefaciens Species 0.000 description 3
- 102000004190 Enzymes Human genes 0.000 description 3
- 108090000790 Enzymes Proteins 0.000 description 3
- 238000000692 Student's t-test Methods 0.000 description 3
- 229910021529 ammonia Inorganic materials 0.000 description 3
- 230000002759 chromosomal effect Effects 0.000 description 3
- 238000013461 design Methods 0.000 description 3
- 238000009472 formulation Methods 0.000 description 3
- 239000000499 gel Substances 0.000 description 3
- 230000010354 integration Effects 0.000 description 3
- 239000012528 membrane Substances 0.000 description 3
- 230000036961 partial effect Effects 0.000 description 3
- 230000010076 replication Effects 0.000 description 3
- 150000003839 salts Chemical class 0.000 description 3
- 238000012353 t test Methods 0.000 description 3
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- 241000894006 Bacteria Species 0.000 description 2
- 108010066133 D-octopine dehydrogenase Proteins 0.000 description 2
- 230000004568 DNA-binding Effects 0.000 description 2
- 241000701484 Figwort mosaic virus Species 0.000 description 2
- 108700007698 Genetic Terminator Regions Proteins 0.000 description 2
- 108010070675 Glutathione transferase Proteins 0.000 description 2
- 102000005720 Glutathione transferase Human genes 0.000 description 2
- 241001477931 Mythimna unipuncta Species 0.000 description 2
- 108020005187 Oligonucleotide Probes Proteins 0.000 description 2
- 108091034057 RNA (poly(A)) Proteins 0.000 description 2
- 238000012300 Sequence Analysis Methods 0.000 description 2
- 241000723792 Tobacco etch virus Species 0.000 description 2
- 108700029229 Transcriptional Regulatory Elements Proteins 0.000 description 2
- 102000004243 Tubulin Human genes 0.000 description 2
- 108090000704 Tubulin Proteins 0.000 description 2
- 108090000848 Ubiquitin Proteins 0.000 description 2
- 102000044159 Ubiquitin Human genes 0.000 description 2
- 230000009471 action Effects 0.000 description 2
- 239000011543 agarose gel Substances 0.000 description 2
- 150000001413 amino acids Chemical class 0.000 description 2
- 238000004166 bioassay Methods 0.000 description 2
- 230000033228 biological regulation Effects 0.000 description 2
- 230000015572 biosynthetic process Effects 0.000 description 2
- 238000007796 conventional method Methods 0.000 description 2
- 235000012343 cottonseed oil Nutrition 0.000 description 2
- 239000002385 cottonseed oil Substances 0.000 description 2
- 238000010586 diagram Methods 0.000 description 2
- 235000013399 edible fruits Nutrition 0.000 description 2
- 210000002257 embryonic structure Anatomy 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 238000002875 fluorescence polarization Methods 0.000 description 2
- 108020002326 glutamine synthetase Proteins 0.000 description 2
- 102000005396 glutamine synthetase Human genes 0.000 description 2
- 230000006872 improvement Effects 0.000 description 2
- 230000000977 initiatory effect Effects 0.000 description 2
- 239000002917 insecticide Substances 0.000 description 2
- 230000000670 limiting effect Effects 0.000 description 2
- 231100000053 low toxicity Toxicity 0.000 description 2
- 238000004519 manufacturing process Methods 0.000 description 2
- 230000035772 mutation Effects 0.000 description 2
- 239000002071 nanotube Substances 0.000 description 2
- 239000002751 oligonucleotide probe Substances 0.000 description 2
- 230000003287 optical effect Effects 0.000 description 2
- 238000009401 outcrossing Methods 0.000 description 2
- 238000003976 plant breeding Methods 0.000 description 2
- 230000010287 polarization Effects 0.000 description 2
- 108090000765 processed proteins & peptides Proteins 0.000 description 2
- 230000001737 promoting effect Effects 0.000 description 2
- 210000001938 protoplast Anatomy 0.000 description 2
- 238000010791 quenching Methods 0.000 description 2
- 230000000171 quenching effect Effects 0.000 description 2
- 238000001228 spectrum Methods 0.000 description 2
- 238000007619 statistical method Methods 0.000 description 2
- 238000001308 synthesis method Methods 0.000 description 2
- 230000008685 targeting Effects 0.000 description 2
- 239000003053 toxin Substances 0.000 description 2
- 231100000765 toxin Toxicity 0.000 description 2
- 108700012359 toxins Proteins 0.000 description 2
- 238000013518 transcription Methods 0.000 description 2
- 230000035897 transcription Effects 0.000 description 2
- 238000011426 transformation method Methods 0.000 description 2
- 230000001131 transforming effect Effects 0.000 description 2
- 108020005065 3' Flanking Region Proteins 0.000 description 1
- 108010020183 3-phosphoshikimate 1-carboxyvinyltransferase Proteins 0.000 description 1
- 108020005029 5' Flanking Region Proteins 0.000 description 1
- 102000007469 Actins Human genes 0.000 description 1
- 108010085238 Actins Proteins 0.000 description 1
- 241000589156 Agrobacterium rhizogenes Species 0.000 description 1
- 241000566547 Agrotis ipsilon Species 0.000 description 1
- 241001136782 Alca Species 0.000 description 1
- 241000483758 Amphipoea oculea Species 0.000 description 1
- 102000007347 Apyrase Human genes 0.000 description 1
- 108010007730 Apyrase Proteins 0.000 description 1
- 241000219194 Arabidopsis Species 0.000 description 1
- 241000219195 Arabidopsis thaliana Species 0.000 description 1
- 108700003918 Bacillus Thuringiensis insecticidal crystal Proteins 0.000 description 1
- 239000002028 Biomass Substances 0.000 description 1
- 101001033883 Cenchritis muricatus Protease inhibitor 2 Proteins 0.000 description 1
- LZZYPRNAOMGNLH-UHFFFAOYSA-M Cetrimonium bromide Chemical compound [Br-].CCCCCCCCCCCCCCCC[N+](C)(C)C LZZYPRNAOMGNLH-UHFFFAOYSA-M 0.000 description 1
- 108010077544 Chromatin Proteins 0.000 description 1
- 241000221635 Cladonia Species 0.000 description 1
- 108091035707 Consensus sequence Proteins 0.000 description 1
- IGXWBGJHJZYPQS-SSDOTTSWSA-N D-Luciferin Chemical compound OC(=O)[C@H]1CSC(C=2SC3=CC=C(O)C=C3N=2)=N1 IGXWBGJHJZYPQS-SSDOTTSWSA-N 0.000 description 1
- YAHZABJORDUQGO-NQXXGFSBSA-N D-ribulose 1,5-bisphosphate Chemical compound OP(=O)(O)OC[C@@H](O)[C@@H](O)C(=O)COP(O)(O)=O YAHZABJORDUQGO-NQXXGFSBSA-N 0.000 description 1
- 238000001712 DNA sequencing Methods 0.000 description 1
- CYCGRDQQIOGCKX-UHFFFAOYSA-N Dehydro-luciferin Natural products OC(=O)C1=CSC(C=2SC3=CC(O)=CC=C3N=2)=N1 CYCGRDQQIOGCKX-UHFFFAOYSA-N 0.000 description 1
- 101100534468 Dictyostelium discoideum staA gene Proteins 0.000 description 1
- 208000035240 Disease Resistance Diseases 0.000 description 1
- 239000004278 EU approved seasoning Substances 0.000 description 1
- 101100434659 Emericella nidulans (strain FGSC A4 / ATCC 38163 / CBS 112.46 / NRRL 194 / M139) alcR gene Proteins 0.000 description 1
- BJGNCJDXODQBOB-UHFFFAOYSA-N Fivefly Luciferin Natural products OC(=O)C1CSC(C=2SC3=CC(O)=CC=C3N=2)=N1 BJGNCJDXODQBOB-UHFFFAOYSA-N 0.000 description 1
- 239000005562 Glyphosate Substances 0.000 description 1
- 241001147381 Helicoverpa armigera Species 0.000 description 1
- 241000255967 Helicoverpa zea Species 0.000 description 1
- 108010034791 Heterochromatin Proteins 0.000 description 1
- 206010020649 Hyperkeratosis Diseases 0.000 description 1
- 206010061217 Infestation Diseases 0.000 description 1
- 108091092195 Intron Proteins 0.000 description 1
- 108010025815 Kanamycin Kinase Proteins 0.000 description 1
- 241000255777 Lepidoptera Species 0.000 description 1
- 108060001084 Luciferase Proteins 0.000 description 1
- 239000005089 Luciferase Substances 0.000 description 1
- DDWFXDSYGUXRAY-UHFFFAOYSA-N Luciferin Natural products CCc1c(C)c(CC2NC(=O)C(=C2C=C)C)[nH]c1Cc3[nH]c4C(=C5/NC(CC(=O)O)C(C)C5CC(=O)O)CC(=O)c4c3C DDWFXDSYGUXRAY-UHFFFAOYSA-N 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 231100000678 Mycotoxin Toxicity 0.000 description 1
- GRYLNZFGIOXLOG-UHFFFAOYSA-N Nitric acid Chemical compound O[N+]([O-])=O GRYLNZFGIOXLOG-UHFFFAOYSA-N 0.000 description 1
- 238000000636 Northern blotting Methods 0.000 description 1
- 108090000417 Oxygenases Proteins 0.000 description 1
- 102000004020 Oxygenases Human genes 0.000 description 1
- 101710091688 Patatin Proteins 0.000 description 1
- 244000046052 Phaseolus vulgaris Species 0.000 description 1
- 235000010627 Phaseolus vulgaris Nutrition 0.000 description 1
- 239000004952 Polyamide Substances 0.000 description 1
- 101710090029 Replication-associated protein A Proteins 0.000 description 1
- 101000693622 Spodoptera frugiperda Allatotropin Proteins 0.000 description 1
- 229920002472 Starch Polymers 0.000 description 1
- 241000098292 Striacosta albicosta Species 0.000 description 1
- 102000004523 Sulfate Adenylyltransferase Human genes 0.000 description 1
- 108010022348 Sulfate adenylyltransferase Proteins 0.000 description 1
- 208000036142 Viral infection Diseases 0.000 description 1
- 241000700605 Viruses Species 0.000 description 1
- 208000027418 Wounds and injury Diseases 0.000 description 1
- 235000007244 Zea mays Nutrition 0.000 description 1
- 239000012190 activator Substances 0.000 description 1
- IRLPACMLTUPBCL-FCIPNVEPSA-N adenosine-5'-phosphosulfate Chemical compound C1=NC=2C(N)=NC=NC=2N1[C@@H]1O[C@@H](CO[P@](O)(=O)OS(O)(=O)=O)[C@H](O)[C@H]1O IRLPACMLTUPBCL-FCIPNVEPSA-N 0.000 description 1
- 239000013566 allergen Substances 0.000 description 1
- 230000004075 alteration Effects 0.000 description 1
- 102000006646 aminoglycoside phosphotransferase Human genes 0.000 description 1
- 238000000137 annealing Methods 0.000 description 1
- 238000013459 approach Methods 0.000 description 1
- 230000001580 bacterial effect Effects 0.000 description 1
- 244000052616 bacterial pathogen Species 0.000 description 1
- 230000010310 bacterial transformation Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000008901 benefit Effects 0.000 description 1
- 230000003115 biocidal effect Effects 0.000 description 1
- 230000001851 biosynthetic effect Effects 0.000 description 1
- 230000015556 catabolic process Effects 0.000 description 1
- 238000004113 cell culture Methods 0.000 description 1
- 238000012512 characterization method Methods 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 239000005081 chemiluminescent agent Substances 0.000 description 1
- 210000003763 chloroplast Anatomy 0.000 description 1
- 108010031100 chloroplast transit peptides Proteins 0.000 description 1
- 210000003483 chromatin Anatomy 0.000 description 1
- 239000013611 chromosomal DNA Substances 0.000 description 1
- 235000005687 corn oil Nutrition 0.000 description 1
- 239000002285 corn oil Substances 0.000 description 1
- 239000002537 cosmetic Substances 0.000 description 1
- 239000012297 crystallization seed Substances 0.000 description 1
- 238000006731 degradation reaction Methods 0.000 description 1
- 238000012217 deletion Methods 0.000 description 1
- 230000037430 deletion Effects 0.000 description 1
- 238000004925 denaturation Methods 0.000 description 1
- 230000036425 denaturation Effects 0.000 description 1
- 238000001784 detoxification Methods 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 235000005911 diet Nutrition 0.000 description 1
- 230000037213 diet Effects 0.000 description 1
- 239000000539 dimer Substances 0.000 description 1
- 210000005069 ears Anatomy 0.000 description 1
- 210000002472 endoplasmic reticulum Anatomy 0.000 description 1
- 230000007613 environmental effect Effects 0.000 description 1
- 238000001976 enzyme digestion Methods 0.000 description 1
- 210000003527 eukaryotic cell Anatomy 0.000 description 1
- 239000004744 fabric Substances 0.000 description 1
- 230000002349 favourable effect Effects 0.000 description 1
- 239000003337 fertilizer Substances 0.000 description 1
- 235000013312 flour Nutrition 0.000 description 1
- 230000037406 food intake Effects 0.000 description 1
- 235000011194 food seasoning agent Nutrition 0.000 description 1
- 238000010353 genetic engineering Methods 0.000 description 1
- XDDAORKBJWWYJS-UHFFFAOYSA-N glyphosate Chemical compound OC(=O)CNCP(O)(O)=O XDDAORKBJWWYJS-UHFFFAOYSA-N 0.000 description 1
- 229940097068 glyphosate Drugs 0.000 description 1
- 210000004458 heterochromatin Anatomy 0.000 description 1
- 230000001976 improved effect Effects 0.000 description 1
- 238000007901 in situ hybridization Methods 0.000 description 1
- 230000001939 inductive effect Effects 0.000 description 1
- 239000012770 industrial material Substances 0.000 description 1
- 208000014674 injury Diseases 0.000 description 1
- 238000011081 inoculation Methods 0.000 description 1
- 230000002452 interceptive effect Effects 0.000 description 1
- 229930027917 kanamycin Natural products 0.000 description 1
- SBUJHOSQTJFQJX-NOAMYHISSA-N kanamycin Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CN)O[C@@H]1O[C@H]1[C@H](O)[C@@H](O[C@@H]2[C@@H]([C@@H](N)[C@H](O)[C@@H](CO)O2)O)[C@H](N)C[C@@H]1N SBUJHOSQTJFQJX-NOAMYHISSA-N 0.000 description 1
- 229960000318 kanamycin Drugs 0.000 description 1
- 229930182823 kanamycin A Natural products 0.000 description 1
- 239000003446 ligand Substances 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 230000014759 maintenance of location Effects 0.000 description 1
- 210000001161 mammalian embryo Anatomy 0.000 description 1
- 235000012054 meals Nutrition 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- 108020004999 messenger RNA Proteins 0.000 description 1
- 230000004060 metabolic process Effects 0.000 description 1
- 108091088140 miR162 stem-loop Proteins 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 239000003147 molecular marker Substances 0.000 description 1
- 239000002636 mycotoxin Substances 0.000 description 1
- 239000002102 nanobead Substances 0.000 description 1
- 229910017604 nitric acid Inorganic materials 0.000 description 1
- 229910052757 nitrogen Inorganic materials 0.000 description 1
- 238000007899 nucleic acid hybridization Methods 0.000 description 1
- 230000031787 nutrient reservoir activity Effects 0.000 description 1
- 210000000056 organ Anatomy 0.000 description 1
- 210000003463 organelle Anatomy 0.000 description 1
- 239000000575 pesticide Substances 0.000 description 1
- 230000005097 photorespiration Effects 0.000 description 1
- 229920002647 polyamide Polymers 0.000 description 1
- 238000012257 pre-denaturation Methods 0.000 description 1
- 102000004196 processed proteins & peptides Human genes 0.000 description 1
- 210000001236 prokaryotic cell Anatomy 0.000 description 1
- 238000012175 pyrosequencing Methods 0.000 description 1
- 238000003908 quality control method Methods 0.000 description 1
- 238000003259 recombinant expression Methods 0.000 description 1
- 230000009467 reduction Effects 0.000 description 1
- 230000002829 reductive effect Effects 0.000 description 1
- 230000001172 regenerating effect Effects 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 229920002477 rna polymer Polymers 0.000 description 1
- NLJMYIDDQXHKNR-UHFFFAOYSA-K sodium citrate Chemical compound O.O.[Na+].[Na+].[Na+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O NLJMYIDDQXHKNR-UHFFFAOYSA-K 0.000 description 1
- 239000001509 sodium citrate Substances 0.000 description 1
- 239000002904 solvent Substances 0.000 description 1
- 238000009331 sowing Methods 0.000 description 1
- 230000009870 specific binding Effects 0.000 description 1
- 230000002269 spontaneous effect Effects 0.000 description 1
- 230000000087 stabilizing effect Effects 0.000 description 1
- 235000019698 starch Nutrition 0.000 description 1
- 239000008107 starch Substances 0.000 description 1
- 239000000758 substrate Substances 0.000 description 1
- 208000024891 symptom Diseases 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 230000009897 systematic effect Effects 0.000 description 1
- 230000002123 temporal effect Effects 0.000 description 1
- 231100000419 toxicity Toxicity 0.000 description 1
- 230000001988 toxicity Effects 0.000 description 1
- 230000017105 transposition Effects 0.000 description 1
- 238000010200 validation analysis Methods 0.000 description 1
- 230000009385 viral infection Effects 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
- 238000009333 weeding Methods 0.000 description 1
Classifications
-
- 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
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
- C12Q1/6895—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for plants, fungi or algae
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01G—HORTICULTURE; CULTIVATION OF VEGETABLES, FLOWERS, RICE, FRUIT, VINES, HOPS OR SEAWEED; FORESTRY; WATERING
- A01G22/00—Cultivation of specific crops or plants not otherwise provided for
- A01G22/20—Cereals
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H1/00—Processes for modifying genotypes ; Plants characterised by associated natural traits
- A01H1/02—Methods or apparatus for hybridisation; Artificial pollination ; Fertility
- A01H1/022—Genic fertility modification, e.g. apomixis
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H1/00—Processes for modifying genotypes ; Plants characterised by associated natural traits
- A01H1/04—Processes of selection involving genotypic or phenotypic markers; Methods of using phenotypic markers for selection
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N57/00—Biocides, pest repellants or attractants, or plant growth regulators containing organic phosphorus compounds
- A01N57/18—Biocides, pest repellants or attractants, or plant growth regulators containing organic phosphorus compounds having phosphorus-to-carbon bonds
- A01N57/20—Biocides, pest repellants or attractants, or plant growth regulators containing organic phosphorus compounds having phosphorus-to-carbon bonds containing acyclic or cycloaliphatic radicals
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01P—BIOCIDAL, PEST REPELLANT, PEST ATTRACTANT OR PLANT GROWTH REGULATORY ACTIVITY OF CHEMICAL COMPOUNDS OR PREPARATIONS
- A01P13/00—Herbicides; Algicides
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K10/00—Animal feeding-stuffs
- A23K10/30—Animal feeding-stuffs from material of plant origin, e.g. roots, seeds or hay; from material of fungal origin, e.g. mushrooms
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L7/00—Cereal-derived products; Malt products; Preparation or treatment thereof
- A23L7/10—Cereal-derived products
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- 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)
- C12N15/8201—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation
- C12N15/8202—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation by biological means, e.g. cell mediated or natural vector
- C12N15/8205—Agrobacterium mediated transformation
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- 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)
- C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
- C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
- C12N15/8271—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
- C12N15/8274—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for herbicide resistance
- C12N15/8277—Phosphinotricin
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- 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)
- C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
- C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
- C12N15/8271—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
- C12N15/8279—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, pathogen resistance, disease resistance
- C12N15/8286—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, pathogen resistance, disease resistance for insect resistance
-
- C—CHEMISTRY; METALLURGY
- C40—COMBINATORIAL TECHNOLOGY
- C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
- C40B40/00—Libraries per se, e.g. arrays, mixtures
- C40B40/04—Libraries containing only organic compounds
- C40B40/06—Libraries containing nucleotides or polynucleotides, or derivatives thereof
-
- 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
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/13—Plant traits
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
- Y02A40/10—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in agriculture
- Y02A40/146—Genetically Modified [GMO] plants, e.g. transgenic plants
Abstract
The invention relates to a nucleic acid sequence of a corn transformation event LG11 and a detection method, wherein the nucleic acid molecule of the corn LG11 comprises a sequence shown in SEQ ID NO. 1 or a reverse complement sequence thereof, or a sequence shown in SEQ ID NO. 2 or a reverse complement sequence thereof. The corn transformation event LG11 has the characteristics of insect resistance and glufosinate herbicide resistance and excellent agronomic characters, and the detection method can accurately and rapidly identify whether the biological sample contains the DNA molecule of the transgenic corn event LG 11.
Description
Technical Field
The invention relates to the technical field of plant biology. In particular, it relates to a nucleic acid sequence and detection method for a maize transformation event LG11, and in particular to a transgenic maize event LG11 that is resistant to insects and to the application of glufosinate herbicide and a nucleic acid sequence and detection method for detecting whether a biological sample contains a particular transgenic maize event LG 11.
Background
The entire growth period of corn is affected by a variety of pests, with corn borers being the major pests in corn production, resulting in about a 10% yield loss each year. In recent years, as global climate is warmed, the damage of corn borers is further aggravated, and meanwhile, the damage of the armyworms in a large area also occurs. Spodoptera frugiperda is a novel agricultural pest invaded into China from Yunnan in 2018, and the number of host plants is 19, wherein the occurrence area of corn accounts for 98.1% of the total occurrence area of crops.
The Cry1Ab protein was developed by Monsanto company for the first time into insect-resistant corn MON810, aiming at improving the resistance to wild rod borer (Ositrinia species) pests such as Asiatic Corn Borer (ACB). Vip3Aa is applied to transgenic corn MIR162 by the company of Santa Cladonia for the first time, and has remarkable control effect on Agrotis ypsilon, corn earmoth Helicoverpa armigera, bean Bai Longqie root worm Loxagrotis albicosta and spodoptera frugiperda Spodoptera frugiperda. The M2cryAb-Vip3A protein is formed by combining main domains of Cry1Ab and Vip3Aa proteins by an artificial synthesis method. The main advantages of the M2cryAb-vip3A protein are: (1) Expanding the insect resistance spectrum, and simultaneously containing the functional domains of two proteins, and expecting to have resistance to corn borer, cotton bollworm, armyworm and spodoptera frugiperda; (2) The method is favorable for the resistance management of target pests, and a large number of researches show that Vip3 has high insecticidal activity on various lepidoptera pests, expands the insecticidal spectrum of Bt proteins, meanwhile, the insecticidal mechanisms of Vip3 toxins and Cry toxins are different, no homology exists in evolution, and the probability of interactive resistance of the pests on the two toxins is small; (3) Reducing the level of mycotoxin in corn ears and kernels and improving the quality of corn kernels; (4) The protein sequence does not contain allergen sequence, and has biological safety while maintaining high-efficiency insecticidal activity.
Weeds in the field compete with crops for water, fertilizer, light energy and growth space, and the yield and quality of the crops are directly affected. Meanwhile, a plurality of weeds are intermediate hosts of crop pathogenic bacteria and pests, and are one of important biological limiting factors for crop yield increase. Therefore, effective control of weeds in the field is one of the important measures for promoting grain yield increase. In addition, the scaling up and mechanization of agricultural planting is a predictable trend, which makes traditional manual weeding approaches impractical. The popularization and use of the herbicide can greatly reduce the labor for cotton field management and labor intensity. The new herbicide product with high efficiency, low toxicity and no residue is developed, and the cost is high, the time consumption is long and the difficulty is high. This difficulty can be overcome by breeding maize with resistance to biocidal herbicides by transgenic technology. The weed problem can be effectively solved by spraying for 1-2 times in the corn growing period, and the dosage and the input cost of herbicide are reduced. Therefore, the herbicide-resistant transgenic corn has very wide application value and market potential.
The invention connects the insect-resistant gene expression cassette and herbicide-resistant expression cassette in series, so that the expression cassette can be efficiently expressed in transgenic corn, has insect-resistant and herbicide-resistant properties, and further enhances the application and economic value of the product.
Expression of exogenous genes in plants is known to be affected by their chromosomal location, possibly due to the proximity of chromatin structures (e.g., heterochromatin) or transcriptional regulatory elements (e.g., enhancers) to the integration site. For this reason, it is often necessary to screen a large number of events to make it possible to identify events that can be commercialized (i.e., events in which the introduced target gene is optimally expressed). For example, it has been observed in plants and other organisms that the expression level of the introduced gene may vary greatly between events; there may also be differences in the spatial or temporal patterns of expression, such as differences in the relative expression of transgenes between different plant tissues, which differences may be manifested in actual expression patterns that are inconsistent with the expression patterns expected from the transcriptional regulatory elements in the introduced gene construct, resulting in differences in the performance of the transformation event. Thus, it is often desirable to generate hundreds or thousands of different events and screen those events for a single event having transgene expression levels and patterns that are expected for commercialization purposes. Events with expected transgene expression levels and expression patterns can be used to introgress transgenes into other genetic backgrounds by sexual outcrossing using conventional breeding methods. The progeny produced by this crossing retain the transgene expression characteristics of the original transformation event. The use of such a strategy can ensure reliable gene expression in many varieties that are well suited to the growth conditions of the locus. Therefore, more transformation events need to be identified and screened for superior transformation events with superior overall trait performance and commercial prospects.
It would be beneficial to be able to detect the presence of a particular event to determine whether the progeny of a sexual cross contain a gene of interest. In addition, methods of detecting specific events will also help to comply with relevant regulations, such as the need for formal approval and marking of foods derived from recombinant crops prior to their being put on the market. It is possible to detect the presence of the transgene by any well known polynucleotide detection method, such as the Polymerase Chain Reaction (PCR). These detection methods are generally focused on commonly used genetic elements such as promoters, terminators, marker genes, and the like. Thus, unless the sequence of chromosomal DNA adjacent to the inserted transgenic DNA ("flanking DNA") is known, such a method as described above cannot be used to distinguish between different events, particularly those generated with the same DNA construct. Therefore, it is common today to identify a transgene specific event by PCR using a pair of primers spanning the junction of the inserted transgene and flanking DNA, specifically a first primer comprising the flanking sequence and a second primer comprising the inserted sequence.
Disclosure of Invention
The invention aims to provide a corn transformation event with excellent insect and herbicide resistance and excellent agronomic characteristics, a nucleic acid sequence for detecting LG11 of corn and a detection method thereof. The transgenic corn event LG11 has good insect resistance and better tolerance to glufosinate herbicide, and the detection method can accurately and quickly identify whether the biological sample contains DNA molecules of the specific transgenic corn event LG 11.
To achieve the above object, the present invention uses pCAMBIA3300+m2cryAb-vip3A expression vector to transform corn HiIIB by agrobacterium-mediated method to obtain more than 400 positive transformants, and after molecular detection, each generation uses corn inbred line Chang 7-2 as recurrent parent to make backcross to obtain BC 5 F 2 Transgenic corn seeds LG 01-LG 20. Through the identification of the insect resistance and herbicide resistance traits, the transformation event LG11 is a transformant with excellent herbicide resistance and insect resistance performance and best agronomic traits, and can be used for improving the insect resistance and herbicide resistance traits of corn.
In order to characterize the identity of LG11, the invention provides a nucleic acid molecule comprising the sequence shown in SEQ ID NO. 1 and/or SEQ ID NO. 2, or the reverse complement thereof.
Further, the nucleic acid sequence comprises the sequence shown in SEQ ID NO. 3 and/or SEQ ID NO. 4, or the reverse complement thereof.
Further, the nucleic acid sequence comprises the sequence shown in SEQ ID NO. 6 and/or SEQ ID NO. 7, or the reverse complement thereof.
Further, the nucleic acid sequence comprises the sequence shown in SEQ ID NO. 5 or the reverse complement thereof.
The invention also provides a probe for detecting corn transformation events, which is characterized by comprising a sequence shown in SEQ ID NO. 1 or SEQ ID NO. 2 or SEQ ID NO. 3 or SEQ ID NO. 4 or SEQ ID NO. 6 or SEQ ID NO. 7 or a fragment or variant or reverse complement thereof.
The invention also provides a primer pair for detecting corn transformation events, which is characterized in that the amplification product of the primer pair comprises a sequence shown as SEQ ID NO. 1 or SEQ ID NO. 2 or SEQ ID NO. 3 or SEQ ID NO. 4 or SEQ ID NO. 6 or SEQ ID NO. 7 or a fragment or variant or reverse complement thereof.
In some embodiments, the primer pair is the sequences set forth in SEQ ID NO. 8 and SEQ ID NO. 9; or SEQ ID NO. 10 and SEQ ID NO. 11.
The invention also provides a kit or microarray for detecting maize transformation events, characterized in that the kit or microarray comprises the probes and/or primer pairs described above.
The invention also provides a method for detecting a maize transformation event, which is characterized by comprising the step of detecting whether the transformation event exists in a sample to be detected by using the probe or the primer pair or the probe and primer pair or the kit or the microarray.
The invention also provides a method for breeding corn, which is characterized by comprising the following steps:
1) Obtaining corn comprising the nucleic acid molecule described above;
2) Obtaining a maize plant, seed, plant cell, progeny plant or plant part from the maize obtained in step 1) by pollen culture, unfertilized embryo culture, doubling culture, cell culture, tissue culture, selfing or crossing or a combination thereof; optionally, the composition may be in the form of a gel,
3) Identifying the progeny plants obtained in step 2) for insect resistance and/or herbicide resistance and detecting the presence or absence of said transformation event using the method described above.
Further, the present invention also provides products, including food, feed or industrial materials, made from the maize plants, seeds, plant cells, progeny plants or plant parts obtained by the above method.
The SEQ ID NO. 1 is a sequence of 22 nucleotides in length, which is positioned near the insertion junction at the 5 '-end of the insertion sequence in the transgenic corn event LG11, the SEQ ID NO. 1 spans the left flank genome DNA sequence of the corn insertion site and the DNA sequence at the 5' -end of the left boundary of the insertion sequence, and the existence of the transgenic corn event LG11 can be identified by comprising the SEQ ID NO. 1 or the reverse complement thereof. The SEQ ID NO. 2 is a sequence of 22 nucleotides in length near the insertion junction at the 3 '-end of the insertion sequence in the transgenic corn event LG11, the SEQ ID NO. 2 spans the DNA sequence at the 3' -end of the right border of the insertion sequence and the right flank genomic DNA sequence of the corn insertion site, and the existence of the transgenic corn event LG11 can be identified by comprising the SEQ ID NO. 2 or the reverse complement thereof.
In the present invention, the nucleic acid sequence may be at least 11 or more contiguous polynucleotides (first nucleic acid sequence) of any portion of the transgene insert sequence in the SEQ ID NO. 3 or reverse complement thereof, or at least 11 or more contiguous polynucleotides (second nucleic acid sequence) of any portion of the 5' left-flanking maize genomic DNA region in the SEQ ID NO. 3 or reverse complement thereof. The nucleic acid sequence may further be homologous or reverse complementary to a portion of the SEQ ID NO. 3 comprising the complete SEQ ID NO. 1 or SEQ ID NO. 6. When the first nucleic acid sequence and the second nucleic acid sequence are used together, these nucleic acid sequences comprise a pair of DNA primers in a DNA amplification method that produces an amplification product. The presence of transgenic maize event LG11 or its progeny can be diagnosed when the amplification product produced in the DNA amplification method using the DNA primer pair is an amplification product comprising SEQ ID NO:1 or SEQ ID NO:3 or SEQ ID NO:6 or its reverse complement.
The SEQ ID NO. 3 is a sequence with the length of 517 nucleotides, which is positioned near the insertion junction at the 5 '-end of the insertion sequence in the transgenic corn event LG11, the SEQ ID NO. 3 consists of 280-nucleotide corn left-side wing genomic DNA sequence (nucleotides 1-280 of SEQ ID NO. 3) and 237-nucleotide glufosinate-resistant first expression cassette 5' -end DNA sequence (nucleotides 281-517 of SEQ ID NO. 3), and the existence of the transgenic corn event LG11 can be identified by the SEQ ID NO. 3 or the reverse complement sequence thereof.
The nucleic acid sequence may be at least 11 or more contiguous polynucleotides (third nucleic acid sequence) of any portion of the transgene insert sequence in the SEQ ID NO. 4 or reverse complement thereof, or at least 11 or more contiguous polynucleotides (fourth nucleic acid sequence) of any portion of the 3' right-flanking maize genomic DNA region in the SEQ ID NO. 4 or reverse complement thereof. The nucleic acid sequence may further be homologous or reverse complementary to a portion of the SEQ ID NO. 4 comprising the complete SEQ ID NO. 2 or SEQ ID NO. 7. When the third nucleic acid sequence and the fourth nucleic acid sequence are used together, these nucleic acid sequences comprise a set of DNA primers in a DNA amplification method that produces an amplification product. The presence of transgenic maize event LG11 or its progeny can be diagnosed when the amplification product produced in the DNA amplification method using the DNA primer pair is an amplification product comprising SEQ ID NO:2 or SEQ ID NO:4 or SEQ ID NO:7 or its reverse complement.
The SEQ ID NO. 4 is a 1631 nucleotide long sequence located near the insertion junction at the 3 'end of the insertion sequence in transgenic corn event LG11, the SEQ ID NO. 4 consists of the 3' end DNA sequence of the second expression cassette of the 575 nucleotide insect-resistant gene (nucleotides 1-575 of SEQ ID NO. 4), the 736 nucleotide pCAMBIA3300+m2cryAb-vip3A construct right border DNA sequence (nucleotides 576-1311 of SEQ ID NO. 4) and the 320 nucleotide corn integration site right flank genomic DNA sequence (nucleotides 1312-1631 of SEQ ID NO. 4), comprising the SEQ ID NO. 4 or its reverse complement can be identified as the presence of transgenic corn event LG 11.
The sequence SEQ ID NO. 5 is a sequence of 7744 nucleotides in length characterizing transgenic maize event LG11, which specifically contains the genome and genetic elements as shown in Table 1. The presence of transgenic maize event LG11 can be identified by comprising said SEQ ID NO 5 or its reverse complement.
Table 1 genome and genetic elements comprised by SEQ ID NO:5
1: the unit bp.
It is well known to those skilled in the art that the first and second nucleic acid sequences or the third and fourth nucleic acid sequences need not consist of only DNA, but may also include RNA, a mixture of DNA and RNA, or a combination of DNA, RNA or other nucleotides or analogues thereof that do not serve as templates for one or more polymerases. Furthermore, the probe or primer of the invention should be at least about 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 or 22 consecutive nucleotides in length, which may be selected from the group consisting of the nucleotides set forth in SEQ ID NO. 1, SEQ ID NO. 2, SEQ ID NO. 3, SEQ ID NO. 4, SEQ ID NO. 5, SEQ ID NO. 6, SEQ ID NO. 7, SEQ ID NO. 8, SEQ ID NO. 9, SEQ ID NO. 10 or SEQ ID NO. 11. When selected from the group consisting of the nucleotides set forth in SEQ ID NO. 3, SEQ ID NO. 4, SEQ ID NO. 5, SEQ ID NO. 6 and SEQ ID NO. 7, the primer may be at least about 21 to about 50 or more consecutive nucleotides in length.
The present invention also provides a method of protecting a maize plant from injury caused by a herbicide comprising applying to a field in which at least one transgenic maize plant comprising in its genome the sequence of SEQ ID NO. 1, the nucleic acid sequences at positions 281-7424 and SEQ ID NO. 2 in that order or the genome of the transgenic maize plant comprising SEQ ID NO. 5, an effective dose of a glufosinate herbicide; the transgenic corn plants have tolerance to glufosinate herbicide.
The present invention also provides a method of protecting a maize plant from insect infestation comprising providing at least one transgenic maize plant cell comprising in its genome the nucleic acid sequence of SEQ ID NO. 1, SEQ ID NO. 5, positions 281-7424 and SEQ ID NO. 2 in that order, or comprising SEQ ID NO. 5 in its genome, in the diet of a target insect; target insects that ingest the cells of the transgenic corn plant are inhibited from further ingestion of the corn plant.
In the nucleic acid sequences and methods of the present invention for detecting corn plants, the following definitions and methods may better define the invention and direct one of ordinary skill in the art to practice the invention, unless otherwise indicated, terms are understood according to one of ordinary skill in the art's conventional usage.
The term "maize" refers to maize (Zea mays) and includes all plant varieties that can be mated to maize, including wild maize varieties.
The term "comprising" means "including but not limited to.
The term "plant" includes whole plants, plant cells, plant organs, plant protoplasts, plant cell tissue cultures from which plants can be regenerated, plant calli, plant clumps (plant cones), and intact plant cells in plants or plant parts, such as embryos, pollen, ovules, seeds, leaves, flowers, branches, fruits, stems, roots, root tips, anthers, and the like. It is to be understood that parts of transgenic plants within the scope of the present invention include, but are not limited to, plant cells, protoplasts, tissues, calli, embryos and flowers, stems, fruits, leaves and roots, which are derived from transgenic plants or their progeny which have been previously transformed with the DNA molecules of the present invention and thus at least partially consist of the transgenic cells.
The term "gene" refers to a nucleic acid fragment that expresses a particular protein, including regulatory sequences preceding (5 'non-coding sequences) and regulatory sequences following (3' non-coding sequences) the coding sequences. "native gene" refers to a gene that is found naturally to have its own regulatory sequences. By "chimeric gene" is meant any gene that is not a native gene, comprising regulatory and coding sequences found in a non-native manner. "endogenous gene" refers to a native gene that is located in its natural location in the genome of an organism. "exogenous gene" is a foreign gene that is present in the genome of an organism and that is not originally present, and also refers to a gene that has been introduced into a recipient cell by a transgenic procedure. The exogenous gene may comprise a native gene or chimeric gene inserted into a non-native organism. A "transgene" is a gene that has been introduced into the genome by a transformation procedure. The site in the plant genome where the recombinant DNA has been inserted may be referred to as an "insertion site" or "target site".
"flanking DNA" may comprise genomic or foreign (heterologous) DNA introduced by a transformation process, such as fragments associated with a transformation event, naturally occurring in an organism such as a plant. Thus, flanking DNA may include a combination of native and foreign DNA. In the present invention, a "flanking region" or "flanking sequence" or "genomic border region" or "genomic border sequence" refers to a sequence of at least 3, 5, 10, 11, 15, 20, 50, 100, 200, 300, 400, 1000, 1500, 2000, 2500, or 5000 base pairs or more that is immediately upstream or downstream of and adjacent to the initial exogenous inserted DNA molecule. When this flanking region is located upstream, it may also be referred to as a "left border flanking" or "5 'genomic flanking region" or "genomic 5' flanking sequence" or the like. When this flanking region is located downstream, it may also be referred to as a "right border flanking" or a "3 'genomic flanking region" or a "genomic 3' flanking sequence", etc.
Transformation procedures that cause random integration of exogenous DNA will result in transformation events that contain different flanking regions that each transformation event specifically contains. When recombinant DNA is introduced into plants by conventional hybridization, its flanking regions are generally not altered. Transformation events will also contain unique junctions between segments of heterologous insert DNA and genomic DNA or between two segments of heterologous DNA. "ligation" is the point at which two specific DNA fragments are ligated. For example, the junction exists where the insert DNA joins the flanking DNA. The junction point is also present in transformed organisms, where the two DNA fragments are joined together in a manner that modifies what is found in the native organism. "adapter DNA" refers to DNA that contains an adapter.
The present invention provides a transgenic corn event designated LG11 and progeny thereof, the transgenic corn event LG11 being a corn LG11 comprising plants and seeds of the transgenic corn event LG11 and plant cells or regenerable parts thereof, plant parts of the transgenic corn event LG11 including, but not limited to, cells, pollen, ovules, flowers, shoots, roots, stems, leaves and products from the corn LG11 such as cotton seed, cottonseed oil, cotton coat, cotton quilt, cotton batting, cotton cloth and biomass left in the corn crop field.
The transgenic corn event LG11 of the invention comprises a DNA construct that, when expressed in a plant cell, confers tolerance to insect and/or glufosinate herbicides to the transgenic corn event LG 11. The DNA construct comprises an expression cassette comprising a suitable promoter for expression in a plant operably linked to an M2cryAb-VIP3A gene having insect resistance and a suitable polyadenylation signal sequence, the nucleic acid sequence of the M2cryAb-VIP3A protein being capable of increasing maize insect resistance. The DNA construct comprises a further expression cassette comprising a suitable promoter for expression in a plant operably linked to a gene bar encoding Phosphinothricin Acetyl Transferase (PAT), the nucleic acid sequence of the PAT protein being tolerant to glufosinate herbicide and a suitable polyadenylation signal sequence. Further, the promoter may be a suitable promoter isolated from plants, including constitutive, inducible, and/or tissue-specific promoters, including, but not limited to, the cauliflower mosaic virus (CaMV) 35S promoter, the Figwort Mosaic Virus (FMV) 35S promoter, the Ubiquitin protein (Ubiquitin) promoter, the Actin (action) promoter, the agrobacterium (Agrobacterium tumefaciens) nopaline synthase (NOS) promoter, the octopine synthase (OCS) promoter, the night yellow leaf curly virus (cestron) promoter, the tuber storage protein (Patatin) promoter, the ribulose-1, 5-bisphosphate carboxylase/oxygenase (rusco) promoter, the Glutathione S Transferase (GST) promoter, the E9 promoter, the GOS promoter, the alcA/alcR promoter, the agrobacterium (Agrobacterium rhizogenes) roller promoter, and the arabidopsis (Arabidopsis thaliana) promoter. The polyadenylation signal sequence may be a suitable polyadenylation signal sequence for functioning in plants, including, but not limited to, polyadenylation signal sequences derived from the Agrobacterium tumefaciens (Agrobacterium tumefaciens) nopaline synthase (NOS) gene, from the cauliflower mosaic virus (CaMV) 35S terminator, from the protease inhibitor II (PIN II) gene, and from the alpha-tubulin (alpha-tubulin) gene.
In addition, the expression cassette may also include other genetic elements including, but not limited to, enhancers and signal peptides/transit peptides. The enhancer may enhance the expression level of a gene, including, but not limited to, tobacco Etch Virus (TEV) translational activator, caMV35S enhancer, and FMV35S enhancer. The signal peptide/transit peptide may direct the transit of the EPSPS protein to a specific organelle or compartment outside or inside the cell, for example, targeting to the chloroplast using a sequence encoding a chloroplast transit peptide, or targeting to the endoplasmic reticulum using a 'KDEL' retention sequence.
The 'glufosinate' refers to a nonselective, broad-spectrum, efficient and low-toxicity organophosphorus herbicide which strongly inhibits the activity of the amino acid biosynthetic enzyme, glutamine synthetase (Glutamine Synthetase, GS), of bacteria and plants. GS plays an important role in plant ammonia assimilation and regulation of ammonia metabolism, and is the only detoxification enzyme in plants, which can detoxify ammonia released by nitric acid reduction, amino acid degradation and photorespiration. Treatment with a "glufosinate herbicide" refers to treatment with any herbicide formulation containing glufosinate. The choice of the rate of use of a certain glufosinate formulation in order to achieve an effective biological dose is not beyond the skills of the average agronomic technician. Treatment of a field containing plant material derived from transgenic corn event LG11 with any herbicide formulation that contains glufosinate will control weed growth in the field and not affect the growth or pest resistance of plant material derived from transgenic corn event LG 11.
The DNA construct is introduced into a plant using transformation methods including, but not limited to, agrobacterium (Agrobacterium) -mediated transformation, gene gun transformation, and pollen tube channel transformation.
The agrobacterium-mediated transformation method is a common method for plant transformation. The foreign DNA to be introduced into the plant is cloned between the left and right border consensus sequences of the vector, i.e., the T-DNA region. The vector is transformed into an agrobacterium cell, which is subsequently used to infect plant tissue, and the T-DNA region of the vector comprising exogenous DNA is inserted into the plant genome. After transformation, the transgenic plants must be regenerated from the transformed plant tissue and offspring with the exogenous DNA selected using appropriate markers.
A DNA construct is a combination of DNA molecules that are linked to one another to provide one or more expression cassettes. The DNA construct is preferably a plasmid capable of self replication in bacterial cells and containing various restriction enzyme sites for the introduction of DNA molecules providing functional genetic elements, i.e. promoters, introns, leader sequences, coding sequences, 3' terminator regions and other sequences. The expression cassette contained in the DNA construct includes the genetic elements necessary to provide for transcription of messenger RNA, and can be designed for expression in prokaryotic or eukaryotic cells. The expression cassette of the invention is designed to be expressed most preferably in plant cells.
A transgenic "event" is obtained by transforming a plant cell with a heterologous DNA construct, i.e., comprising at least one nucleic acid expression cassette containing a gene of interest, inserting into the plant genome by transgenic means to produce a plant population, regenerating the plant population, and selecting a particular plant having the characteristics of being inserted into a particular genomic locus. The term "event" refers to both the original transformation event comprising heterologous DNA and the progeny of the transformation event. The term "event" also refers to the progeny of a sexual cross between a transformation event and other species of individuals containing heterologous DNA, even after repeated backcrosses with a backcross parent, the inserted DNA and flanking genomic DNA from the transformation event parent are present at the same chromosomal location in the hybrid progeny. The term "event" also refers to a DNA sequence from an original transformation event that comprises an inserted DNA and flanking genomic sequences immediately adjacent to the inserted DNA, which DNA sequence is expected to be transferred into progeny resulting from sexual crossing of a parental line containing the inserted DNA (e.g., progeny resulting from the original transformation event and its selfing) with a parental line not containing the inserted DNA, and which progeny received the inserted DNA comprising the gene of interest.
"recombinant" in the context of the present invention refers to forms of DNA and/or proteins and/or organisms that are not normally found in nature and are therefore produced by manual intervention. Such manual intervention may result in recombinant DNA molecules and/or recombinant plants. The "recombinant DNA molecule" is obtained by artificially combining two otherwise isolated sequence segments, for example by chemical synthesis or by manipulation of isolated nucleic acid segments by genetic engineering techniques. Techniques for performing nucleic acid manipulations are well known.
The term "transgene" includes any cell, cell line, callus, tissue, plant part or plant, the genotype of which is altered by the presence of a heterologous nucleic acid, and includes the transgene originally so altered as well as progeny individuals produced from the original transgene by sexual crosses or asexual propagation. In the present invention, the term "transgene" does not include genomic (chromosomal or extrachromosomal) alterations by conventional plant breeding methods or naturally occurring events such as random allofertilisation, non-recombinant viral infection, non-recombinant bacterial transformation, non-recombinant transposition or spontaneous mutation.
By "heterologous" in the present invention is meant that the first molecule is not normally found in combination with the second molecule in nature. For example, a molecule may originate from a first species and be inserted into the genome of a second species. Such molecules are thus heterologous to the host and are artificially introduced into the genome of the host cell.
A transgenic corn event LG11 having insect-resistant properties and tolerance to glufosinate herbicide is grown by the steps of: first sexually crossing a first parent corn plant consisting of a corn plant grown from a transgenic corn event LG11 and its progeny obtained by transformation with an expression cassette of the invention that is insect-resistant and tolerant to a glufosinate herbicide, with a second parent corn plant lacking insect-resistant properties or tolerant to a glufosinate herbicide, thereby producing a multiplicity of first generation progeny plants; progeny plants that are tolerant to the glufosinate herbicide are then selected and maize plants that are tolerant to the glufosinate herbicide can be grown. These steps may further include backcrossing the insect-and glufosinate-tolerant progeny plant with the second parent corn plant or the third parent corn plant, and then selecting the progeny by application with the glufosinate herbicide or by identification of a molecular marker associated with the trait (e.g., a DNA molecule comprising the junction site identified at the 5 'and 3' ends of the insertion sequence in transgenic corn event LG 11) to produce a corn plant with insect-resistant properties and tolerance to the glufosinate herbicide.
It will also be appreciated that two different transgenic plants can also be crossed to produce offspring containing two independent, separately added exogenous genes. Selfing of appropriate offspring can result in offspring plants that are homozygous for both added exogenous genes. Backcrossing of parent plants and outcrossing with non-transgenic plants as previously described are also contemplated, as are asexual propagation.
The term "probe" is an isolated nucleic acid molecule to which a conventional detectable label or reporter molecule, e.g., a radioisotope, ligand, chemiluminescent agent, or enzyme, is attached. Such a probe is complementary to one strand of the target nucleic acid, and in the present invention, the probe is complementary to one strand of DNA from the genome of transgenic maize event LG11, whether the genomic DNA is from transgenic maize event LG11 or seed or a plant or seed or extract derived from transgenic maize event LG 11. Probes of the present invention include not only deoxyribonucleic acid or ribonucleic acid, but also polyamides and other probe materials that specifically bind to a target DNA sequence and can be used to detect the presence of the target DNA sequence.
The term "primer" is an isolated nucleic acid molecule that binds to a complementary target DNA strand by nucleic acid hybridization, anneals to form a hybrid between the primer and the target DNA strand, and then extends along the target DNA strand under the action of a polymerase (e.g., DNA polymerase). The primer pairs of the invention relate to their use in the amplification of a target nucleic acid sequence, for example, by the Polymerase Chain Reaction (PCR) or other conventional nucleic acid amplification methods. The length of the primer is generally 11 polynucleotides or more, preferably 18 polynucleotides or more, more preferably 24 polynucleotides or more, and most preferably 30 polynucleotides or more. Such primers hybridize specifically to the target sequence under highly stringent hybridization conditions. Although primers which are different from the target DNA sequence and which maintain the ability to hybridize to the target DNA sequence can be designed by conventional methods, it is preferred that the primers of the present invention have complete DNA sequence identity to consecutive nucleic acids of the target sequence.
As used herein, "kit" or "microarray" refers to a set of reagents or chips for the purpose of identification and/or detection of maize transformation events in a biological sample. For the purpose of quality control (e.g. purity of seed lot), detection of events in or containing plant material or materials derived from plant material, such as but not limited to food or feed products, kits or chips may be used and their components may be specifically tailored.
Primers and probes based on flanking genomic DNA and insert sequences of the invention can be determined by conventional methods, for example, by isolating the corresponding DNA molecule from plant material derived from transgenic corn event LG11 and determining the nucleic acid sequence of the DNA molecule. The DNA molecule comprises a transgene insert and maize genomic flanking regions, and fragments of the DNA molecule may be used as primers and probes.
The primers and probes of the invention hybridize to the target DNA sequence under stringent conditions. Any conventional amplification method can be used to identify the presence of DNA in the sample derived from transgenic corn event LG 11. The nucleic acid molecule or fragment thereof is capable of specifically hybridizing to other nucleic acid molecules under certain conditions. As used herein, two nucleic acid molecules can be said to specifically hybridize to each other if they are capable of forming antiparallel double-stranded nucleic acid structures. Two nucleic acid molecules are said to be "complements" of one nucleic acid molecule if they exhibit complete complementarity. As used herein, a nucleic acid molecule is said to exhibit "complete complementarity" when each nucleotide of the two molecules is complementary to a corresponding nucleotide of the other nucleic acid molecule. Two nucleic acid molecules are said to be "minimally complementary" if they are capable of hybridizing to each other with sufficient stability such that they anneal and bind to each other under at least conventional "low stringency" conditions. Similarly, two nucleic acid molecules are said to have "complementarity" if they are capable of hybridizing to each other with sufficient stability such that they anneal and bind to each other under conventional "highly stringent" conditions. Deviations from complete complementarity are permissible provided that such deviations do not completely prevent the formation of double-stranded structures by the two molecules. In order to enable a nucleic acid molecule to act as a primer or probe, it is only necessary to ensure sufficient complementarity in sequence to allow the formation of a stable double-stranded structure at the particular solvent and salt concentration employed.
As used herein, a substantially homologous sequence is a nucleic acid molecule that is capable of specifically hybridizing under highly stringent conditions to the complementary strand of a matching other nucleic acid molecule. Suitable stringent conditions for promoting DNA hybridization, for example, treatment with 6.0 XSSC/sodium citrate (SSC) at about 45℃followed by washing with 2.0 XSSC at 50℃are well known to those skilled in the art. For example, the salt concentration in the washing step may be selected from about 2.0 XSSC at low stringency conditions, about 0.2 XSSC at 50℃to high stringency conditions, about 50 ℃. In addition, the temperature conditions in the washing step may be raised from about 22 ℃ at room temperature under low stringency conditions to about 65 ℃ under high stringency conditions. The temperature conditions and salt concentration may both be varied, or one may remain unchanged while the other variable is varied. Preferably, a nucleic acid molecule of the invention can specifically hybridize under moderately stringent conditions, e.g., at about 2.0 XSSC and about 65℃to one or more of SEQ ID NO. 1, SEQ ID NO. 2, SEQ ID NO. 3, SEQ ID NO. 4, SEQ ID NO. 5, SEQ ID NO. 6 and SEQ ID NO. 7, or to a complement thereof, or to any fragment of the foregoing. More preferably, a nucleic acid molecule of the invention hybridizes specifically under highly stringent conditions to one or more of SEQ ID NO. 1, SEQ ID NO. 2, SEQ ID NO. 3, SEQ ID NO. 4, SEQ ID NO. 5, SEQ ID NO. 6 and SEQ ID NO. 7 or to the complement thereof, or to any fragment of the above sequences. In the present invention, preferred marker nucleic acid molecules have SEQ ID NO. 1, SEQ ID NO. 2, SEQ ID NO. 3, SEQ ID NO. 4, SEQ ID NO. 6 or SEQ ID NO. 7 or the complement thereof, or any fragment of the above sequences.
Another preferred marker nucleic acid molecule of the invention has 80% to 100% or 90% to 100% sequence identity with SEQ ID NO. 1, SEQ ID NO. 2, SEQ ID NO. 3, SEQ ID NO. 4, SEQ ID NO. 6 or SEQ ID NO. 7 or the complement thereof, or any fragment of the above sequences. SEQ ID NO. 1, SEQ ID NO. 2, SEQ ID NO. 3, SEQ ID NO. 4, SEQ ID NO. 6 and SEQ ID NO. 7 can be used as markers in plant breeding methods to identify offspring of genetic crosses. Hybridization of the probe to the target DNA molecule may be detected by any method known to those skilled in the art, including, but not limited to, fluorescent labels, radiolabels, antibody-based labels, and chemiluminescent labels.
With respect to amplification (e.g., by PCR) of a target nucleic acid sequence using specific amplification primers, "stringent conditions" refer to conditions that allow hybridization of only the primer pair to the target nucleic acid sequence in a DNA thermal amplification reaction, and primers having a wild-type sequence (or its complement) corresponding to the target nucleic acid sequence are capable of binding to the target nucleic acid sequence and preferably produce a unique amplification product, i.e., an amplicon.
The term "specific binding (target sequence)" means that the primer hybridizes only to the target sequence in a sample containing the target sequence under stringent hybridization conditions.
As used herein, "amplified DNA" or "amplicon" refers to the nucleic acid amplification product of a target nucleic acid sequence that is part of a nucleic acid template. For example, to determine whether a maize plant is produced by sexual hybridization from a maize sample containing the transgenic maize event LG11 of the invention, or whether a maize sample collected from a field contains the transgenic maize event LG11, or whether a maize extract, such as cotton, cotton seed oil, contains the transgenic maize event LG11, DNA extracted from a maize plant tissue sample or extract can be amplified by a nucleic acid amplification method using a primer pair to produce an amplicon that is diagnostic for the presence of DNA of the transgenic maize event LG 11. The primer pair includes a first primer derived from a flanking sequence in the genome of the plant adjacent to the insertion site of the inserted foreign DNA, and a second primer derived from the inserted foreign DNA. The amplicon has a length and sequence that is also diagnostic for the transgenic corn event LG 11.
The length of the amplicon may range from the combined length of the primer pair plus one nucleotide base pair, preferably plus about fifty nucleotide base pairs, more preferably plus about two hundred fifty nucleotide base pairs, and most preferably plus about four hundred fifty nucleotide base pairs or more.
Alternatively, the primer pair may be derived from flanking genomic sequences flanking the inserted DNA to produce an amplicon comprising the entire inserted nucleotide sequence. One of the primer pairs derived from the plant genomic sequence may be located at a distance from the inserted DNA sequence that may range from one nucleotide base pair to about twenty thousand nucleotide base pairs. The use of the term "amplicon" specifically excludes primer dimers formed in the DNA thermal amplification reaction.
The nucleic acid amplification reaction may be accomplished by any nucleic acid amplification reaction method known in the art, including the Polymerase Chain Reaction (PCR). Various methods of nucleic acid amplification are well known to those skilled in the art. PCR amplification methods have been developed to amplify 22kb genomic DNA and 42kb phage DNA. These methods, as well as other DNA amplification methods in the art, may be used in the present invention. The inserted exogenous DNA sequence and flanking DNA sequences from transgenic corn event LG11 can be amplified from the genome of transgenic corn event LG11 by using the provided primer sequences, and standard DNA sequencing can be performed on the PCR amplicon or cloned DNA after amplification.
DNA detection kits based on DNA amplification methods contain DNA primer molecules that hybridize specifically to the target DNA under appropriate reaction conditions and amplify the diagnostic amplicon. The kit may provide agarose gel-based detection methods or a number of methods known in the art for detecting diagnostic amplicons. Kits comprising DNA primers homologous or reverse complementary to any portion of the maize genomic region of SEQ ID NO. 3 or SEQ ID NO. 4 and homologous or reverse complementary to any portion of the transgene insertion region of SEQ ID NO. 5 are provided by the invention. In particular, primer pairs identified as useful in DNA amplification methods are SEQ ID NO. 8 and SEQ ID NO. 9, which amplify a diagnostic amplicon homologous to a portion of the 5' transgene/genomic region of transgenic maize event LG11, wherein the amplicon comprises SEQ ID NO. 1. Primer pairs useful in DNA amplification methods are also identified as comprising SEQ ID NO. 10 and SEQ ID NO. 11, which amplify a diagnostic amplicon homologous to a portion of the 3' transgene/genomic region of transgenic maize event LG11, wherein the amplicon comprises SEQ ID NO. 2. Other DNA molecules used as DNA primers may be selected from SEQ ID NO. 5.
Amplicons produced by these methods can be detected by a variety of techniques. One of the methods is Genetic Bit Analysis, which designs a DNA oligonucleotide strand that spans the insert DNA sequence and adjacent flanking genomic DNA sequences. The oligonucleotide strand is immobilized in a microwell of a microwell plate, and after PCR amplification of the target region (using one primer in each of the insert sequence and adjacent flanking genomic sequences), the single-stranded PCR product can hybridize to the immobilized oligonucleotide strand and serve as a template for a single base extension reaction using DNA polymerase and ddNTPs specifically labeled for the next desired base. The results may be obtained by fluorescence or ELISA-like methods. The signal represents the presence of an insertion/flanking sequence, which indicates that the amplification, hybridization and single base extension reactions were successful.
Another method is Pyrosequencing technology. The method contemplates an oligonucleotide strand spanning the insertion DNA sequence and adjacent genomic DNA binding sites. The oligonucleotide strand and the single stranded PCR product of the target region (using one primer in each of the insert sequence and adjacent flanking genomic sequences) are hybridized and then incubated with DNA polymerase, ATP, sulfurylase, luciferase, apyrase, adenosine-5' -phosphosulfate and luciferin. dNTPs are added separately and the resulting optical signal is measured. The optical signal represents the presence of an insertion/flanking sequence, which indicates that amplification, hybridization, and single base or multiple base extension reactions were successful.
Fluorescence polarization is also one method that can be used to detect the amplicons of the invention (Chen X, levine L, and Kwok PY. Fluorescence polarization in homogeneous nucleic acid analysis [ J ]. Genome Res,1999,9 (5): 492-8.). The use of this method requires the design of an oligonucleotide strand spanning the insertion DNA sequence and adjacent genomic DNA binding sites. The oligonucleotide strand is hybridized to a single stranded PCR product of the target region (using one primer in each of the insert sequence and adjacent flanking genomic sequences) and then incubated with DNA polymerase and a fluorescent labeled ddNTP. Single base extension will result in insertion of ddNTP. Such an insertion can measure the change in its polarization using a fluorometer. The change in polarization represents the presence of an insertion/flanking sequence, which indicates that amplification, hybridization, and single base extension reactions were successful.
Taqman is described as a method for detecting and quantifying the presence of a DNA sequence, which is described in detail in the instructions provided by the manufacturer. Briefly, a FRET oligonucleotide probe is designed that spans the intervening DNA sequence and adjacent genomic flanking binding sites, as described below. The FRET probe and PCR primers (one primer in each of the insert sequence and adjacent flanking genomic sequences) are cycled in the presence of a thermostable polymerase and dNTPs. Hybridization of the FRET probe results in cleavage of the fluorescent moiety and the quencher moiety on the FRET probe and release of the fluorescent moiety. The generation of a fluorescent signal is representative of the presence of the insertion/flanking sequences, which indicates that amplification and hybridization were successful.
Suitable techniques for detecting plant material derived from transgenic maize event LG11 can also include Southern blot hybridization, northern blot hybridization, and in situ hybridization based on the hybridization principle. In particular, the suitable technique includes incubating the probe and sample, washing to remove unbound probe and detecting whether the probe has hybridized. The detection method depends on the type of label attached to the probe, for example, radiolabeled probes can be detected by X-ray exposure and development, or enzymatically labeled probes can be detected by substrate conversion to effect a color change.
The sequences can also be detected using molecular markers (Tyagi S and Kramer F R.molecular beacons: probes that fluoresce upon hybridization [ J ]. Nat. Biotechnol,1996,14 (3): 303-8.). A FRET oligonucleotide probe is designed that spans the inserted DNA sequence and adjacent genomic flanking binding sites. The unique structure of the FRET probe results in it containing a secondary structure that is capable of retaining both the fluorescent moiety and the quenching moiety in close proximity. The FRET probe and PCR primers (one primer in each of the insert sequence and adjacent flanking genomic sequences) are cycled in the presence of a thermostable polymerase and dNTPs. Upon successful PCR amplification, hybridization of the FRET probe to the target sequence results in a loss of secondary structure of the probe, thereby spatially separating the fluorescent moiety from the quenching moiety, producing a fluorescent signal. The generation of a fluorescent signal is representative of the presence of the insertion/flanking sequences, which indicates that amplification and hybridization were successful.
Other described methods, such as microfluidics (microfluidics), provide methods and apparatus for isolating and amplifying DNA samples. The photodyes are used to detect and determine specific DNA molecules. A nano tube (nano tube) device comprising an electronic sensor for detecting DNA molecules or a nano bead binding to a specific DNA molecule and thus being detectable is useful for detecting the DNA molecules of the invention.
DNA detection kits may be developed using the compositions of the present invention and methods described in or known to the DNA detection arts. The kit facilitates the identification of the presence or absence of DNA for transgenic corn event LG11 in a sample and can also be used to develop corn plants containing DNA for transgenic corn event LG 11. The kit may contain DNA primers or probes homologous or reverse complementary to at least a portion of SEQ ID NO. 1, 2, 3, 4, 5, 6 or 7, or other DNA primers or probes homologous or complementary to DNA contained in the transgenic genetic element of DNA, which DNA sequences may be used in DNA amplification reactions or as probes in DNA hybridization methods. The DNA structure of the transgene insert contained in the corn genome and the binding site to the corn genome illustrated in fig. 1 and table 1 comprises: a left flanking genomic region of maize LG11 at the 5' end of the transgene insert, a portion of the insert from the left border region (LB) of agrobacterium, the first expression cassette being the 35S promoter (CaMV 35S promoter (enhanced)) of cauliflower mosaic virus, operably linked to the glufosinate resistance gene sequence (bar), and operably linked to the 35S terminator (CaMV poly (a)) of cauliflower mosaic virus; the second expression cassette consisted of the 35S promoter of cauliflower mosaic virus (CaMV 35 Spromoter), operably linked to the insect-resistant gene m2cryAb-vip3A, and operably linked to the nopaline synthase gene terminator (NOS terminator), a portion of the insert sequence from the right border Region (RB) of Agrobacterium, and the maize LG11 right flanking genomic region (SEQ ID NO: 5) at the 3' end of the transgene insert sequence. In the DNA amplification method, the DNA molecule used as a primer may be any part derived from the transgene insert sequence in the transgenic corn event LG11, or any part derived from the DNA region of the flanking corn genome in the transgenic corn event LG 11.
Transgenic corn event LG11 can be combined with other transgenic corn varieties, such as herbicide (e.g., glufosinate, glyphosate, etc.) tolerant corn, or transgenic corn varieties carrying an insect-resistant gene. Various combinations of all of these different transgenic events, when bred with transgenic corn event LG11 of the present invention, can provide improved hybrid transgenic corn varieties that are resistant to insects and multiple herbicides. These varieties may exhibit superior characteristics of insect resistance, resistance to various herbicides, and the like, compared to non-transgenic varieties and transgenic varieties of single traits.
The invention provides a nucleic acid sequence for detecting corn plants and a detection method thereof, and transgenic corn event LG11 has the effects of improving insect resistance and tolerating glufosinate herbicide. Maize plants of this trait express the M2cryAb-vip3A protein and Phosphinothricin Acetyltransferase (PAT) protein, which confers insect resistance and tolerance to glufosinate to plants. Meanwhile, in the detection method, SEQ ID NO. 1 or a reverse complement thereof, SEQ ID NO. 2 or a reverse complement thereof, SEQ ID NO. 3 or a reverse complement thereof, SEQ ID NO. 4 or a reverse complement thereof, SEQ ID NO. 6 or a reverse complement thereof, or SEQ ID NO. 7 or a reverse complement thereof can be used as a DNA primer or a probe to generate an amplification product for diagnosing the transgenic corn event LG11 or a progeny thereof, and the existence of plant materials derived from the transgenic corn event LG11 can be rapidly, accurately and stably identified.
The transgenic corn event LG11 has strong glufosinate tolerance and outstanding insect resistance. These characteristics allow the LG11 transformant to be used to improve glufosinate herbicide tolerance and pest resistance traits of corn, thereby breeding new varieties of corn resistant to pest and herbicide.
The technical scheme of the invention is further described in detail through the drawings and the embodiments.
Drawings
FIG. 1 is a schematic structural diagram of a binding site between a transgene insert sequence and a maize genome.
FIG. 2 physical map of recombinant expression vector pCAMBIA3300+m2cryAb-vip 3A. The English and abbreviations of the elements are listed below:
T-DNA left border sequence of LB Agrobacterium.
35S terminator of CaMV poly (A) cauliflower mosaic virus (CaMV).
bar codes PAT protein and releases glufosinate toxicity.
35S promoter of CaMV 35S promoter (enhanced) cauliflower mosaic virus, and initiates transcription of target gene
Terminator of NOS terminator nopaline synthase gene.
m2cryAb-vip3A M2cryAb-vip3A protein coding gene.
35S promoter of CaMV 35S promter cauliflower mosaic virus (CaMV).
T-DNA right border sequence of Agrobacterium RB.
plasmid stabilizing site of pVS1 staA pVS1 plasmid.
Replication initiation site of pVS1 RepA pVS1 plasmid.
The bom site of the bom pBR322 plasmid.
Replication initiation site of ori pBR322 plasmid.
kanR encodes an aminoglycoside phosphotransferase protein, conferring kanamycin resistance on bacteria.
FIG. 3LG11 event and control Chang 7-2 plant against glufosinate herbicide. A: chang 7-2 at 0 times dosage; b: chang 7-2 at 1 time dose; c: LG11 at 0-fold dose; d: LG11 at 1-fold dose; e: LG11 at 2-fold dose; f: LG11 at 4-fold dose.
FIG. 4 shows the performance of the LG11 event against insect resistance against control Chang 7-2. A: non-transgenic control Chang 7-2 maize plants; b: LG11 transformed event maize plants.
FIG. 5LG11 transformation event specific PCR validation results. M: marker, size label side (unit: bp); n: blank control; CK: negative control (Chang 7-2 and Zhengdan 958 mixture) genomic DNA;1-3: different generation transformation event LG11 genomic DNA;4: novel transgenic corn hybrid LG 11-Zhengdan 958 genomic DNA. A: the expected size of the left border PCR fragment is 286bp; b: the right border PCR fragment was expected to be 1447bp in size.
FIG. 6Southern hybridization cleavage and probe position.
FIG. 7 Southern blot hybridization of the insertion copy number of the LG11 gene of interest m2cryAb-vip3A
A: hindIII cleavage hybridization map; b: bamHI cleavage hybridization map; c: schematic of the cleavage site of the probe and HindIII; d: the probe and BamHI cleavage sites are schematically shown. The lower line segment indicates the probe position and the right arrow of the picture indicates the exogenous strip.
M: DNA Marker, the size of the band is marked beside, the unit is bp;
CK: negative control Chang 7-2;
1: positive control plasmid;
2-4; transformants LG11 of different generations.
FIG. 8 Southern blot hybridization of the bar insertion copy number of LG11 gene of interest
A: hindIII cleavage hybridization map; b: bamHI cleavage hybridization map; c: schematic of the cleavage site of the probe and HindIII; d: the probe and BamHI cleavage sites are schematically shown. The lower line segment indicates the probe position and the right arrow of the picture indicates the exogenous strip.
M: DNAMmarker, the size of the band is marked beside, and the unit is bp;
CK: negative control Chang 7-2;
1: positive control plasmid;
2-4; transformants LG11 of different generations.
Detailed Description
The transformation event LG11 refers to a maize plant obtained by crossing and backcrossing maize HiIIB serving as a receptor with recurrent parent maize inbred line Chang 7-2 and selfing to insert an exogenous gene insert (T-DNA insert) between specific genome sequences. In a specific example, the expression vector used for the transgene has the physical map shown in FIG. 2, and the resulting T-DNA insert has the sequence shown in nucleotides 281-7424 of SEQ ID NO. 5. Transformation event LG11 may refer to this transgenic process, or to T-DNA inserts within the genome resulting from this process, or to a combination of T-DNA inserts and flanking sequences, or to maize plants resulting from this transgenic process. In specific examples, the event is also applicable to plants obtained by transforming other recipient varieties with the same expression vector, thereby inserting a T-DNA insert into the same genomic location. The transformation event LG11 may also refer to progeny plants resulting from asexual, sexual, double or double propagation of the above plants or combinations thereof.
Example 1 acquisition and characterization of transformation events
The M2cryAb-Vip3A protein is formed by combining main structural domains of Cry1Ab and Vip3Aa proteins by an artificial synthesis method, and the Cry1Ab and Vip3Aa have remarkable control effects on corn borer, spodoptera frugiperda and other pests; the bar gene codes phosphinothricin acetyl transferase, which can improve the tolerance of plants to glufosinate herbicide. The invention uses pCAMBIA3300+m2cryAb-vip3A expression vector (the physical diagram of the vector is shown in figure 2, and comprises an m2cryAb-vip3A gene expression cassette and a bar gene expression cassette), converts receptor HiIIB by an agrobacterium-mediated method to obtain more than 400 positive transformants, and after molecular detection, each generation uses maize inbred line Chang 7-2 as recurrent parent to carry out backcross to obtain BC 5 F 2 The transgenic corn seeds LG 01-LG 20 are used for screening and identifying herbicide tolerance, insect resistance and related agronomic traits of the transformed seedlings.
1. Screening transformants excellent in insect-resistant and herbicide-resistant traits
(1) Herbicide resistance screening
By taking recurrent parent Chang 7-2 as a reference, transformants with better herbicide tolerance are screened by a method of spraying glufosinate with the quantity 1 times of the recommended concentration in the field. The results show that only 9 transformation events were significantly more tolerant to glufosinate herbicide than the control (table 2).
TABLE 2 herbicide resistance manifestation
Values are from the mean ± standard deviation of 3 biological replicates. Statistical analysis multiple comparisons (α=0.05) were made using LSD, with different letters indicating the significance of the same column data difference at the same herbicide concentration.
(2) Insect resistance
Transformants with better resistance to insects were selected from the 9 transformation events described above by the leaf-chamber method using the recurrent parent Chang 7-2 as a reference. The materials were evaluated for insect resistance using ex vivo corn leaf feeding to the first larvae of Asian corn borer and Spodoptera frugiperda. Mortality of corn borers and spodoptera frugiperda caused by 9 transformant leaves was significantly higher than that of the control (table 3), with LG04, LG06, LG09, LG11 being resistant to both corn borers and spodoptera frugiperda at high levels and the remainder being medium or resistant.
TABLE 3 indoor biological assay
Values are expressed as mean ± standard deviation of 4 biological replicates, and the significance of the differences in the same column of data was analyzed using LSD method (α=0.05).
(3) Agronomic trait investigation
The identification of resistance traits of several transformants was accompanied by detailed recordings of their agronomic traits (e.g., plant height, leaf size, ear size, grain weight, etc.). When data statistics were carried out, it was unexpectedly found that the plant heights and hundred grain weights of the transformants LG01, LG03, LG04, LG05, LG06, LG08, LG09 and LG14 were significantly lower than those of the control Chang 7-2, and that only the agronomic traits (plant heights and hundred grain weights) of the transformant LG11 were not significantly different from those of the control (Table 4).
TABLE 4 results of partial agronomic trait investigation
Values are from the mean ± standard deviation of 3 biological replicates. Statistical analysis multiple comparisons (α=0.05) were made using LSD, with different letters representing the significance of the same column data difference over the same period.
Taken together, LG11 is a transformant that is excellent in herbicide resistance, insect resistance, and agronomic traits.
2. Insertion site flanking sequence analysis of exogenous sequences on maize genome
To further define the insertion site for the transformation event LG11, the present invention analyzes flanking sequences of the LG11 exogenous sequence at the insertion site on the maize genome.
100mg plant leaves are taken, and after liquid nitrogen is rapidly ground, total DNA is extracted by adopting a CTAB method. After concentration measurement of genomic DNA, the total amount of DNA was ensured to be > 2. Mu.g. By utilizing genome re-sequencing (completed by Wuhan biosamples library Co., ltd.), each Reads is 150bp in length to obtain at least 20Gb data, and the data quality index Q30 is more than or equal to 80% (namely, the ratio of the base with the sequencing error rate being more than 0.1% is lower than 20%). According to the genome re-sequencing result, BWA software is used for carrying out sequence homology comparison screening (BWA, http:// bio-bwa.sourceforge.net/, default) with the T-DNA sequence of the transgenic vector serving as a template and the total sequence obtained by sequencing. And further splicing and screening the sequences obtained by screening, and removing the Reads with all the sequences being vector sequences to finally obtain a type of Reads sequences, wherein one half of the Reads are genome sequences and the other half of the Reads are vector sequences. And (3) according to the obtained genome sequence, performing Blast sequence comparison on a corn genome website https:// www.maizegdb.org /), and obtaining a specific position of the genome sequence on a genome, namely a possible insertion site.
And respectively comparing the sequencing result with a reference genome and an exogenous T-DNA sequence to obtain the information of the insertion position of the exogenous insertion fragment. Then, forward and reverse primers are designed on the genomic flanking sequence and the exogenous insertion sequence at the left and right boundaries of the insertion site, the insertion site is verified by a PCR amplification method, and the PCR product is sequenced and analyzed. The results show that the exogenous fragment of transformant LG11 was inserted forward between the maize genome chr5:177554037-177554053 bp.
Subsequently, primer design was performed using Primerblast software (https:// Blast. NCBI. Lm. Nih. Gov/Blast) from NCBI website at the genomic and T-DNA regions at the left and right boundaries of the insertion site, and the amplified product fused a portion of the maize genomic sequence and a portion of the T-DNA sequence.
PCR amplification was performed using transgenic maize strain genomic DNA as template. The PCR reaction was performed in a 20. Mu.L system. The amplification cycle program is: pre-denaturation at 94℃for 3min; denaturation at 94℃for 30s, annealing for 30s, extension at 72℃for a certain time (set according to the size of the product fragment), 35 cycles; extending at 72℃for 5min.
Based on the flanking sequence and the results of the insertion position, the LG11 transformation event was PCR amplified using the genomic upstream primer (SEQ ID NO: 8) and the vector left border primer (SEQ ID NO: 9) and the vector right border primer (SEQ ID NO: 10) and the genomic downstream primer (SEQ ID NO: 11) to verify the foreign fragment insertion position. The results are shown in FIG. 5. The result proves that the LG11 exogenous fragment is stably inserted into the position of chr5:177554037 bp of the corn genome, and the inserted sequence and upstream and downstream genome flanking sequence fragments are further obtained through overlapping PCR amplification and sequencing analysis, and the sequence assembly is shown in figure 6. The sequence analysis showed that the insert size was 7144bp.
By analyzing the border sequences on the left and right sides, the insertion of the exogenous sequence causes the sequence mutation of 15bp of the corn genome, meanwhile, the left border sequence of the vector is deleted by 388bp (comprising partial sequence of herbicide-resistant gene bar and complete terminator sequence), and the right border sequence is deleted by 22bp.
3. Systematic identification of LG11 herbicide tolerance and insect resistance
According to the invention, seeds of a corn transformant LG11 and a control Chang 7-2 are sown in summer 2022 at a plant site of Longshan office of Zhangshan in Jiku district of Shandong province, and herbicide tolerance and insect resistance of the transformant LG11 are identified by spraying glufosinate with different concentration doses and artificial insect grafting in a field.
(1) Herbicide tolerance identification
The glufosinate-ammonium spraying time was 18 days after sowing, and the number of plants (including plants without phytotoxicity) and the plant height of each phytotoxicity grade were examined at 1 week, 2 weeks and 4 weeks after spraying, respectively, and the results are shown in table 6 and fig. 3. All plants of the control Chang 7-2 show 4-5 grade phytotoxicity in 1 week after the glufosinate is sprayed, most of the plants die, the seedling rate is 0.00%, and the damage rate is 100.00%; LG11 can form seedlings at 100.00% at different doses, but certain phytotoxicity is generated, the phytotoxicity rate reaches 4.57% -4.69%, and the phytotoxicity symptoms disappear after 2 weeks; the strain height performance was further investigated, and the strain heights of the transformants were not significantly different at different doses.
The field herbicide tolerance identification result shows that the transformant LG11 can tolerate glufosinate-ammonium in the dosage of 4 times of the field recommended dosage, and the agronomic characters such as plant height and the like are not obviously different from those of the control. In general, partial sequence and terminator deletions affect gene expression, resulting in loss of gene function, but surprisingly, the herbicide tolerance phenotype of LG11 transformants is not affected, significantly stronger than the control, as determined by the results of the identification.
TABLE 6LG11 tolerance to glufosinate herbicide
Values are expressed as mean ± standard deviation of 3 biological replicates. 0×, 1×, 2×, 4× represent the multiples of the recommended dose of glufosinate to be sprayed, respectively. The same column data difference significance analysis was compared using LSD analysis (α=0.05). "-" means not investigated.
(2) Insect resistance identification
Reference to agricultural rural part 953 bulletin-10-2007 transgenic plants and environmental safety detection of their products, section 1: insect resistance. The insect-catching method and the investigation method are executed according to NY/T1248.5 corn disease resistance identification technical Specification. And (5) artificially inoculating insects in the corn leaf period, and investigating insect pest conditions after 2-3 weeks of insect inoculation.
Results of field resistance identification on corn borers (table 7 and fig. 4):
The heart leaf period investigation result shows that the leaf eating grade of Chang 7-2 is 7.73+/-0.31, the insect pest grade is 7, the resistance grade is sensitive, the material is a local pest sensing material, and the quality of the artificial pest grafting can meet the requirement of resistance identification; meanwhile, the leaf rating of LG11 is 1.33+/-0.24, which is significantly lower than that of the control. Insect pest grade 1, indicating a high resistance grade.
The ear period investigation result shows that the female ear damage level of the Chang 7-2 is 6.50+/-0.71, the resistance level is sensitive, the material is a local pest sensing material, and the quality of the artificial pest grafting can meet the requirement of resistance identification; the female ear harm grade of LG11 is 1.40+/-0.06, which is obviously lower than that of the control, and the resistance grade is high.
TABLE 7 identification of LG11 field resistance to corn borers
Values are expressed as mean ± standard deviation of 3 biological replicates, and the significance of the differences in the same column of data was analyzed using the t-test method (α=0.05). The transformant in the heart leaf stage is inoculated with 25 strains of insects, and 15 strains of control; the transformant at the laying stage was inoculated with 68 strains and control 15 strains.
Results of field resistance identification on spodoptera frugiperda (table 8 and fig. 4):
the heart leaf period investigation result shows that the leaf eating grade of Chang 7-2 is 7.83+/-0.24, the insect pest grade is 7, the resistance grade is sensitive, the material is a local pest sensing material, and the quality of artificial pest grafting can meet the requirement of resistance identification; while the leaf rating of LG11 was 1.09±0.09, significantly lower than the control. The transformant had a pest grade of 1 and a resistance grade of high resistance.
The ear period investigation result shows that the female ear damage grade of the Chang 7-2 is 6.20+/-0.57, the resistance grade is sensitive, the material is a local pest sensing material, and the quality of artificial pest grafting can meet the resistance identification requirement; while the leaf rating of LG11 is 1.41+ -0.41, significantly lower than the control, the resistance rating is high.
TABLE 8 Spodoptera frugiperda field biological assay
Values are expressed as mean ± standard deviation of 3-4 biological replicates, and the significance of the differences in the same column of data was analyzed using the t-test method (α=0.05). The transformant in the heart leaf stage is inoculated with 55 strains and a control 17 strain; the transformant was inoculated with 22 strains and 10 strains were compared in the laying period.
The field insect resistance identification result shows that the transformant LD11 can reach high resistance level to corn borers and spodoptera frugiperda, and the agronomic characters such as plant height and the like have no obvious difference from the contrast. Thus, LG11 transformants can be used to improve glufosinate herbicide tolerance and pest resistance traits of maize, thereby breeding new varieties of herbicide resistant maize.
Example 2 transformation event LG11 exogenous sequence copy number analysis
The copy number of the exogenous sequence was determined by Southern blot hybridization. In the Southern hybridization detection, two restriction enzymes which are arranged on a T-DNA region and are not arranged in a hybridization region are selected to digest genome DNA, each insertion copy in the genome is hybridized to form a single and specific band, and after the genome DNA is subjected to restriction enzyme digestion, a region to be detected is selected as a probe to carry out Southern imprinting hybridization experiment.
Southern hybridization the restriction enzymes HindIII and BamHI were selected to digest the positive control plasmid, control Change 7-2 and LG11 transformant genomic DNA, and the sequence fragments of the genes of interest m2cryAb-vip3A and bar were selected as probes, and the schematic of the probe and cleavage sites is shown in FIG. 6. The specific sequences of the probe primers are shown in Table 9.
TABLE 9 probes for Southern hybridization experiments
1: the unit bp.
The insertion copy number hybridization detection of the target gene m2cryAb-vip3A selects restriction enzymes HindIII and BamHI to cleave positive control plasmid, negative control Chang 7-2 genome DNA and LG11 transformant genome DNA. After running the gel and transferring the membrane, the membrane is marked by an m2cryAb-vip3A gene probe, and the hybridization result is shown in FIG. 7A and FIG. 7B. The probe position of the exogenous gene m2cryAb-vip3A and the restriction enzyme cleavage site are shown in FIG. 7C and FIG. 7D. From the hybridization results, the m2cryAb-vip3A gene of LG11 was inserted into the maize genome in a single copy.
The inserted copy number hybridization of the target gene bar is detected by selecting restriction enzymes HindIII and BamHI to cleave positive control plasmid, negative control Chang 7-2 genome DNA and LG11 transformant genome DNA. After running gel and transferring membrane, the bar gene probe is used for marking, and the hybridization result is shown in FIG. 8A and FIG. 8B. The probe position of the target gene bar and the restriction enzyme cleavage site are shown in FIG. 8C and FIG. 8D. From the hybridization results, the bar gene of LG11 was inserted in a single copy into the maize genome.
Example 3 methods for producing insect and herbicide resistant corn Using transformation event LG11
The early test shows that the transformation event LG11 has excellent resistance and other agronomic characters, so that the event can be used for rapid transformation and improvement of backbone parents of the same group in production to improve the insect resistance and herbicide tolerance of the backbone parents. In addition, the transformation event LG11 can be directly used for assembling hybrid groups, and the insect-resistant herbicide-resistant transgenic corn LG 11-Zhengdan 958 is directly assembled by taking Zheng 58 as a female parent and taking the transformation event LG11 as a male parent; LG 11-Zhengdan 958 increases the insect resistance and glufosinate resistance properties on the basis of retaining the original good properties (Table 10).
The method provided by the invention can be used for detecting the LG11 in the parent improvement and hybridization combination assembly process so as to provide a molecular auxiliary means for breeding varieties, and can also be used for identifying whether the corn varieties contain the LG11 transformation event. The following method is a specific example of identifying LG11.
Molecular detection of transformation event LG11, a new hybrid of transgenic maize LG 11-Zhengdan 958 was performed to confirm the inclusion of transformation event LG11 in the material. PCR primer pairs are designed according to the gene sequences, and the existence of the transformation event LG11 is determined by detecting the existence of the left boundary and the right boundary of the transformation event.
One of the detection methods is as follows: detecting specific boundary sequences in the transformation event LG11 maize plants by using a PCR method, wherein the used PCR primer pairs are SEQ ID NO. 8, SEQ ID NO. 9, SEQ ID NO. 10 and SEQ ID NO. 11 respectively, and a PCR reaction system:
the reaction procedure is:
94 ℃ for 5min; (94 ℃,30sec;55 ℃,30sec;72 ℃,90 sec). Times.35 cycles; 72 ℃ for 5min;4 ℃ for 5min.
PCR products were taken and detected electrophoretically in 1% (w/v) 1 XTAE agarose gel. The expected target bands can be amplified in LG11 transformation event, and the sizes are 286bp (SEQ ID NO: 6) and 1447bp (SEQ ID NO: 7), respectively, and the results are shown in FIG. 5. Moreover, the PCR method can track the existence of transformation events, so that the PCR method is applied to breeding work.
TABLE 10 identification of insect resistance (corn borer and spodoptera frugiperda) and herbicide tolerance
/>
Values are expressed as mean ± standard deviation, and the significance of the differences in the same column of data was analyzed using the t-test method (α=0.05).
Example 4 detection method of transformation event LG11
New varieties can be raised from transgenic corn event LG11 and produce agricultural or commodity products. If a sufficient amount is detected in the agricultural product or commodity, the agricultural product or commodity is expected to contain a nucleotide sequence capable of diagnosing the presence of transgenic corn event LG11 material in the agricultural product or commodity. Such agricultural products or commodities include, but are not limited to, corn oil, corn flour, corn meal, corn paste, starch, and other seasonings or any other food product that is a food source for consumption by an animal, or cosmetics, industrial products, and the like. Nucleic acid detection methods and/or kits based on probe or primer pairs can be developed to detect a transgenic corn event LG11 nucleotide sequence such as shown in SEQ ID NO. 1 or SEQ ID NO. 2 in a biological sample, wherein the probe sequence or primer amplification sequence is selected from the sequences shown in SEQ ID NO. 1, SEQ ID NO. 2, SEQ ID NO. 3, SEQ ID NO. 4, SEQ ID NO. 5, SEQ ID NO. 6 and SEQ ID NO. 7 to diagnose the presence of a transgenic corn event LG 11.
In summary, the transgenic corn event LG11 of the present invention can increase plant pest resistance and have higher tolerance to glufosinate herbicide, and can be used to improve other corn germplasm and create new corn hybrid combinations. The detection method can accurately and rapidly identify whether the biological sample contains the DNA molecule of the transgenic corn event LG 11.
Finally, it should be noted that the above-mentioned embodiments are merely for illustrating the technical solution of the present invention and not for limiting the same, and although the present invention has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications and equivalents may be made to the technical solution of the present invention without departing from the spirit and scope of the technical solution of the present invention.
Claims (4)
1. A nucleic acid molecule for detecting transgenic maize event LG11, characterized in that the sequence of the nucleic acid molecule is any of the following groups i) -iv):
i) SEQ ID NO. 1 and SEQ ID NO. 2 or the reverse complement thereof;
ii) the sequences shown in SEQ ID NO. 3 and SEQ ID NO. 4 or the reverse complement thereof;
iii) SEQ ID NO. 6 and SEQ ID NO. 7 or the reverse complement thereof;
iv) the sequence shown in SEQ ID No. 5 or its reverse complement;
The transgenic corn event LG11 refers to the insertion of an exogenous fragment at the position chr 5:177554037bp of the corn genome; the exogenous fragment has a sequence shown as 281-7424 nucleotide of SEQ ID NO. 5.
2. The primer group for detecting the transgenic corn event LG11 is characterized in that the primer group is a primer pair with sequences shown in SEQ ID NO. 8 and SEQ ID NO. 9; and a primer pair of the sequences shown in SEQ ID NO. 10 and SEQ ID NO. 11;
the transgenic corn event LG11 refers to the insertion of an exogenous fragment at the position chr 5:177554037bp of the corn genome; the exogenous fragment has a sequence shown as 281-7424 nucleotide of SEQ ID NO. 5.
3. A kit or microarray for detecting transgenic maize event LG11, characterized in that the kit or microarray comprises the nucleic acid molecule of claim 1 and/or the primer set of claim 2;
the transgenic corn event LG11 refers to the insertion of an exogenous fragment at the position chr 5:177554037bp of the corn genome; the exogenous fragment has a sequence shown as 281-7424 nucleotide of SEQ ID NO. 5.
4. A method for detecting a transgenic maize event LG11 comprising detecting the presence or absence of said transgenic maize event LG11 in a test sample using any of the following i) -iv):
i) The nucleic acid molecule of claim 1;
ii) the primer set of claim 2;
iii) The nucleic acid molecule of claim 1 and the primer set of claim 2;
iv) a kit or microarray according to claim 3;
the transgenic corn event LG11 refers to the insertion of an exogenous fragment at the position chr 5:177554037bp of the corn genome; the exogenous fragment has a sequence shown as 281-7424 nucleotide of SEQ ID NO. 5.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2022116066569 | 2022-12-13 | ||
| CN202211606656.9A CN115725571A (en) | 2022-12-13 | 2022-12-13 | Nucleic acid sequence of corn transformation event LG11 and detection method thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN116200529A CN116200529A (en) | 2023-06-02 |
| CN116200529B true CN116200529B (en) | 2024-03-12 |
Family
ID=85301316
Family Applications (2)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202211606656.9A Pending CN115725571A (en) | 2022-12-13 | 2022-12-13 | Nucleic acid sequence of corn transformation event LG11 and detection method thereof |
| CN202310139736.6A Active CN116200529B (en) | 2022-12-13 | 2023-02-16 | Nucleic acid sequence of corn transformation event LG11 and detection method thereof |
Family Applications Before (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202211606656.9A Pending CN115725571A (en) | 2022-12-13 | 2022-12-13 | Nucleic acid sequence of corn transformation event LG11 and detection method thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (2) | CN115725571A (en) |
Citations (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103923999A (en) * | 2014-04-25 | 2014-07-16 | 浙江省农业科学院 | Qualitative PCR detecting method for transgenic insect-resistant herbicide-tolerant rice G6H1 and derived varieties thereof |
| CN104830847A (en) * | 2015-04-30 | 2015-08-12 | 北京大北农科技集团股份有限公司 | Nucleic acid sequence for detecting corn plant DBN9936, and detection method thereof |
| CN104878094A (en) * | 2015-04-30 | 2015-09-02 | 北京大北农科技集团股份有限公司 | Maize plant DBN9888 nucleic acid sequence for detecting weedicide tolerance and detection method thereof |
| CN109971880A (en) * | 2019-04-09 | 2019-07-05 | 北京大北农生物技术有限公司 | For detecting the nucleic acid sequence and its detection method of corn plant DBN9508 |
| CN111518941A (en) * | 2020-05-13 | 2020-08-11 | 四川农业大学 | Nucleic acid sequence for detecting corn plant AN1 and detection method thereof |
| CN112852801A (en) * | 2021-01-27 | 2021-05-28 | 隆平生物技术(海南)有限公司 | Transgenic maize event LP007-1 and methods of detecting same |
| CN113201531A (en) * | 2021-04-27 | 2021-08-03 | 隆平生物技术(海南)有限公司 | Transgenic corn event LW2-1 and methods for detecting same |
| CN113462690A (en) * | 2021-07-02 | 2021-10-01 | 河南大学 | Application of soybean gene promoters pRPS28 and pRPS28-I in soybeans, arabidopsis thaliana and tobaccos |
| CN114107344A (en) * | 2021-11-15 | 2022-03-01 | 山东省农业科学院 | Insect-resistant fusion gene M2CryAb-VIP3A, expression vector and product thereof, and application thereof |
| CN115449521A (en) * | 2021-06-09 | 2022-12-09 | 杭州芳韵生物科技有限公司 | Binary vector for simultaneously expressing insect-resistant gene and herbicide-resistant gene and application thereof |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP2992090A4 (en) * | 2013-04-30 | 2017-01-11 | Basf Se | Plants having increased tolerance to herbicides |
-
2022
- 2022-12-13 CN CN202211606656.9A patent/CN115725571A/en active Pending
-
2023
- 2023-02-16 CN CN202310139736.6A patent/CN116200529B/en active Active
Patent Citations (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103923999A (en) * | 2014-04-25 | 2014-07-16 | 浙江省农业科学院 | Qualitative PCR detecting method for transgenic insect-resistant herbicide-tolerant rice G6H1 and derived varieties thereof |
| CN104830847A (en) * | 2015-04-30 | 2015-08-12 | 北京大北农科技集团股份有限公司 | Nucleic acid sequence for detecting corn plant DBN9936, and detection method thereof |
| CN104878094A (en) * | 2015-04-30 | 2015-09-02 | 北京大北农科技集团股份有限公司 | Maize plant DBN9888 nucleic acid sequence for detecting weedicide tolerance and detection method thereof |
| CN109971880A (en) * | 2019-04-09 | 2019-07-05 | 北京大北农生物技术有限公司 | For detecting the nucleic acid sequence and its detection method of corn plant DBN9508 |
| CN111518941A (en) * | 2020-05-13 | 2020-08-11 | 四川农业大学 | Nucleic acid sequence for detecting corn plant AN1 and detection method thereof |
| CN112852801A (en) * | 2021-01-27 | 2021-05-28 | 隆平生物技术(海南)有限公司 | Transgenic maize event LP007-1 and methods of detecting same |
| CN113201531A (en) * | 2021-04-27 | 2021-08-03 | 隆平生物技术(海南)有限公司 | Transgenic corn event LW2-1 and methods for detecting same |
| CN115449521A (en) * | 2021-06-09 | 2022-12-09 | 杭州芳韵生物科技有限公司 | Binary vector for simultaneously expressing insect-resistant gene and herbicide-resistant gene and application thereof |
| CN113462690A (en) * | 2021-07-02 | 2021-10-01 | 河南大学 | Application of soybean gene promoters pRPS28 and pRPS28-I in soybeans, arabidopsis thaliana and tobaccos |
| CN114107344A (en) * | 2021-11-15 | 2022-03-01 | 山东省农业科学院 | Insect-resistant fusion gene M2CryAb-VIP3A, expression vector and product thereof, and application thereof |
Non-Patent Citations (1)
| Title |
|---|
| 商品化转基因植物的外源基因及其检测技术;李俊等;中国农业科技导报;第10卷(第3期);31-39 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN115725571A (en) | 2023-03-03 |
| CN116200529A (en) | 2023-06-02 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN113201531B (en) | Transgenic corn event LW2-1 and detection method thereof | |
| KR20120004964A (en) | Transgenic rice event 17314 and methods of use thereof | |
| CN112831584B (en) | Transgenic corn event LP007-2 and detection method thereof | |
| CN112831585B (en) | Transgenic corn event LP007-4 and detection method thereof | |
| CN112852991B (en) | Transgenic corn event LP007-7 and detection method thereof | |
| CN116144817B (en) | Transgenic corn event LP026-4 and detection method thereof | |
| CN113151533B (en) | Transgenic corn event LP007-6 and detection method thereof | |
| CN116144818A (en) | Transgenic corn event LP026-2 and detection method thereof | |
| CN112877454B (en) | Transgenic corn event LP007-3 and detection method thereof | |
| CN109971880B (en) | Nucleic acid sequence for detecting corn plant DBN9508 and detection method thereof | |
| CN116287384B (en) | Nucleic acid molecule of insect-resistant herbicide-resistant corn transformation event LD05, detection method and application thereof | |
| CN110881367A (en) | Corn event Ttrans-4 and methods of use thereof | |
| CN112662799B (en) | Nucleic acid molecule for detecting corn plant ND4401 and detection method thereof | |
| CN113278721B (en) | Transgenic corn event LW2-2 and detection method thereof | |
| CN116200529B (en) | Nucleic acid sequence of corn transformation event LG11 and detection method thereof | |
| CN116694813B (en) | Transgenic soybean event LP086-1 and detection method thereof | |
| CN112574987B (en) | Nucleic acid molecule for detecting corn plant ND6603 and detection method thereof | |
| CN116656870B (en) | Transgenic soybean event LP086-3 and detection method thereof | |
| CN116694812B (en) | Transgenic soybean event LP086-2 and detection method thereof | |
| CN116716434B (en) | Transgenic soybean event LP012-3 and detection method thereof | |
| CN112481412B (en) | Nucleic acid molecule for detecting corn plant ND4403 and detection method thereof | |
| CN116694815B (en) | Transgenic soybean event LP012-2 and detection method thereof | |
| CN116694814B (en) | Transgenic soybean event LP012-1 and detection method thereof | |
| CN115820630A (en) | Exogenous insertion sequence of maize transformation event LD02 and application thereof | |
| CN116640761B (en) | Transgenic maize event LP018-1 and detection method thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |