CN112662799A - Nucleic acid molecule for detecting corn plant ND4401 and detection method thereof - Google Patents
Nucleic acid molecule for detecting corn plant ND4401 and detection method thereof Download PDFInfo
- Publication number
- CN112662799A CN112662799A CN202011552975.7A CN202011552975A CN112662799A CN 112662799 A CN112662799 A CN 112662799A CN 202011552975 A CN202011552975 A CN 202011552975A CN 112662799 A CN112662799 A CN 112662799A
- Authority
- CN
- China
- Prior art keywords
- seq
- sequence
- dna
- plant
- corn
- 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.)
- Granted
Links
- 150000007523 nucleic acids Chemical class 0.000 title claims abstract description 77
- 102000039446 nucleic acids Human genes 0.000 title claims abstract description 72
- 108020004707 nucleic acids Proteins 0.000 title claims abstract description 72
- 241001057636 Dracaena deremensis Species 0.000 title claims abstract description 22
- 238000001514 detection method Methods 0.000 title abstract description 27
- 240000008042 Zea mays Species 0.000 claims abstract description 150
- 235000002017 Zea mays subsp mays Nutrition 0.000 claims abstract description 133
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 claims abstract description 125
- 230000009261 transgenic effect Effects 0.000 claims abstract description 82
- 229910052757 nitrogen Inorganic materials 0.000 claims abstract description 63
- 235000016383 Zea mays subsp huehuetenangensis Nutrition 0.000 claims abstract description 49
- 235000009973 maize Nutrition 0.000 claims abstract description 49
- 230000000295 complement effect Effects 0.000 claims abstract description 41
- 230000007812 deficiency Effects 0.000 claims abstract description 27
- 241000196324 Embryophyta Species 0.000 claims description 96
- 235000005824 Zea mays ssp. parviglumis Nutrition 0.000 claims description 84
- 235000005822 corn Nutrition 0.000 claims description 84
- 238000000034 method Methods 0.000 claims description 61
- 239000000523 sample Substances 0.000 claims description 55
- 230000009466 transformation Effects 0.000 claims description 54
- 108090000623 proteins and genes Proteins 0.000 claims description 53
- 239000002773 nucleotide Substances 0.000 claims description 37
- 125000003729 nucleotide group Chemical group 0.000 claims description 37
- 108091028043 Nucleic acid sequence Proteins 0.000 claims description 31
- 238000003199 nucleic acid amplification method Methods 0.000 claims description 31
- 230000014509 gene expression Effects 0.000 claims description 29
- 230000003321 amplification Effects 0.000 claims description 27
- 230000001965 increasing effect Effects 0.000 claims description 18
- 239000012634 fragment Substances 0.000 claims description 15
- 238000009395 breeding Methods 0.000 claims description 7
- 238000012360 testing method Methods 0.000 claims description 7
- 230000001488 breeding effect Effects 0.000 claims description 6
- 235000013305 food Nutrition 0.000 claims description 6
- 238000002493 microarray Methods 0.000 claims description 5
- 239000002689 soil Substances 0.000 claims description 4
- 210000001161 mammalian embryo Anatomy 0.000 claims description 3
- 238000004113 cell culture Methods 0.000 claims description 2
- 239000012770 industrial material Substances 0.000 claims 1
- 108020004414 DNA Proteins 0.000 abstract description 105
- 102000053602 DNA Human genes 0.000 abstract description 17
- 239000012472 biological sample Substances 0.000 abstract description 6
- 239000013615 primer Substances 0.000 description 58
- 238000003752 polymerase chain reaction Methods 0.000 description 25
- 108700019146 Transgenes Proteins 0.000 description 23
- 210000004027 cell Anatomy 0.000 description 22
- 238000003780 insertion Methods 0.000 description 22
- 230000037431 insertion Effects 0.000 description 22
- 238000009396 hybridization Methods 0.000 description 19
- 108091093088 Amplicon Proteins 0.000 description 14
- 238000006243 chemical reaction Methods 0.000 description 14
- 230000002068 genetic effect Effects 0.000 description 13
- 241000701489 Cauliflower mosaic virus Species 0.000 description 12
- 239000002987 primer (paints) Substances 0.000 description 12
- 239000000463 material Substances 0.000 description 10
- 102000040430 polynucleotide Human genes 0.000 description 10
- 108091033319 polynucleotide Proteins 0.000 description 10
- 239000002157 polynucleotide Substances 0.000 description 10
- 239000013598 vector Substances 0.000 description 10
- 108020001019 DNA Primers Proteins 0.000 description 9
- 230000004544 DNA amplification Effects 0.000 description 9
- 239000003155 DNA primer Substances 0.000 description 9
- 210000001519 tissue Anatomy 0.000 description 9
- 108010076504 Protein Sorting Signals Proteins 0.000 description 8
- 230000015572 biosynthetic process Effects 0.000 description 8
- 108091034117 Oligonucleotide Proteins 0.000 description 7
- 238000002866 fluorescence resonance energy transfer Methods 0.000 description 7
- 108010058731 nopaline synthase Proteins 0.000 description 7
- 239000013612 plasmid Substances 0.000 description 7
- 230000008488 polyadenylation Effects 0.000 description 7
- 235000018102 proteins Nutrition 0.000 description 7
- 102000004169 proteins and genes Human genes 0.000 description 7
- 230000001568 sexual effect Effects 0.000 description 7
- 238000003786 synthesis reaction Methods 0.000 description 7
- 108090000848 Ubiquitin Proteins 0.000 description 6
- 238000013461 design Methods 0.000 description 6
- 238000005516 engineering process Methods 0.000 description 6
- 239000003550 marker Substances 0.000 description 6
- 239000000618 nitrogen fertilizer Substances 0.000 description 6
- 230000008569 process Effects 0.000 description 6
- 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 description 5
- 241000589158 Agrobacterium Species 0.000 description 5
- 239000005561 Glufosinate Substances 0.000 description 5
- 238000012408 PCR amplification Methods 0.000 description 5
- 235000007244 Zea mays Nutrition 0.000 description 5
- 235000013339 cereals Nutrition 0.000 description 5
- 239000003623 enhancer Substances 0.000 description 5
- 239000013604 expression vector Substances 0.000 description 5
- 102000016928 DNA-directed DNA polymerase Human genes 0.000 description 4
- 108010014303 DNA-directed DNA polymerase Proteins 0.000 description 4
- 229910002651 NO3 Inorganic materials 0.000 description 4
- NHNBFGGVMKEFGY-UHFFFAOYSA-N Nitrate Chemical compound [O-][N+]([O-])=O NHNBFGGVMKEFGY-UHFFFAOYSA-N 0.000 description 4
- 108090000836 Nitrate Transporters Proteins 0.000 description 4
- 108020004511 Recombinant DNA Proteins 0.000 description 4
- 102000044159 Ubiquitin Human genes 0.000 description 4
- 101150103518 bar gene Proteins 0.000 description 4
- 230000027455 binding Effects 0.000 description 4
- 239000003337 fertilizer Substances 0.000 description 4
- 230000001105 regulatory effect Effects 0.000 description 4
- 241000894007 species Species 0.000 description 4
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 3
- FWMNVWWHGCHHJJ-SKKKGAJSSA-N 4-amino-1-[(2r)-6-amino-2-[[(2r)-2-[[(2r)-2-[[(2r)-2-amino-3-phenylpropanoyl]amino]-3-phenylpropanoyl]amino]-4-methylpentanoyl]amino]hexanoyl]piperidine-4-carboxylic acid Chemical compound C([C@H](C(=O)N[C@H](CC(C)C)C(=O)N[C@H](CCCCN)C(=O)N1CCC(N)(CC1)C(O)=O)NC(=O)[C@H](N)CC=1C=CC=CC=1)C1=CC=CC=C1 FWMNVWWHGCHHJJ-SKKKGAJSSA-N 0.000 description 3
- 241000589155 Agrobacterium tumefaciens Species 0.000 description 3
- 241000219195 Arabidopsis thaliana Species 0.000 description 3
- 108091029865 Exogenous DNA Proteins 0.000 description 3
- 206010020649 Hyperkeratosis Diseases 0.000 description 3
- 238000010521 absorption reaction Methods 0.000 description 3
- 238000012271 agricultural production Methods 0.000 description 3
- 238000004458 analytical method Methods 0.000 description 3
- 230000002759 chromosomal effect Effects 0.000 description 3
- 210000002257 embryonic structure Anatomy 0.000 description 3
- 235000013312 flour Nutrition 0.000 description 3
- 238000010353 genetic engineering Methods 0.000 description 3
- 230000012010 growth Effects 0.000 description 3
- 239000004009 herbicide Substances 0.000 description 3
- 230000001976 improved effect Effects 0.000 description 3
- 230000010354 integration Effects 0.000 description 3
- 239000000203 mixture Substances 0.000 description 3
- 230000014075 nitrogen utilization Effects 0.000 description 3
- 108010082527 phosphinothricin N-acetyltransferase Proteins 0.000 description 3
- 230000010076 replication Effects 0.000 description 3
- 150000003839 salts Chemical class 0.000 description 3
- 238000012163 sequencing technique Methods 0.000 description 3
- 238000007671 third-generation sequencing Methods 0.000 description 3
- 230000001131 transforming effect Effects 0.000 description 3
- 238000011144 upstream manufacturing Methods 0.000 description 3
- 238000005406 washing Methods 0.000 description 3
- 102000007469 Actins Human genes 0.000 description 2
- 108010085238 Actins Proteins 0.000 description 2
- 241000589156 Agrobacterium rhizogenes Species 0.000 description 2
- 108091026890 Coding region Proteins 0.000 description 2
- 229920002261 Corn starch Polymers 0.000 description 2
- 108010066133 D-octopine dehydrogenase Proteins 0.000 description 2
- 241000701484 Figwort mosaic virus Species 0.000 description 2
- 108091092195 Intron Proteins 0.000 description 2
- 108020005187 Oligonucleotide Probes Proteins 0.000 description 2
- KWYUFKZDYYNOTN-UHFFFAOYSA-M Potassium hydroxide Chemical compound [OH-].[K+] KWYUFKZDYYNOTN-UHFFFAOYSA-M 0.000 description 2
- 101100484967 Solanum tuberosum PVS1 gene Proteins 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
- 238000009825 accumulation Methods 0.000 description 2
- 239000011543 agarose gel Substances 0.000 description 2
- 238000000137 annealing Methods 0.000 description 2
- 230000008859 change Effects 0.000 description 2
- 210000000349 chromosome Anatomy 0.000 description 2
- 238000007796 conventional method Methods 0.000 description 2
- 235000005687 corn oil Nutrition 0.000 description 2
- 239000002285 corn oil Substances 0.000 description 2
- 239000008120 corn starch Substances 0.000 description 2
- 239000002537 cosmetic Substances 0.000 description 2
- 230000002950 deficient Effects 0.000 description 2
- 238000011161 development Methods 0.000 description 2
- 235000013399 edible fruits Nutrition 0.000 description 2
- 230000007613 environmental effect Effects 0.000 description 2
- 230000004720 fertilization Effects 0.000 description 2
- 238000002875 fluorescence polarization Methods 0.000 description 2
- 230000006870 function Effects 0.000 description 2
- RWSXRVCMGQZWBV-WDSKDSINSA-N glutathione Chemical compound OC(=O)[C@@H](N)CCC(=O)N[C@@H](CS)C(=O)NCC(O)=O RWSXRVCMGQZWBV-WDSKDSINSA-N 0.000 description 2
- 230000002363 herbicidal effect Effects 0.000 description 2
- 208000015181 infectious disease Diseases 0.000 description 2
- 229930027917 kanamycin Natural products 0.000 description 2
- SBUJHOSQTJFQJX-NOAMYHISSA-N kanamycin Chemical class 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 2
- 235000012054 meals Nutrition 0.000 description 2
- 230000001404 mediated effect Effects 0.000 description 2
- 239000002071 nanotube Substances 0.000 description 2
- 238000007899 nucleic acid hybridization Methods 0.000 description 2
- 239000002751 oligonucleotide probe Substances 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
- 229940072033 potash Drugs 0.000 description 2
- BWHMMNNQKKPAPP-UHFFFAOYSA-L potassium carbonate Substances [K+].[K+].[O-]C([O-])=O BWHMMNNQKKPAPP-UHFFFAOYSA-L 0.000 description 2
- 235000015320 potassium carbonate Nutrition 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
- 238000012175 pyrosequencing Methods 0.000 description 2
- 238000010791 quenching Methods 0.000 description 2
- 230000000171 quenching effect Effects 0.000 description 2
- 230000009467 reduction Effects 0.000 description 2
- 238000012216 screening Methods 0.000 description 2
- 230000008685 targeting Effects 0.000 description 2
- 238000011426 transformation method Methods 0.000 description 2
- 101150084750 1 gene Proteins 0.000 description 1
- 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
- 241001136782 Alca Species 0.000 description 1
- 102000007347 Apyrase Human genes 0.000 description 1
- 108010007730 Apyrase Proteins 0.000 description 1
- 241000894006 Bacteria Species 0.000 description 1
- 239000002028 Biomass Substances 0.000 description 1
- 208000003643 Callosities Diseases 0.000 description 1
- 101001033883 Cenchritis muricatus Protease inhibitor 2 Proteins 0.000 description 1
- 241001480010 Cestrum Species 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
- 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
- 238000007400 DNA extraction Methods 0.000 description 1
- 238000001712 DNA sequencing Methods 0.000 description 1
- 230000004568 DNA-binding Effects 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
- 239000005504 Dicamba 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
- 102000004190 Enzymes Human genes 0.000 description 1
- 108090000790 Enzymes 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
- 108700007698 Genetic Terminator Regions Proteins 0.000 description 1
- 108010024636 Glutathione Proteins 0.000 description 1
- 108010068370 Glutens Proteins 0.000 description 1
- 239000005562 Glyphosate Substances 0.000 description 1
- 206010053759 Growth retardation Diseases 0.000 description 1
- 108010034791 Heterochromatin Proteins 0.000 description 1
- 241000238631 Hexapoda Species 0.000 description 1
- 108010025815 Kanamycin Kinase Proteins 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
- 238000000636 Northern blotting Methods 0.000 description 1
- 108020004711 Nucleic Acid Probes Proteins 0.000 description 1
- 240000007594 Oryza sativa Species 0.000 description 1
- 235000007164 Oryza sativa Nutrition 0.000 description 1
- 108090000417 Oxygenases Proteins 0.000 description 1
- 102000004020 Oxygenases Human genes 0.000 description 1
- 229910019142 PO4 Inorganic materials 0.000 description 1
- 101710091688 Patatin Proteins 0.000 description 1
- OAICVXFJPJFONN-UHFFFAOYSA-N Phosphorus Chemical compound [P] OAICVXFJPJFONN-UHFFFAOYSA-N 0.000 description 1
- 239000004952 Polyamide Substances 0.000 description 1
- ZLMJMSJWJFRBEC-UHFFFAOYSA-N Potassium Chemical compound [K] ZLMJMSJWJFRBEC-UHFFFAOYSA-N 0.000 description 1
- 244000061456 Solanum tuberosum Species 0.000 description 1
- 235000002595 Solanum tuberosum Nutrition 0.000 description 1
- 238000002105 Southern blotting Methods 0.000 description 1
- 102000004523 Sulfate Adenylyltransferase Human genes 0.000 description 1
- 108010022348 Sulfate adenylyltransferase Proteins 0.000 description 1
- 241000209140 Triticum Species 0.000 description 1
- 235000021307 Triticum Nutrition 0.000 description 1
- 208000036142 Viral infection Diseases 0.000 description 1
- 241000700605 Viruses Species 0.000 description 1
- MMDJDBSEMBIJBB-UHFFFAOYSA-N [O-][N+]([O-])=O.[O-][N+]([O-])=O.[O-][N+]([O-])=O.[NH6+3] Chemical compound [O-][N+]([O-])=O.[O-][N+]([O-])=O.[O-][N+]([O-])=O.[NH6+3] MMDJDBSEMBIJBB-UHFFFAOYSA-N 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 239000012190 activator Substances 0.000 description 1
- 239000013543 active substance Substances 0.000 description 1
- 230000009418 agronomic effect Effects 0.000 description 1
- 238000005904 alkaline hydrolysis reaction Methods 0.000 description 1
- 102000006646 aminoglycoside phosphotransferase Human genes 0.000 description 1
- 229930002877 anthocyanin Natural products 0.000 description 1
- 235000010208 anthocyanin Nutrition 0.000 description 1
- 239000004410 anthocyanin Substances 0.000 description 1
- 150000004636 anthocyanins Chemical class 0.000 description 1
- 230000011681 asexual reproduction Effects 0.000 description 1
- 238000013465 asexual reproduction Methods 0.000 description 1
- 230000001580 bacterial effect Effects 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
- 230000033228 biological regulation Effects 0.000 description 1
- 230000036978 cell physiology Effects 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
- 238000003776 cleavage reaction Methods 0.000 description 1
- 239000002299 complementary DNA Substances 0.000 description 1
- 239000012297 crystallization seed Substances 0.000 description 1
- 238000012258 culturing Methods 0.000 description 1
- 238000004925 denaturation Methods 0.000 description 1
- 230000036425 denaturation Effects 0.000 description 1
- IWEDIXLBFLAXBO-UHFFFAOYSA-N dicamba Chemical compound COC1=C(Cl)C=CC(Cl)=C1C(O)=O IWEDIXLBFLAXBO-UHFFFAOYSA-N 0.000 description 1
- 239000000539 dimer Substances 0.000 description 1
- MWEQTWJABOLLOS-UHFFFAOYSA-L disodium;[[[5-(6-aminopurin-9-yl)-3,4-dihydroxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-oxidophosphoryl] hydrogen phosphate;trihydrate Chemical compound O.O.O.[Na+].[Na+].C1=NC=2C(N)=NC=NC=2N1C1OC(COP(O)(=O)OP([O-])(=O)OP(O)([O-])=O)C(O)C1O MWEQTWJABOLLOS-UHFFFAOYSA-L 0.000 description 1
- 210000005069 ears Anatomy 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 210000002472 endoplasmic reticulum Anatomy 0.000 description 1
- 238000003912 environmental pollution Methods 0.000 description 1
- 238000001976 enzyme digestion Methods 0.000 description 1
- 235000021321 essential mineral Nutrition 0.000 description 1
- 210000003527 eukaryotic cell Anatomy 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 238000009313 farming Methods 0.000 description 1
- 238000007672 fourth generation sequencing Methods 0.000 description 1
- 229960003180 glutathione Drugs 0.000 description 1
- 235000021312 gluten Nutrition 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
- 239000008187 granular material Substances 0.000 description 1
- 231100000001 growth retardation Toxicity 0.000 description 1
- 210000004458 heterochromatin Anatomy 0.000 description 1
- 238000007901 in situ hybridization Methods 0.000 description 1
- 230000001939 inductive effect Effects 0.000 description 1
- 239000004615 ingredient Substances 0.000 description 1
- 230000000977 initiatory effect Effects 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
- 230000007774 longterm Effects 0.000 description 1
- 230000014759 maintenance of location Effects 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 230000035800 maturation Effects 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 108020004999 messenger RNA Proteins 0.000 description 1
- 239000011707 mineral Substances 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
- 238000009740 moulding (composite fabrication) Methods 0.000 description 1
- 230000035772 mutation Effects 0.000 description 1
- 239000002102 nanobead Substances 0.000 description 1
- 230000000422 nocturnal effect Effects 0.000 description 1
- 239000002853 nucleic acid probe Substances 0.000 description 1
- 230000031787 nutrient reservoir activity Effects 0.000 description 1
- 239000003921 oil Substances 0.000 description 1
- 235000019198 oils Nutrition 0.000 description 1
- 230000003287 optical effect Effects 0.000 description 1
- 210000000056 organ Anatomy 0.000 description 1
- 210000003463 organelle Anatomy 0.000 description 1
- 244000052769 pathogen Species 0.000 description 1
- 239000010452 phosphate Substances 0.000 description 1
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 description 1
- 239000002686 phosphate fertilizer Substances 0.000 description 1
- 229910052698 phosphorus Inorganic materials 0.000 description 1
- 239000011574 phosphorus Substances 0.000 description 1
- 230000008635 plant growth Effects 0.000 description 1
- 229920002647 polyamide Polymers 0.000 description 1
- 239000011591 potassium Substances 0.000 description 1
- 229910052700 potassium Inorganic materials 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
- 238000000746 purification Methods 0.000 description 1
- 238000003908 quality control method Methods 0.000 description 1
- 230000002285 radioactive effect Effects 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 238000003259 recombinant expression Methods 0.000 description 1
- 230000001172 regenerating effect Effects 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 230000004044 response Effects 0.000 description 1
- 108091008146 restriction endonucleases Proteins 0.000 description 1
- 235000009566 rice Nutrition 0.000 description 1
- 229920002477 rna polymer Polymers 0.000 description 1
- 230000007017 scission Effects 0.000 description 1
- 239000001509 sodium citrate Substances 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
- 239000002904 solvent Substances 0.000 description 1
- 230000002269 spontaneous effect Effects 0.000 description 1
- 230000006641 stabilisation Effects 0.000 description 1
- 238000011105 stabilization Methods 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 239000000758 substrate Substances 0.000 description 1
- 230000009469 supplementation Effects 0.000 description 1
- 230000001502 supplementing effect Effects 0.000 description 1
- 230000002123 temporal effect Effects 0.000 description 1
- 235000012184 tortilla Nutrition 0.000 description 1
- 230000001988 toxicity Effects 0.000 description 1
- 231100000419 toxicity Toxicity 0.000 description 1
- 238000013518 transcription Methods 0.000 description 1
- 230000035897 transcription 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
- 238000004383 yellowing Methods 0.000 description 1
Images
Classifications
-
- 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
Landscapes
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention relates to a nucleic acid molecule for detecting a corn plant ND4401 and a detection method thereof, wherein the nucleic acid molecule sequence of the corn plant comprises a sequence shown in SEQ ID NO. 1 or a complementary sequence thereof, or a sequence shown in SEQ ID NO. 2 or a complementary sequence thereof. The maize plant ND4401 of the invention has high yield and/or nitrogen deficiency tolerance traits, and the detection method can accurately and rapidly identify whether a biological sample contains the DNA molecule of the transgenic maize event ND 4401.
Description
Technical Field
The invention relates to the technical field of plant biology. In particular to a nucleic acid molecule for detecting a corn plant ND4401 and a detection method thereof, in particular to a transgenic corn event ND4401 with high yield and/or nitrogen deficiency tolerance characters, a nucleic acid molecule for detecting whether a biological sample contains the specific transgenic corn event ND4401 and a detection method thereof.
Background
Nitrogen is one of the essential mineral nutrients of plants and an essential component for the synthesis of proteins, nucleic acids and many biologically active substances. Nitrogen deficiency usually leads to Plant growth retardation, green-yellow mature leaves with Accumulation of anthocyanins (Diaz C, Salibani V, Loudet O, et al, leaf Yellowing and Anthocynin Accumulation area Two genetic introduction Strategies in Response to Nitrogen Limitation in Arabidopsis thaliana [ J ]. Plant & Cell Physiology,2006,47(1): 74-83.). The large use of nitrogen fertilizer in agricultural production greatly improves crop yield. The current global annual application of about 8-9 million tons of nitrogen fertilizer is predicted to increase to 2.4 million tons in 2050 (Timman D. Global environmental impacts of agricultural expansion: the connected for stable and effective pathogens [ J ]. Proc Natl Acad Sci U S.1999, 96(11): 5995-. The increase of energy cost leads to the continuous increase of the price of the nitrogen fertilizer, thereby increasing the agricultural production cost, and simultaneously the loss of the nitrogen fertilizer gradually deteriorates the ecological environment. Based on double considerations of economic benefit and environmental protection, planting nitrogen-efficient crop varieties in modern agricultural production is an effective way for solving the problems of low utilization rate of nitrogen fertilizer, reduction of production cost and reduction of environmental pollution, and is also a basic requirement for agricultural sustainable development and enhancement of international competitiveness of products. The traditional breeding of new species is a very long-term process, and nowadays, the continuous maturation of genetic engineering makes it possible to rapidly obtain nitrogen-efficient species by means of genetic engineering. The maize ZmNRT1.1 gene encodes a nitrate transporter with low affinity, which can influence the absorption of nitrate nitrogen by the root system of a plant, thereby improving the utilization efficiency of nitrogen (Quaggiotti S, Ruperti B, Pizzeghello D, et al. Effect of low molecular size maize substructures on nitrate uptake and expression of genes involved in nitrate transport in mail (Zea Mays L.) [ J ]. Journal of Experimental Boy, 2004,55(398):816 and 823.), and can be used for improving the tolerance of maize under the condition of nitrogen deficiency and finally increasing the yield.
It is known that expression of foreign genes in plants is influenced by their chromosomal location, possibly due to chromatin structure (e.g., heterochromatin) or the proximity of transcriptional regulatory elements (e.g., enhancers) to the integration site. For this reason, it is often necessary to screen a large number of events in order to be able to identify a commercializable event (i.e., an event in which the introduced gene of interest is optimally expressed). For example, it has been observed in plants and other organisms that the amount of expression of an introduced gene may vary greatly between events; there may also be differences in the spatial or temporal pattern of expression, such as differences in the relative expression of the transgene between different plant tissues, in that the actual expression pattern may not be consistent with the expression pattern expected from the transcriptional regulatory elements in the introduced gene construct, resulting in differences in the performance of the transformation event in the trait. Thus, it is often necessary to generate hundreds to thousands of different events and to screen those events for a single event with the amount and pattern of transgene expression expected for commercial purposes. Events with expected expression levels and patterns of transgenes can be used to introgress the transgenes into other genetic backgrounds by sexual outcrossing using conventional breeding methods. Progeny produced by this crossing pattern retain the transgene expression characteristics of the original transformation event. The use of this strategy ensures reliable gene expression in many varieties that are well adapted to local growth conditions. Therefore, there is a need for trait identification and screening of more transformation events to obtain superior transformation events with superior overall trait performance and commercial prospects.
It would be beneficial to be able to detect the presence of particular events to determine whether progeny of a sexual cross contain a gene of interest. Furthermore, methods of detecting specific events will also help to comply with relevant regulations, such as that foods derived from recombinant crops need to be officially approved and labeled before being placed on the market. It is possible to detect the presence of a transgene by any well-known polynucleotide detection method, such as Polymerase Chain Reaction (PCR) or DNA hybridization using polynucleotide probes. These detection methods usually focus 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, this method described above cannot be used to distinguish between different events, particularly those produced with the same DNA construct. Therefore, it is now common to identify specific events of a transgene 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 yield traits and/or nitrogen deficiency tolerance traits, a nucleic acid molecule for detecting a corn plant ND4401 and a detection method thereof. Transgenic corn event ND4401 exhibits high yield and/or nitrogen deficiency tolerance traits due to its high nitrogen utilization efficiency, and the detection method can accurately and rapidly identify whether a biological sample contains the DNA molecule of a particular transgenic corn event ND 4401.
In order to realize the purpose, 45 ZmNRTT 1.1A and bar gene-transferred maize transformation events are obtained by transforming the pCAMBIA 1301-ZmNRTT 1.1A expression vector into maize y822 immature embryos by utilizing an agrobacterium infection technology. Among them, ND4401 shows stable generation inheritance, nitrogen deficiency tolerance and outstanding yield traits, and has good application prospects.
In order to characterize the identity of ND4401, the invention provides a nucleic acid molecule comprising the sequence shown in SEQ ID NO. 1 and/or SEQ ID NO. 2, or the complement thereof.
Further, the nucleic acid molecule sequence comprises a sequence shown as SEQ ID NO. 3, or a complementary sequence thereof.
Further, the nucleic acid molecule sequence comprises the sequence shown in SEQ ID NO. 4 or a complementary sequence thereof.
In another aspect, the invention provides a probe for detecting a maize transformation event, comprising the sequence shown in SEQ ID No. 1 or SEQ ID No. 2 or SEQ ID No. 3 or SEQ ID No. 4 or a fragment or variant or complement thereof.
The invention also provides a primer pair for detecting a corn transformation event, which is characterized by comprising:
a primer which specifically recognizes the 1 st to 625 nd nucleotide sequences of the sequence shown by SEQ ID NO. 4 and a primer which specifically recognizes the 626-4920 nd nucleotide sequences of the sequence shown by SEQ ID NO. 4; and/or
A primer which specifically recognizes the 626-4920 nucleotide sequence of the sequence shown by SEQ ID NO. 4 and a primer which specifically recognizes the 4921-5052 nucleotide sequence of the sequence shown by SEQ ID NO. 4;
in some embodiments, the amplification product of the primer pair comprises the sequence of claim 2;
in some embodiments, the primer pair is the sequences shown in SEQ ID NO 5 and SEQ ID NO 6 or complements thereof.
The invention also provides a kit or microarray for detecting a maize transformation event, characterized in that it comprises the probe described above and/or the primer pair described above.
The invention also provides a method for detecting corn transformation events, which is characterized by detecting whether the transformation events exist in a sample to be detected by using the probe or the primer pair or the probe and primer pair or the kit or 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;
2) subjecting the corn obtained in step 1) to pollen culture, unfertilized embryo culture, doubling culture, cell culture, tissue culture, selfing or crossing or a combination thereof to obtain a corn plant, seed, plant cell, progeny plant or plant part; and optionally also (c) a second set of one or more of,
3) subjecting the progeny plants obtained in step 2) to yield trait identification and/or nitrogen deficiency tolerance identification and testing for the presence of said transformation event therein using the methods described above.
Further, the invention also provides products made of the corn plants, seeds, plant cells, progeny plants or plant parts obtained by the method, including food, feed or industrial raw materials.
In the present invention, the nucleic acid molecule sequence may be at least 11 or more contiguous polynucleotides of any portion of the transgene insert sequence in said SEQ ID NO:1 or the complement thereof (first nucleic acid molecule) or at least 11 or more contiguous polynucleotides of any portion of the 5' left flank corn genomic DNA region in said SEQ ID NO:1 or the complement thereof (second nucleic acid molecule). The nucleic acid molecule may further be homologous or complementary to a portion of said SEQ ID NO. 3 comprising the entire said SEQ ID NO. 5. When the first nucleic acid molecule and the second nucleic acid molecule are used together, these nucleic acid molecules comprise a DNA primer pair in a DNA amplification method that produces an amplification product. The presence of transgenic maize event ND4401 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 4.
The SEQ ID NO:1 or its complement is a 1363 nucleotide long sequence located near the insertion junction at the 5' end of the insertion in transgenic maize event ND4401, the SEQ ID NO:1 or its complement consists of a 625 nucleotide maize left flank genomic DNA sequence (nucleotides 1-625 of SEQ ID NO: 1), a 69 nucleotide pCAMBIA1301-ZmNRT1.1A construct left border DNA sequence (nucleotides 626-694 of SEQ ID NO: 1) and a 669 nucleotide first expression cassette DNA sequence of the glufosinate-resistant gene (nucleotides 695-1363 of SEQ ID NO: 1), comprising the SEQ ID NO:1 or its complement is identifiable as the presence of transgenic maize event ND 4401.
The nucleic acid molecule sequence may be at least 11 or more contiguous polynucleotides of any portion of the transgene insert sequence in SEQ ID No. 2 or its complement (third nucleic acid molecule) or at least 11 or more contiguous polynucleotides of any portion of the 3' right flanking corn genomic DNA region in SEQ ID No. 2 or its complement (fourth nucleic acid molecule). When the third nucleic acid molecule and the fourth nucleic acid molecule are used together, these nucleic acid molecules comprise a set of DNA primers in a DNA amplification method that produces an amplification product. The presence of transgenic maize event ND4401 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.
The SEQ ID NO 2 or its complement is a 720 nucleotide long sequence located near the insertion junction at the 3 'end of the insert in transgenic maize event ND4401, the SEQ ID NO 2 or its complement consists of the 3' end DNA sequence of the second cassette for a 581 nucleotide nitrogen efficient utilization gene (nucleotides 1-581 of SEQ ID NO: 2), the 7 nucleotide pCAMBIA 1301-ZmNRTT 1.1A construct right border DNA sequence (nucleotides 582-588 of SEQ ID NO: 2) and the 132 nucleotide genomic DNA sequence right flanking the maize integration site (nucleotides 589-720 of SEQ ID NO: 2), comprising the SEQ ID NO:2 or its complement identifies the presence of transgenic maize event ND 4401.
4 or the complement thereof is a 5052 nucleotide long sequence that characterizes transgenic maize event ND4401, specifically comprising genomic and genetic elements as shown in table 1. The presence of transgenic maize event ND4401 can be identified by comprising said SEQ ID No. 4 or the complement thereof.
TABLE 1 genomic and genetic elements contained in SEQ ID NO. 4
1: the unit bp.
It is well known to those skilled in the art that the first and second nucleic acid molecules or the third and fourth nucleic acid molecules 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 analogs thereof that do not serve as templates for one or more polymerases. In addition, 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 contiguous nucleotides in length, which may be selected from the nucleotides set forth in SEQ ID NO 1, SEQ ID NO 2, SEQ ID NO 3 or SEQ ID NO 4. When selected from the nucleotides set forth in SEQ ID NO. 1, SEQ ID NO. 2, SEQ ID NO. 3, or SEQ ID NO. 4, the probes and primers may be contiguous nucleotides of at least about 21 to about 50 or more in length.
The present invention also provides a method for increasing maize yield and/or nitrogen deficiency tolerance, comprising growing in soil at least one transgenic maize plant comprising in its genome the nucleic acid sequence at position 2165-5052 of SEQ ID NO 4 or comprising in its genome SEQ ID NO 4; the transgenic maize plants have high yield and/or nitrogen deficiency tolerance traits.
The invention also provides a method for improving the yield and/or the nitrogen deficiency tolerance of corn, which is characterized in that an expression cassette for expressing a nitrogen high-efficiency utilization gene with a sequence shown as the 2165-4913 nucleotide of SEQ ID NO. 4 is introduced into the corn genome.
In the nucleic acid molecules and methods for detecting maize plants of the present invention, the following definitions and methods may better define the invention and guide those of ordinary skill in the art in the practice of the invention, unless otherwise indicated, the terms are understood according to their conventional usage by those of ordinary skill in the art.
The "corn" refers to maize (Zea mays) and includes all plant species that can be mated with corn, including wild corn species.
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 granules), and plant cells intact in plants or plant parts such as embryos, pollen, ovules, seeds, leaves, flowers, branches, fruits, stalks, roots, root tips, anthers, and the like. It is to be understood that parts of transgenic plants within the scope of the present invention, which are derived from transgenic plants or progeny thereof which have been previously transformed with a DNA molecule of the invention and thus consist at least in part of transgenic cells, include, but are not limited to, plant cells, protoplasts, tissue, callus, embryos, and flowers, stems, fruits, leaves, and roots.
The term "gene" refers to a nucleic acid fragment that expresses a particular protein, including regulatory sequences preceding the coding sequence (5 'non-coding sequences) and regulatory sequences following the coding sequence (3' non-coding sequences). "native gene" refers to a gene that is naturally found to have its own regulatory sequences. "chimeric gene" refers to any gene that is not a native gene, comprising regulatory and coding sequences not found in nature. "endogenous gene" refers to a native gene that is located in its natural location in the genome of an organism. "foreign gene" is a foreign gene that exists in the genome of an organism and does not originally exist, and also refers to a gene that has been introduced into a recipient cell through a transgenic step. The foreign gene may comprise a native gene or a 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 at which the recombinant DNA has been inserted may be referred to as an "insertion site" or "target site".
"flanking DNA" may comprise the genome as it occurs naturally in an organism such as a plant or foreign (heterologous) DNA introduced by the transformation process, such as a fragment associated with the transformation event. Thus, flanking DNA may include a combination of native and foreign DNA. In the present invention, a "flanking region" or "flanking sequence" or "genomic boundary region" or "genomic boundary 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 in length, which is located directly upstream or downstream of and adjacent to the originally exogenously inserted DNA molecule. When the flanking region is located upstream, it may also be referred to as "left border flanking" or "5 'genomic flanking region" or "genomic 5' flanking sequence" or the like. When the flanking region is located downstream, it may also be referred to as "right border flanking" or "3 'genomic flanking region" or "genomic 3' flanking sequence" or the like.
Transformation procedures that result in random integration of the exogenous DNA will result in transformation events that contain different flanking regions that are specific for each transformation event. When the recombinant DNA is introduced into a plant by conventional crossing, its flanking regions are not usually altered. Transformation events will also contain unique junctions between the heterologous insert DNA and segments of genomic DNA or between two segments of heterologous DNA. "junction" is the point at which two specific DNA fragments are joined. For example, the junction is present where the insert DNA joins the flanking DNA. A junction point is also present in a transformed organism where two DNA fragments are joined together in a manner that is modified in the way found in the native organism. "junction DNA" refers to DNA comprising a junction site.
The present invention provides a transgenic corn event designated ND4401, and progeny thereof, said transgenic corn event ND4401 being a corn plant ND4401, comprising plants and seeds of transgenic corn event ND4401 and plant cells thereof or regenerable parts thereof, said plant parts of transgenic corn event ND4401, including but not limited to cells, pollen, ovules, flowers, buds, roots, stems, silks, inflorescences, ears, leaves, and products from corn plant ND4401, such as corn flour, corn oil, corn steep liquor, corn silks, corn starch, and biomass left in a corn crop field.
Transgenic maize event ND4401 of the invention comprises a DNA construct which when expressed in a plant cell, confers enhanced yield and/or nitrogen deficiency tolerance traits to said transgenic maize event ND 4401. The DNA construct comprises an expression cassette comprising a suitable promoter for expression in plants operably linked to a nucleic acid molecule encoding a maize nitrate transporter gene ZmNAR1.1A, said ZmNAR1.1A being tolerant to nitrogen deficiency and a suitable polyadenylation signal sequence. The DNA construct comprises a further expression cassette comprising a suitable promoter for expression in plants operably linked to the bar gene encoding Phosphinothricin Acetyltransferase (PAT) protein whose nucleic acid molecule is tolerant to glufosinate herbicides and which can be used as a selection marker for the transformation stage, and a suitable polyadenylation signal sequence. Further, the promoter may be a suitable promoter isolated from a plant, including constitutive, inducible and/or tissue specific promoters, suitable promoters include, but are not limited to, the cauliflower mosaic virus (CaMV)35S promoter, the Figwort Mosaic Virus (FMV)35S promoter, the Ubiquitin protein (Ubiquitin) promoter, the Actin (Actin) promoter, the Agrobacterium tumefaciens (Agrobacterium tumefaciens) nopaline synthase (NOS) promoter, the octopine synthase (OCS) promoter, the nocturnal (Cestrum) yellow leaf curly virus promoter, the potato tuber storage protein (Patatin) promoter, the ribulose-1, 5-bisphosphate carboxylase/oxygenase (rusco) promoter, the glutathione thiotransferase (GST) promoter, the E9 promoter, the GOS promoter, the alcA/alcR promoter, the Agrobacterium rhizogenes (Agrobacterium rhizogenes) RolD promoter, and the Arabidopsis thaliana (Arabidopsis thaliana) Suc2 promoter. The polyadenylation signal sequence may be a suitable polyadenylation signal sequence that functions in plants, including, but not limited to, polyadenylation signal sequence derived from the Agrobacterium tumefaciens nopaline synthase (NOS) gene, cauliflower mosaic virus (CaMV)35S terminator, polyadenylation signal sequence derived from the protease inhibitor II (PIN II) gene, and polyadenylation signal sequence derived 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/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 transportation of the EPSPS protein to a particular organelle or compartment outside or within the cell, for example, targeting the chloroplast using a sequence encoding a chloroplast transit peptide, or targeting the endoplasmic reticulum using a 'KDEL' retention sequence.
The DNA construct is introduced into a plant using transformation methods including, but not limited to, Agrobacterium-mediated transformation, biolistic transformation, and pollen tube channel transformation.
The Agrobacterium-mediated transformation method is a commonly used 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. Said vector is transformed into an agrobacterium cell, which is subsequently used to infect plant tissue, said T-DNA region of the vector comprising the foreign DNA being inserted into the plant genome.
After transformation, transgenic plants must be regenerated from the transformed plant tissue and progeny with exogenous DNA selected using appropriate markers.
A DNA construct is a combination of DNA molecules linked together to provide one or more expression cassettes. The DNA construct is preferably a plasmid capable of autonomous replication in bacterial cells and containing different 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, which includes the genetic elements necessary to provide for transcription of messenger RNA, can be designed for expression in prokaryotic or eukaryotic cells. The expression cassette of the invention is designed to be most preferably expressed 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 population of plants, regenerating said population of plants, and selecting for a particular plant characterized by the insertion of a particular genomic locus. The term "event" refers to both the original transformation event comprising the heterologous DNA and the progeny of the transformation event. The term "event" also refers to progeny resulting from sexual crosses between a transformation event and individuals of other varieties containing heterologous DNA, where the inserted DNA and flanking genomic DNA from the parent of the transformation event are present in the same chromosomal location in the progeny of the cross, even after repeated backcrosses with the backcross parents. 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 to progeny that result 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 that does not contain 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 a form of DNA and/or protein and/or organism that is not normally found in nature and is therefore produced by human intervention. Such manual intervention may result in recombinant DNA molecules and/or recombinant plants. Such "recombinant DNA molecules" are obtained by artificially combining two otherwise isolated sequence segments, for example, by chemical synthesis or by manipulating an isolated nucleic acid segment 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 heterologous nucleic acid, including the transgene body that was originally so altered, as well as progeny individuals generated from the original transgene body by sexual crossing or asexual reproduction. In the present invention, the term "transgene" does not include changes (chromosomal or extra-chromosomal) in the genome by conventional plant breeding methods or naturally occurring events such as random allofertilization, non-recombinant viral infection, non-recombinant bacterial transformation, non-recombinant transposition, or spontaneous mutation.
By "heterologous" in the context of 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 be derived from a first species and inserted into the genome of a second species. Such molecules are therefore heterologous to the host and are artificially introduced into the genome of the host cell.
Culturing a transgenic maize event ND4401 that is highly productive and/or tolerant to nitrogen deficiency by: first sexually crossing a first parent corn plant consisting of a corn plant bred from transgenic corn event ND4401 and its progeny obtained by transformation with a nitrogen efficient use expression cassette of the present invention with a second parent corn plant lacking high yield and/or nitrogen deficiency tolerance, thereby producing a multiplicity of first generation progeny plants; progeny plants that are tolerant to nitrogen deficiency or have increased yield are then selected and maize plants that are tolerant to nitrogen deficiency or have increased yield can be grown. These steps can further include backcrossing the high-yielding and/or nitrogen-deficient tolerant progeny plants with the second or third parent corn plant, and then selecting the progeny through identification of a trait-related molecular marker (e.g., a DNA molecule comprising the junction site identified at the 5 'end and 3' end of the insert sequence in transgenic corn event ND 4401) to produce yield-improved and/or nitrogen-deficient tolerant corn plants.
It is also understood that two different transgenic plants may also be crossed to produce progeny containing two separate, separately added exogenous genes. Selfing of appropriate progeny can yield progeny plants that are homozygous for both added exogenous genes. Backcrossing of parental plants and outcrossing with non-transgenic plants as described above is also contemplated, as is asexual propagation.
The term "probe" is an isolated nucleic acid molecule having a conventional detectable label or reporter molecule, e.g., a radioisotope, ligand, chemiluminescent agent or enzyme, bound thereto. Such a probe is complementary to one strand of the target nucleic acid, and in the present invention, the probe is complementary to one DNA strand from the genome of transgenic corn event ND4401, whether the genomic DNA is from transgenic corn event ND4401 or seed or a plant or seed or extract derived from transgenic corn event ND 4401. Probes of the invention include not only deoxyribonucleic or ribonucleic acids, 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, annealing, forming a hybrid between the primer and the target DNA strand, and then extending along the target DNA strand under the action of a polymerase (e.g., a DNA polymerase). The primer pairs of the present invention are directed to their use in the amplification of a target nucleic acid molecule, for example, by Polymerase Chain Reaction (PCR) or other conventional nucleic acid amplification methods.
The length of the probes and primers 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 probes and primers hybridize specifically to the target sequence under highly stringent hybridization conditions. Although probes that are different from and maintain the ability to hybridize to the target DNA sequence can be designed by conventional methods, it is preferred that the probes and primers of the present invention have complete DNA sequence identity to the contiguous nucleic acid 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 corn transformation events in a biological sample. For the purpose of quality control (e.g. purity of seed lot), detection of events in or in a material comprising or derived from plant material, such as, but not limited to, food or feed products, kits or chips may be used, and components thereof may be specifically adjusted.
Primers and probes based on the flanking genomic DNA and insertion sequences of the present invention can be determined by conventional methods, for example, by isolating the corresponding DNA molecule from plant material derived from transgenic maize event ND4401 and determining the nucleic acid sequence of the DNA molecule. The DNA molecule comprises a transgene insert and a maize genomic flanking region, and fragments of the DNA molecule can be used as primers or probes.
The nucleic acid probes and primers of the invention hybridize to a target DNA sequence under stringent conditions. Any conventional nucleic acid hybridization or amplification method can be used to identify the presence of DNA derived from transgenic corn event ND4401 in a sample. Nucleic acid molecules or fragments thereof are capable of specifically hybridizing to other nucleic acid molecules under certain circumstances. As used herein, two nucleic acid molecules can be said to be capable of specifically hybridizing to each other if they are capable of forming an antiparallel, double-stranded nucleic acid structure. Two nucleic acid molecules are said to be "complements" of one another if they exhibit complete complementarity. As used herein, a nucleic acid molecule is said to exhibit "perfect complementarity" when each nucleotide of the nucleic acid molecule is complementary to a corresponding nucleotide of another 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 to allow them to 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 to allow them to anneal and bind to each other under conventional "highly stringent" conditions. Deviations from perfect complementarity may be tolerated as long as such deviations do not completely prevent the formation of a double-stranded structure by the two molecules. In order to allow 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 in the particular solvent and salt concentrations employed.
As used herein, a substantially homologous sequence is a nucleic acid molecule that specifically hybridizes under highly stringent conditions to the complementary strand of a matched nucleic acid molecule. Suitable stringency conditions for promoting DNA hybridization include, for example, treatment with 6.0 XSSC/sodium citrate (SSC) at about 45 ℃ followed by a wash with 2.0 XSSC at 50 ℃, as is well known to those skilled in the art. For example, the salt concentration in the washing step can be selected from the group consisting of about 2.0 XSSC for low stringency conditions, 50 ℃ to about 0.2 XSSC for high stringency conditions, 50 ℃. In addition, the temperature conditions in the washing step can be raised from about 22 ℃ at room temperature for low stringency conditions to about 65 ℃ for high stringency conditions. Both the temperature conditions and the salt concentration may be varied, or one may be held constant while the other is varied. Preferably, a nucleic acid molecule of the invention can specifically hybridize to one or more of 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, or complements thereof, or any fragment thereof, under moderately stringent conditions, such as at about 2.0 XSSC and about 65 ℃. More preferably, a nucleic acid molecule of the invention specifically hybridizes under highly stringent conditions to one or more of SEQ ID NO 1, SEQ ID NO 2, SEQ ID NO 3 and SEQ ID NO 4 or to a complementary sequence thereof, or to a fragment of any of the foregoing. In the context of the present invention, preferred marker nucleic acid molecules have SEQ ID NO 1, SEQ ID NO 2, SEQ ID NO 3 or SEQ ID NO 4 or the complement thereof or any fragment of the aforementioned 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, 2, 3 or 4 or the complement thereof or any fragment thereof. SEQ ID NO 1, SEQ ID NO 2, SEQ ID NO 3 or SEQ ID NO 4 can be used as markers in plant breeding methods to identify progeny of a genetic cross. Hybridization of the probe to the target DNA molecule can be detected by any method known to those skilled in the art, including, but not limited to, fluorescent, radioactive, antibody-based, and chemiluminescent labels.
With respect to amplification of a target nucleic acid molecule using a particular amplification primer (e.g., by PCR), "stringent conditions" refer to conditions that allow only hybridization of the primer to the target nucleic acid molecule in a DNA thermal amplification reaction, a primer having a sequence corresponding to the target nucleic acid molecule (or its complement) that is capable of binding to the target nucleic acid molecule and preferably producing a unique amplification product, i.e., an amplicon.
The term "specifically binds (target sequence)" means that the probe or primer hybridizes only to the target molecule in a sample containing the target molecule under stringent hybridization conditions.
As used herein, "amplified DNA" or "amplicon" refers to the nucleic acid amplification product of a target nucleic acid molecule that is part of a nucleic acid template. For example, to determine whether a corn plant was produced by sexual hybridization comprising the transgenic corn event ND4401 of the present invention, or whether a corn sample collected from a field comprises the transgenic corn event ND4401, or whether a corn extract, such as meal, flour, or oil, comprises the transgenic corn event ND4401, DNA extracted from a corn plant tissue sample or extract can be subjected to a nucleic acid amplification method using a primer pair to produce an amplicon diagnostic for the presence of DNA of the transgenic corn event ND 4401. The primer pair includes a first primer derived from a flanking sequence adjacent to the insertion site of the inserted foreign DNA in the plant genome, 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 ND 4401.
The amplicon can range in length from the bound 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, primer pairs may be derived from flanking genomic sequences flanking the inserted DNA to produce amplicons that include the entire inserted nucleotide sequence. One of the primer pairs derived from a 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 carried out by any of the nucleic acid amplification reaction methods known in the art, including the Polymerase Chain Reaction (PCR). Various nucleic acid amplification methods are well known to those skilled in the art. The PCR amplification method has been developed to amplify 22kb of genomic DNA and 42kb of phage DNA. These methods, as well as other DNA amplification methods known in the art, can be used in the present invention. The inserted exogenous DNA sequence and flanking DNA sequences from transgenic maize event ND4401 can be obtained by amplifying the genome of transgenic maize event ND4401 using the provided primer sequences, followed by standard DNA sequencing of the PCR amplicons or cloned DNA.
DNA detection kits based on DNA amplification methods contain DNA primer molecules that specifically hybridize to the target DNA and amplify the diagnostic amplicon under appropriate reaction conditions. The kit may provide an agarose gel based detection method or a number of methods known in the art for detecting diagnostic amplicons. Kits containing DNA primers homologous or complementary to any portion of the maize genomic region of SEQ ID NO 1 or SEQ ID NO 2 and to any portion of the transgene insert region of SEQ ID NO 4 are provided by the present invention. Specifically identifying primer pairs useful in DNA amplification methods are SEQ ID NO 5 and SEQ ID NO 6 which amplify a diagnostic amplicon homologous to a portion of the 5' transgene/genomic region of transgenic maize event ND4401, wherein the amplicon comprises SEQ ID NO 3.
Amplicons produced by these methods can be detected by a variety of techniques. One such method is Genetic Bit Analysis, which designs a DNA oligonucleotide strand spanning an intervening DNA sequence and adjacent flanking genomic DNA sequences. The oligonucleotide strand is immobilized within a microwell of a microwell plate, and after PCR amplification of the target region (using one primer in each of the intervening sequence and adjacent flanking genomic sequence), a 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 can be obtained by fluorescence or ELISA-like methods. The signal represents the presence of the inserted/flanking sequence, indicating that the amplification, hybridization and single base extension reactions were successful.
Another method is Pyrosequencing (Pyrosequencing) technology. The method designs an oligonucleotide strand that spans the junction of the inserted DNA sequence and the adjacent genomic DNA. The oligonucleotide strand is hybridized to the single-stranded PCR product of the target region (using one primer in each of the intervening and adjacent flanking genomic sequences), and then incubated with DNA polymerase, ATP, sulfurylase, luciferase, apyrase, adenosine-5' -phosphothioate, and luciferin. dNTPs were added separately and the resulting optical signals were measured. The light signal represents the presence of the inserted/flanking sequence, indicating that the amplification, hybridization, and single or multiple base extension reactions were successful.
Fluorescence polarization is also a method that can be used to detect the amplicons of the invention (Chen X, Levine L, and Kwok P Y. fluorescence polarization in genetic nucleic acid analysis [ J ]. Genome Res,1999,9(5): 492-8.). Using this method requires the design of an oligonucleotide strand that spans the intervening DNA sequence and adjacent genomic DNA binding sites. The oligonucleotide strand is hybridized to the single stranded PCR product of the target region (using one primer each within the insert sequence and adjacent flanking genomic sequence) and then incubated with DNA polymerase and a fluorescently labeled ddNTP. Single base extension will result in insertion of ddNTPs. This insertion can be measured for changes in its polarization using a fluorometer. The change in polarization represents the presence of the inserted/flanking sequence, indicating that the amplification, hybridization and single base extension reactions were successful.
Taqman is described as a method for detecting and quantifying the presence of DNA sequences, which is described in detail in the instructions provided by the manufacturer. Briefly, as illustrated below, a FRET oligonucleotide probe is designed to span the inserted DNA sequence and adjacent genomic flanking binding sites. The FRET probe and PCR primers (one primer for each of the flanking genomic sequences within the insert and adjacent) are cycled in the presence of a thermostable polymerase and dNTPs. Hybridization of the FRET probe results in cleavage of the fluorescent and quenching moieties on the FRET probe and release of the fluorescent moiety. The generation of a fluorescent signal represents the presence of the inserted/flanking sequence, indicating that amplification and hybridization were successful.
Suitable techniques for detecting plant material derived from transgenic maize event ND4401 can also include Southern blot hybridization, Northern blot hybridization and in situ hybridization based on hybridization principles. In particular, the suitable techniques include incubating the probe and sample, washing to remove unbound probe and detecting whether the probe has hybridised. The detection method depends on the type of label attached to the probe, for example, a radiolabeled probe can be detected by X-ray exposure and development, or an enzymatically labeled probe can be detected by a color change achieved by substrate conversion.
Sequences can also be detected using molecular markers (Tyagi S and Kramer F R. molecular beacons: probes that are fluorescent upon hybridization [ J ]. Nat Biotechnol,1996,14(3): 303-8.). A FRET oligonucleotide probe is designed that spans the inserted DNA sequence and the 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 a fluorescent moiety and a quenching moiety in close proximity. The FRET probe and PCR primers (one primer for each of the flanking genomic sequences within the insert and adjacent) 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 loss of the secondary structure of the probe, thereby spatially separating the fluorescent moiety from the quencher moiety to produce a fluorescent signal. The generation of a fluorescent signal represents the presence of the inserted/flanking sequence, indicating that amplification and hybridization were successful.
Other described methods, such as microfluidics (microfluidics), provide methods and apparatus for isolating and amplifying DNA samples. The fluorochromes are used to detect and measure specific DNA molecules. A nano-tube (nanotube) device comprising an electronic sensor for detecting DNA molecules or nano-beads binding to specific DNA molecules and thus being detectable is useful for detecting the DNA molecules of the present invention.
The DNA detection kit can be developed using the compositions described herein and methods described or known in the DNA detection art. The kit is useful for identifying the presence of DNA of transgenic corn event ND4401 in a sample, and can also be used for breeding corn plants containing DNA of transgenic corn event ND 4401. The kit may contain DNA primers or probes homologous or complementary to at least a portion of SEQ ID NO 1, 2, 3 or 4, 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 site of the maize genome containing the transgene insert and illustrated in table 1 comprises: a maize ND4401 left flanking genomic region located 5' to the transgene insert, a portion of the insert from the left border region of the vector, a first expression cassette consisting of a cauliflower mosaic virus 35S promoter (CaMV 35S promoter), operably linked to a glufosinate (glufosinate) resistance gene sequence (bar), and operably linked to a cauliflower mosaic virus 35S terminator (CaMV 35S polyA); the second expression cassette consists of a maize ubiquitin gene promoter (ubiquitin promoter), operably linked to the maize nitrate transporter gene ZmNAR1.1A, and operably linked to a nopaline synthase gene terminator (NOs terminator), a portion of the insertion sequence from the right border region of the vector, and the maize plant ND4401 right wing genomic region at the 3' end of the transgene insertion sequence (SEQ ID NO: 4). In the DNA amplification method, the DNA molecule that serves as a primer can be any portion of the DNA sequence derived from the transgene insertion sequence in transgenic corn event ND4401, or any portion of the DNA region flanking the corn genome in transgenic corn event ND 4401.
Transgenic corn event ND4401 can be combined with other transgenic corn varieties, such as herbicide (e.g., glyphosate, dicamba, etc.) tolerant corn, or transgenic corn varieties carrying insect-resistant genes. All of these various combinations of different transgenic events, when bred with transgenic corn event ND4401 of the invention, can provide improved hybrid transgenic corn varieties with multiple traits. These varieties can exhibit more excellent characteristics such as an increase in yield as compared with non-transgenic varieties and single-trait transgenic varieties.
The invention provides a nucleic acid molecule for detecting corn plants and a detection method thereof, wherein a transgenic corn event ND4401 has high-yield and/or nitrogen-deficiency tolerance traits. The maize plants of this trait express the nitrate transporter zmnar1.1a protein, which confers to plants a nitrogen efficient use trait to increase yield and/or nitrogen deficiency tolerance traits (herbicide resistance gene bar as a selection marker at the transformation stage). Meanwhile, the nucleic acid molecules of SEQ ID NO. 1 or a complementary sequence thereof, SEQ ID NO. 2 or a complementary sequence thereof, SEQ ID NO. 3 or a complementary sequence thereof, and SEQ ID NO. 4 or a complementary sequence thereof in the detection method of the present invention can be used as DNA primers or probes to generate an amplification product diagnosed as transgenic corn event ND4401 or its progeny, and the presence of plant material derived from transgenic corn event ND4401 can be identified rapidly, accurately and stably.
The technical solution of the present invention is further described in detail by the accompanying drawings and embodiments.
Drawings
FIG. 1 physical map of recombinant expression vector pCAMBIA 1301-ZmNRT1.1A. The English and abbreviated meanings of each element are listed as follows:
T-Border (left) T-DNA left border sequence.
35S terminator of CaMV35S polyA cauliflower mosaic virus (CaMV).
bar codes PAT protein and relieves glufosinate toxicity.
The 35S promoter of CaMV35S promoter cauliflower mosaic virus (CaMV).
The terminator of the nos terminator nopaline synthase gene.
ZmNAR1.1A AnVP1 gene CDS.
A promoter of a ubiquitin promoter maize ubiquitin gene.
The terminator of the nos terminator nopaline synthase gene.
T-Border (right) T-DNA right border sequence.
Plasmid stabilization site of PVS1 sta pVS1 plasmid.
The replication initiation site of the PVS1 rep pVS1 plasmid.
The bom site of the PBR322 bom pBR322 plasmid.
Origin of replication of the PBR322 ori pBR322 plasmid.
kanamycins (R) encode aminoglycoside phosphotransferase proteins that confer kanamycin resistance to bacteria.
FIG. 2 transformation event specific PCR validation results. M: DL2000 DNA marker; t: ND4401 corn material; CK: non-transgenic maize. The PCR fragment was expected to be 1069bp in size.
Detailed Description
The transformation event ND4401 refers to a maize plant which is obtained by taking a maize inbred line Y822 as a receptor and performing genetic transformation to insert an exogenous gene insert (T-DNA insert) between specific genome sequences. In a specific embodiment, the expression vector used for the transgene has the physical map shown in FIG. 1, and the resulting T-DNA insert has the sequence shown in nucleotide 695 and 4913 of SEQ ID NO. 4. Transformation event ND4401 may refer to this transgenic process, may also refer to the combination of a T-DNA insert and flanking sequences within the genome resulting from this process, or may refer to a maize plant resulting from this transgenic process. In a specific example, the event is also applicable to plants obtained by transforming other recipient varieties with the same expression vector and inserting the T-DNA insert into the same genomic position. Transformation event ND4401 may also refer to progeny plants derived from the above plants by vegetative propagation, sexual propagation, doubling or doubling, or a combination thereof.
Example 1 acquisition and screening of transformation events
The cDNA sequence of ZmNRT1.1A gene is cloned from corn, and is inserted into an intermediate vector pCAMBIA1301-Ubi through enzyme digestion connection, the intermediate vector already contains bar gene, and finally the intermediate vector is constructed into a pCAMBIA1301-ZmNRT1.1A expression vector, and the physical map of the vector is shown in figure 1. The recombinant plasmid is efficiently transformed into the young embryo of the corn y822, a selected line of agricultural biotechnology research institute of agricultural academy of sciences of Jilin province by utilizing an agrobacterium infection technology, and 45 transformation events of the corn with the ZmNRT1.1A and bar genes are obtained. The transgenic corn event ND4401 contains exogenous genes ZmNRT1.1A and bar, shows stable generation inheritance and outstanding yield traits and nitrogen deficiency tolerance, and has a good application prospect.
The detection method of the exogenous ZmNRT1.1A and bar genes comprises the following steps: the genome DNA of corn is used as a template, and PCR amplification is carried out by using specific primer pairs of ZmNRT1.1A and bar to respectively obtain the materials of strips with 452bp size (ZmNRT1.1A gene) and 441bp size (bar gene) as plants containing the ZmNRT1.1A gene and the bar gene.
TABLE 2 PCR detection primer sequences for the ZmNRT1.1A and bar genes
Example 2 nitrogen efficient utilization and yield trait identification of transformation event ND4401
The corn ZmNRT1.1A protein has the function of promoting nitrate absorption. The ZmNRT1.1A protein is expressed in the corn in a constitutive expression mode, and the nitrogen utilization efficiency of the corn is improved by improving the absorption capacity of the corn nitrate, so that the yield is finally improved. The invention identifies the field growth form and physiological characters of the ND4401 plants under different nitrogen fertilizer gradients.
The test site is a transgenic test base of agricultural scientific institute of Jilin province, four Ping city, principals, and four. The test soil is sandy loam with flat terrain, the content of alkaline hydrolysis nitrogen in a plough layer (20cm) soil is 125.94mg/kg, the content of quick-acting phosphorus is 23.96mg/kg, the content of quick-acting potassium is 149.24mg/kg, and the content of organic matters is 25.22 g/kg. The row length of each cell is 5m, the row spacing is 0.60m, the planting spacing is 0.25m, and each cell has 6 rows.
And (3) testing conditions are as follows: according to large formula and fertilization suggestions (2013) of three large grain crop regions, namely wheat, corn and rice, issued by rural parts in agriculture, the high nitrogen treatment (HN) is applied with 38 kg/mu of formula fertilizer according to the fertilization amount of normal farming; medium nitrogen treatment (MN): applying 70% of formula fertilizer, and supplementing phosphate fertilizer and potash fertilizer as same as high-nitrogen treatment; low nitrogen treatment (LN) no formulated fertilizer is applied, and phosphate and potash supplementation is the same as high nitrogen treatment.
Experiment design: the test adopts a random block design, 10 corns with approximately same growth vigor are taken in each cell, and relevant forms and physiological indexes are determined, wherein the specific indexes comprise: ear weight, ear length, ear row, hundred grain weight and total nitrogen content of the overground part of the plant.
The result shows that the ear weight, ear length and hundred-grain weight average of the corn ND4401 positive plant are remarkably higher than those of the negative plant under various nitrogen supply conditions, and the yield increasing effect is remarkable. Specifically, the weight of the spike is increased by 22.8%, the length of the spike is increased by 23.3% and the weight of the hundred grains is increased by 15.4% under the condition of low nitrogen. Under the condition of medium nitrogen, the weight of the spike is increased by 36.9%, the length of the spike is increased by 26.3%, the row number of the spike is increased by about 1 row, and the weight of the hundred grains is increased by 8.5%. Under high nitrogen conditions, the ear weight was increased by 37.1%, the ear length was increased by 24.9%, and the grain weight was increased by 31.4% (see table 3 for details). Yield of ND4401 negative plants under low nitrogen (nitrogen deficiency) conditions was only 76% (132.62/173.92) under high nitrogen (nitrogen sufficiency) conditions, while yield of ND4401 positive plants under low nitrogen (nitrogen deficiency) conditions reached 85% (202.70/238.48) under high nitrogen (nitrogen sufficiency) conditions, indicating that ND4401 positive plants were better tolerant to nitrogen deficiency conditions.
TABLE 3 comparison of agronomic traits for ND4401 and controls
From the results of the total nitrogen content of the plant stalks under different nitrogen conditions, the total nitrogen content of the ND4401 plants under different nitrogen conditions is lower than that of the receptor control material. Especially under high nitrogen conditions, the total nitrogen content of the control stalk is 3.00g/kg, while the total nitrogen content of ND4401 is only 2.17g/kg (Table 4). This indicates that the nitrogen utilization efficiency of ND4401 was higher than that of the wild-type control, and this is also the reason why ND4401 was produced in a higher amount.
TABLE 4 measurement of total nitrogen content of plant stalks by ND4401 and receptor control
Example 3 flanking sequences and maize genomic insertion position of transformation event ND4401 exogenous sequence
To clarify the identity of transformation event ND4401, the present invention further identifies the insertion site of the ND4401 exogenous sequence on the maize genome.
The invention adopts a third generation sequencing method to position the T-DNA insertion position of the transgenic corn ND 4401. The third generation sequencing technology utilizes Nanopore sequencing technology of Oxford Nanopore Technologies, ONT for short, in England to perform re-sequencing on the transformant ND4401, the sequencing depth is 10 times, the average read length of data is 20kb, the longest read length is 60kb, and the accuracy is 85%.
BLAST search alignment of the third generation sequencing results using the T-DNA foreign sequence revealed that a contig with a read length of approximately 14kb contained the target sequence, indicating that the foreign sequence in ND4401 was a single copy insertion. The 5 'end 625bp and the 3' end 132bp genome flanking sequences of the exogenous sequence obtained by sequencing are subjected to BLAST nucleic acid-nucleic acid alignment in a maize public genome database (http:// ensemble. gram. org/Zea _ mays/Info/association/# assembly), and the exogenous sequence is integrated at the position of maize chromosome 5 38327236-38327270.
At the left border of the insertion site, 625bp and an exogenous insertion sequence on the genome are selected to design a primer pair, so that an F primer can be combined with the genome sequence, an R primer can be combined with the exogenous insertion sequence, and an amplification product is fused with a part of corn genome sequence and a part of exogenous insertion sequence. And carrying out PCR detection on ND4401 by using the designed primer. And carrying out PCR amplification by using the genome DNA of the transgenic corn strain as a template. The PCR reaction was carried out in a 20. mu.L system. The amplification cycle program was: pre-denaturation at 94 ℃ for 3 min; denaturation at 94 ℃ for 30s, TmAnnealing for 30s, extending for a certain time (according to the size of a product fragment) at 72 ℃, and performing 35 cycles; extension at 72 ℃ for 5 min.
The ND4401 transformation event was PCR amplified using amplification of the genome upstream primer (SEQ ID NO:5) and the vector left border primer (SEQ ID NO:6) based on the results of the flanking sequences and the insertion positions to verify the foreign fragment insertion position. The results are shown in FIG. 2. The results demonstrated that the ND4401 foreign fragment was stably inserted into chromosome 5 at position 38327236-38327270.
Example 4 detection method of transformation event ND4401
Breeding can be performed from transgenic maize event ND4401 and producing, for example, an agricultural or commodity product from the bred new variety. If a sufficient amount is detected in the agricultural or commodity product, the agricultural or commodity product is expected to contain a nucleotide sequence capable of diagnosing the presence of transgenic corn event ND4401 material in the agricultural or commodity product. Such agricultural or commercial products include, but are not limited to, corn oil, corn meal, corn flour, corn gluten, corn tortillas, corn starch, and any other food product intended for consumption by an animal as a food source, or otherwise for cosmetic use as an ingredient in a bulking or cosmetic composition, and the like. A probe or primer pair based nucleic acid detection method and/or kit can be developed to detect a nucleic acid analysis of whether a transgenic corn event ND4401 is contained in a biological sample, wherein the probe sequence or primer amplification sequence is selected from the sequences as set forth in SEQ ID No. 1, SEQ ID No. 2, SEQ ID No. 3 or SEQ ID No. 4, to diagnose the presence of transgenic corn event ND 4401.
One detection method comprises the following steps:
the genome DNA of a corn sample is extracted by adopting a common CTAB method, and the specific operation process is implemented according to No. 1485 bulletin-4-2010 of Ministry of agriculture, and the DNA extraction and purification of the transgenic plants and the product components thereof. Wherein the corn sample comprises corn ND4401 to be transgenic and non-transgenic corn y 822. Detecting the specific boundary sequence in the ND4401 plant by using a PCR method, wherein the used PCR primer pairs are respectively SEQ ID NO. 5 and SEQ ID NO. 6, and the PCR reaction system comprises:
the reaction procedure is as follows:
and 2, step 4: circulating for 35 times
The PCR product was electrophoretically detected on a 1% (w/v)1 XTAE agarose gel, and the results are shown in FIG. 2. The expected target band (SEQ ID NO:3) was amplified in the ND4401 transformation event. Moreover, the PCR method can track the existence of transformation events, thereby being applied to breeding work.
In summary, the transgenic corn event ND4401 of the invention has higher yield and/or tolerance to nitrogen deficiency, and the detection method can accurately and rapidly identify whether the biological sample contains the DNA molecule of the transgenic corn event ND 4401.
Finally, it should be noted that the above embodiments are only for illustrating the technical solutions of the present invention and not for limiting, and although the present invention is described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications or equivalent substitutions may be made on the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention.
Sequence listing
<110> Jilin province academy of agricultural sciences
<120> nucleic acid molecule for detecting corn plant ND4401 and detection method thereof
<130> 1
<160> 6
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1363
<212> DNA
<213> Artificial Synthesis (unknown)
<400> 1
tcgcccgacc tcaggcttcg gcctcgggag gaggtctccg cctcgcctga cccccggact 60
cggcctcaac ctcggtctcg ggaggaatcg cgtcctcgcc cgaccccggc ctcggcctca 120
agagaagtct tcgcctcgcc cgaccttggg ctcggaccga ccgcgccaac aggggataca 180
tcattaccct atctctagcc tgctcaggct acaaggaaca agaccggtgt cccatctggc 240
ttaccccggc gacgggtaat gatggtgccc tgcgtgcacc atgacgccgg tggctctcag 300
ccccttacgg cagcaaggag acgtcagcag gacccttgcc gcactgccag ctgtgctcct 360
acagggctca ggcgcttctc cgacggccac actatcatgt gtatagggct caagctctcc 420
cccgacagcc acgttggcat gtacacaagg cttaggcatc tctctgccga acacgttagc 480
gcatagctac aaccccattg tacgactgga ccctctcctt gcgtctataa aagggaggtc 540
cagggccatc ctggaggggg gttcgcgcga gggaaggagt ggacgaacat gctccctctc 600
tctctttctc tccctctcat gacatgctta gacaacttaa taacacattg cggacgtttt 660
taatgtactg aattaacgcc gaattaattc gggggatctg gattttagta ctggattttg 720
gttttaggaa ttagaaattt tattgataga agtattttac aaatacaaat acatactaag 780
ggtttcttat atgctcaaca catgagcgaa accctatagg aaccctaatt cccttatctg 840
ggaactactc acacattatt atggagaaac tcgagtcaaa tctcggtgac gggcaggacc 900
ggacggggcg gtaccggcag gctgaagtcc agctgccaga aacccacgtc atgccagttc 960
ccgtgcttga agccggccgc ccgcagcatg ccgcgggggg catatccgag cgcctcgtgc 1020
atgcgcacgc tcgggtcgtt gggcagcccg atgacagcga ccacgctctt gaagccctgt 1080
gcctccaggg acttcagcag gtgggtgtag agcgtggagc ccagtcccgt ccgctggtgg 1140
cggggggaga cgtacacggt cgactcggcc gtccagtcgt aggcgttgcg tgccttccag 1200
gggcccgcgt aggcgatgcc ggcgacctcg ccgtccacct cggcgacgag ccagggatag 1260
cgctcccgca gacggacgag gtcgtccgtc cactcctgcg gttcctgcgg ctcggtacgg 1320
aagttgaccg tgcttgtctc gatgtagtgg ttgacgatgg tgc 1363
<210> 2
<211> 720
<212> DNA
<213> Artificial Synthesis (unknown)
<400> 2
ctgcagaagt aacaccaaac aacagggtga gcatcgacaa aagaaacagt accaagcaaa 60
taaatagcgt atgaaggcag ggctaaaaaa atccacatat agctgctgca tatgccatca 120
tccaagtata tcaagatcaa aataattata aaacatactt gtttattata atagataggt 180
actcaaggtt agagcatatg aatagatgct gcatatgcca tcatgtatat gcatcagtaa 240
aacccacatc aacatgtata cctatcctag atcgatattt ccatccatct taaactcgta 300
actatgaaga tgtatgacac acacatacag ttccaaaatt aataaataca ccaggtagtt 360
tgaaacagta ttctactccg atctagaacg aatgaacgac cgcccaacca caccacatca 420
tcacaaccaa gcgaacaaaa agcatctctg tatatgcatc agtaaaaccc gcatcaacat 480
gtatacctat cctagatcga tatttccatc catcatcttc aattcgtaac tatgaatatg 540
tatggcacac acatacagat ccaaaattaa taaatccacc acccctcccc cctggtgcag 600
gataatacaa ggcttgtccc acctcttgtg ttccatcccg cgccaaccca tctgggcagg 660
ggcacgtagc gacaatctca ctcgtcggtc cagggacccc cgggtccgaa acgccgacaa 720
<210> 3
<211> 1069
<212> DNA
<213> Artificial Synthesis (unknown)
<400> 3
tctcagcccc ttacggcagc aaggagacgt cagcaggacc cttgccgcac tgccagctgt 60
gctcctacag ggctcaggcg cttctccgac ggccacacta tcatgtgtat agggctcaag 120
ctctcccccg acagccacgt tggcatgtac acaaggctta ggcatctctc tgccgaacac 180
gttagcgcat agctacaacc ccattgtacg actggaccct ctccttgcgt ctataaaagg 240
gaggtccagg gccatcctgg aggggggttc gcgcgaggga aggagtggac gaacatgctc 300
cctctctctc tttctctccc tctcatgaca tgcttagaca acttaataac acattgcgga 360
cgtttttaat gtactgaatt aacgccgaat taattcgggg gatctggatt ttagtactgg 420
attttggttt taggaattag aaattttatt gatagaagta ttttacaaat acaaatacat 480
actaagggtt tcttatatgc tcaacacatg agcgaaaccc tataggaacc ctaattccct 540
tatctgggaa ctactcacac attattatgg agaaactcga gtcaaatctc ggtgacgggc 600
aggaccggac ggggcggtac cggcaggctg aagtccagct gccagaaacc cacgtcatgc 660
cagttcccgt gcttgaagcc ggccgcccgc agcatgccgc ggggggcata tccgagcgcc 720
tcgtgcatgc gcacgctcgg gtcgttgggc agcccgatga cagcgaccac gctcttgaag 780
ccctgtgcct ccagggactt cagcaggtgg gtgtagagcg tggagcccag tcccgtccgc 840
tggtggcggg gggagacgta cacggtcgac tcggccgtcc agtcgtaggc gttgcgtgcc 900
ttccaggggc ccgcgtaggc gatgccggcg acctcgccgt ccacctcggc gacgagccag 960
ggatagcgct cccgcagacg gacgaggtcg tccgtccact cctgcggttc ctgcggctcg 1020
gtacggaagt tgaccgtgct tgtctcgatg tagtggttga cgatggtgc 1069
<210> 4
<211> 5052
<212> DNA
<213> Artificial Synthesis (unknown)
<400> 4
tcgcccgacc tcaggcttcg gcctcgggag gaggtctccg cctcgcctga cccccggact 60
cggcctcaac ctcggtctcg ggaggaatcg cgtcctcgcc cgaccccggc ctcggcctca 120
agagaagtct tcgcctcgcc cgaccttggg ctcggaccga ccgcgccaac aggggataca 180
tcattaccct atctctagcc tgctcaggct acaaggaaca agaccggtgt cccatctggc 240
ttaccccggc gacgggtaat gatggtgccc tgcgtgcacc atgacgccgg tggctctcag 300
ccccttacgg cagcaaggag acgtcagcag gacccttgcc gcactgccag ctgtgctcct 360
acagggctca ggcgcttctc cgacggccac actatcatgt gtatagggct caagctctcc 420
cccgacagcc acgttggcat gtacacaagg cttaggcatc tctctgccga acacgttagc 480
gcatagctac aaccccattg tacgactgga ccctctcctt gcgtctataa aagggaggtc 540
cagggccatc ctggaggggg gttcgcgcga gggaaggagt ggacgaacat gctccctctc 600
tctctttctc tccctctcat gacatgctta gacaacttaa taacacattg cggacgtttt 660
taatgtactg aattaacgcc gaattaattc gggggatctg gattttagta ctggattttg 720
gttttaggaa ttagaaattt tattgataga agtattttac aaatacaaat acatactaag 780
ggtttcttat atgctcaaca catgagcgaa accctatagg aaccctaatt cccttatctg 840
ggaactactc acacattatt atggagaaac tcgagtcaaa tctcggtgac gggcaggacc 900
ggacggggcg gtaccggcag gctgaagtcc agctgccaga aacccacgtc atgccagttc 960
ccgtgcttga agccggccgc ccgcagcatg ccgcgggggg catatccgag cgcctcgtgc 1020
atgcgcacgc tcgggtcgtt gggcagcccg atgacagcga ccacgctctt gaagccctgt 1080
gcctccaggg acttcagcag gtgggtgtag agcgtggagc ccagtcccgt ccgctggtgg 1140
cggggggaga cgtacacggt cgactcggcc gtccagtcgt aggcgttgcg tgccttccag 1200
gggcccgcgt aggcgatgcc ggcgacctcg ccgtccacct cggcgacgag ccagggatag 1260
cgctcccgca gacggacgag gtcgtccgtc cactcctgcg gttcctgcgg ctcggtacgg 1320
aagttgaccg tgcttgtctc gatgtagtgg ttgacgatgg tgcagaccgc cggcatgtcc 1380
gcctcggtgg cacggcggat gtcggccggg cgtcgttctg ggctcatggt agactcgaga 1440
gagatagatt tgtagagaga gactggtgat ttcagcgtgt cctctccaaa tgaaatgaac 1500
ttccttatat agaggaaggt cttgcgaagg atagtgggat tgtgcgtcat cccttacgtc 1560
agtggagata tcacatcaat ccacttgctt tgaagacgtg gttggaacgt cttctttttc 1620
cacgatgctc ctcgtgggtg ggggtccatc tttgggacca ctgtcggcag aggcatcttg 1680
aacgatagcc tttcctttat cgcaatgatg gcatttgtag gtgccacctt ccttttctac 1740
tgtccttttg atgaagtgac agatagctgg gcaatggaat ccgaggaggt ttcccgatat 1800
taccctttgt tgaaaagtct caatagccct ttggtcttct gagactgtat ctttgatatt 1860
cttggagtag acgagagtgt cgtgctccac catgttggca agctgctcta gccaatacgc 1920
aaaccgcctc tccccgcgcg ttggccgatt cattaatgca gctggcacga caggtttccc 1980
gactggaaag cgggcagtga gcgcaacgca attaatgtga gttagctcac tcattaggca 2040
ccccaggctt tacactttat gcttccggct cgtatgttgt gtggaattgt gagcggataa 2100
caatttcaca caggaaacag ctatgacatg attacgaatt ctcatgtttg acagcttatc 2160
atcggatcta gtaacataga tgacaccgcg cgcgataatt tatcctagtt tgcgcgctat 2220
attttgtttt ctatcgcgta ttaaatgtat aattgcggga ctctaatcat aaaaacccat 2280
ctcataaata acgtcatgca ttacatgtta attattacat gcttaacgta attcaacaga 2340
aattatatga taatcatcgc aagaccggca acaggattca atcttaagaa actttattgc 2400
caaatgtttg aacgatcgat ccactagtca atcactagtg aattcgagct cgagctcggt 2460
acccggggat cctcagtgga gcgtgggctc gtcggccatc tcgacgccgt tgacgctgcc 2520
gtcggcgccg gggcggcccg ccttgtactt gtaccagcgg gcggcgacga ggtagactag 2580
taggttggcg aggcagacgg cggcgagcag ccagtagaag ttgtcgaggc ggcccttgtt 2640
gaggtcgttg gcgatccagg ggtggcggtc gcccgtgacc ctgtgcacgg cggcgacgag 2700
cgcggagctg acgaagaatc ccagcgacag ggtgctgagg aacagccccg tgctcatggt 2760
cttcatcccc ttggggcact cgcgcaggaa gaagtcgagc tggccgatgt acgtgaacgc 2820
ctcgcccgcc cccacgagga agaactgcgg gatgagccag aacacggaca tgggcacgac 2880
gcctccggag gcggactcgg aggaatcgcg cgcgacgcgg aggcggcgga cctccgtgag 2940
cgcggcgccc gccatggcga cgacggagag cgcgaggccg acggcgatcc gctgcagcgg 3000
ggtgaggccg tgcgggttgc cgctgacgcg gcgcgcgacg ggcaccacca ggcggtcgta 3060
gacgggcacg gtgagcagga tggagccgac gaagaagacg gtgagggagc ccgcggggat 3120
ctggaacgag cccccgacgc ggcggtccat ggtggtggcc tgcgacaccg agaaggtggt 3180
catctgcgcg tacaccgtcc agaacatgat cgtggtcgcc cagatcggca gcatccgcgc 3240
caccgtcttc acctcctcca cgtccgtcag cgtcgccagc cgccacttgc tgctgctgct 3300
cgcccccgcc gccggatcct cgttgatcgc cgcgtggtcc aggaagcgga actggtcggt 3360
gtgggggagg cgctccttgc gcttgctctt cttgctggag gacccatcct cgacggcggc 3420
ggccttgccg acgtcgacgt cgtagagcat ggcggggtcg gcggggagag ggaggcggcg 3480
cttgcgccag gcggcgacga cgacggcggc gatctgcgtg agggggctgc cggccagctt 3540
cttgaagcgg tacctgcgtg tgccggccag gaagacgagg aggcccgcgg cgatggcgca 3600
ggcgcaggcg ccgtagcccc agcgcctgcc caggttgtcc tggacgtaca ccagcacggt 3660
gacggccagc agcgacccca gcgagatgaa gaagaagaac cagttgaaga agcgcatcat 3720
ctgccgcttc tccccgccgt ccgactcgtc gaactggtcc gacccgaacc ccgacacgct 3780
cgactttagc ccacccgtgc ccagcgccgt caggtacagc gccaggtaca gcacccccag 3840
ctgcgccccc gacgcccgcg cgcactcccc gacgacgcct ccgccggtcg cggagcagga 3900
cgccggccgt agccccggcg ccgccgtcga gatcgtcagg atcgtcacgc ccgaggcctg 3960
gacggcggtg aagatggcga tggtgaggta gcggccgagg aaggagtcgg cgacgaagcc 4020
gccgaggagg cagagcatga aggaggtgcc catgaagttg gtgacgacgt tggcggactc 4080
ggcgttgccc aggtgcatgg tgcccgtcag gtacgtcacc aggttcacgg cgatgcccag 4140
cgtcgtcagc cgctcgttca gctccgccac taggatcatg gcggcggcgc cccagcggcc 4200
ggtggtggcg cgcggggccg gccggccctt gaagtcccag gcgtcgagga ggacgtccgt 4260
ctccgccgcg gcattggtct cggggaggag tccgaccatg agctcggtac ccggggatcc 4320
tctagagtcg acctgcagaa gtaacaccaa acaacagggt gagcatcgac aaaagaaaca 4380
gtaccaagca aataaatagc gtatgaaggc agggctaaaa aaatccacat atagctgctg 4440
catatgccat catccaagta tatcaagatc aaaataatta taaaacatac ttgtttatta 4500
taatagatag gtactcaagg ttagagcata tgaatagatg ctgcatatgc catcatgtat 4560
atgcatcagt aaaacccaca tcaacatgta tacctatcct agatcgatat ttccatccat 4620
cttaaactcg taactatgaa gatgtatgac acacacatac agttccaaaa ttaataaata 4680
caccaggtag tttgaaacag tattctactc cgatctagaa cgaatgaacg accgcccaac 4740
cacaccacat catcacaacc aagcgaacaa aaagcatctc tgtatatgca tcagtaaaac 4800
ccgcatcaac atgtatacct atcctagatc gatatttcca tccatcatct tcaattcgta 4860
actatgaata tgtatggcac acacatacag atccaaaatt aataaatcca ccacccctcc 4920
cccctggtgc aggataatac aaggcttgtc ccacctcttg tgttccatcc cgcgccaacc 4980
catctgggca ggggcacgta gcgacaatct cactcgtcgg tccagggacc cccgggtccg 5040
aaacgccgac aa 5052
<210> 5
<211> 18
<212> DNA
<213> Zea mays
<400> 5
tctcagcccc ttacggca 18
<210> 6
<211> 18
<212> DNA
<213> Artificial Synthesis (unknown)
<400> 6
gcaccatcgt caaccact 18
Claims (9)
1. A nucleic acid molecule, wherein the sequence comprises any one of:
i) comprises the sequence shown in SEQ ID NO. 1 and/or SEQ ID NO. 2, or the complementary sequence thereof;
ii) comprises the sequence shown in SEQ ID NO. 3, or a complementary sequence thereof;
iii) comprises the sequence shown in SEQ ID NO. 4, or the complement thereof.
2. A probe for detecting a maize transformation event comprising the sequence shown as SEQ ID NO 1 or 2 or 3 or 4 or a fragment or variant or complement thereof.
3. A primer pair for detecting a corn transformation event, wherein the primer pair comprises:
a primer which specifically recognizes the 1 st to 625 nd nucleotide sequences of the sequence shown by SEQ ID NO. 4 and a primer which specifically recognizes the 626-4920 nd nucleotide sequences of the sequence shown by SEQ ID NO. 4; and/or
A primer which specifically recognizes the 626-4920 nucleotide sequence of the sequence shown by SEQ ID NO. 4 and a primer which specifically recognizes the 4921-5052 nucleotide sequence of the sequence shown by SEQ ID NO. 4;
optionally, the amplification product of the primer pair comprises the sequence of claim 2;
optionally, the primer pair is a sequence shown in SEQ ID NO. 5 and SEQ ID NO. 6 or a complementary sequence thereof.
4. A kit or microarray for detecting a corn transformation event comprising the probe of claim 2 and/or the primer pair of claim 3.
5. A method for detecting a corn transformation event, comprising detecting the presence of said transformation event in a test sample using:
i) the probe of claim 2;
ii) the primer pair of claim 3;
iii) the probe of claim 2 and the primer pair of claim 3; or
iv) the kit or microarray of claim 4.
6. A method of breeding maize, comprising the steps of:
1) obtaining maize comprising the nucleic acid molecule of claim 1;
2) subjecting the corn obtained in step 1) to pollen culture, unfertilized embryo culture, doubling culture, cell culture, tissue culture, selfing or crossing or a combination thereof to obtain a corn plant, seed, plant cell, progeny plant or plant part; and optionally also (c) a second set of one or more of,
3) subjecting the progeny plant obtained in step 2) to nitrogen deficiency tolerance identification and/or yield trait identification and detecting the presence or absence of the transformation event therein using the method of claim 5.
7. A product made from the corn plant, seed, plant cell, progeny plant or plant part obtained by the method of claim 6, comprising a food, feed or industrial material.
8. A method for increasing maize yield and/or nitrogen deficiency tolerance comprising growing in soil at least one transgenic maize plant comprising in its genome the nucleic acid sequence at position 2165-5052 of SEQ ID NO 4 or comprising in its genome SEQ ID NO 4; the transgenic maize plants have high yield and/or nitrogen deficiency tolerance traits.
9. A method for increasing the yield and/or nitrogen deficiency tolerance of maize, characterized in that an expression cassette for a nitrogen high efficiency utilizing gene having the sequence shown in nucleotide 2165-4913 of SEQ ID NO. 4 is introduced into the maize genome.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202011552975.7A CN112662799B (en) | 2020-12-24 | 2020-12-24 | Nucleic acid molecule for detecting corn plant ND4401 and detection method thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202011552975.7A CN112662799B (en) | 2020-12-24 | 2020-12-24 | Nucleic acid molecule for detecting corn plant ND4401 and detection method thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN112662799A true CN112662799A (en) | 2021-04-16 |
| CN112662799B CN112662799B (en) | 2023-12-22 |
Family
ID=75408502
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202011552975.7A Active CN112662799B (en) | 2020-12-24 | 2020-12-24 | Nucleic acid molecule for detecting corn plant ND4401 and detection method thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN112662799B (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN116926069A (en) * | 2023-06-28 | 2023-10-24 | 华中农业大学 | Nucleic acid sequence for detecting cotton OE-3 and detection method thereof |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2014164074A1 (en) * | 2013-03-13 | 2014-10-09 | Pioneer Hi-Bred International, Inc. | Enhanced nitrate uptake and nitrate translocation by over-expressing maize functional low-affinity nitrate transporters in transgenic maize |
| WO2016173540A1 (en) * | 2015-04-30 | 2016-11-03 | 北京大北农科技集团股份有限公司 | Herbicide-tolerant maize plant dbn9888, and nucleotide sequence and method for detecting same |
| CN111206031A (en) * | 2020-03-11 | 2020-05-29 | 中国农业科学院生物技术研究所 | Nucleic acid sequence for detecting corn plant NAZ-4 and detection method thereof |
| 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 |
-
2020
- 2020-12-24 CN CN202011552975.7A patent/CN112662799B/en active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2014164074A1 (en) * | 2013-03-13 | 2014-10-09 | Pioneer Hi-Bred International, Inc. | Enhanced nitrate uptake and nitrate translocation by over-expressing maize functional low-affinity nitrate transporters in transgenic maize |
| WO2016173540A1 (en) * | 2015-04-30 | 2016-11-03 | 北京大北农科技集团股份有限公司 | Herbicide-tolerant maize plant dbn9888, and nucleotide sequence and method for detecting same |
| CN111206031A (en) * | 2020-03-11 | 2020-05-29 | 中国农业科学院生物技术研究所 | Nucleic acid sequence for detecting corn plant NAZ-4 and detection method thereof |
| 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 |
Non-Patent Citations (2)
| Title |
|---|
| QUAGGIOTTI S ET AL: "Effect of low molecularsize humic substances on nitrate uptake and expression of genes involved innitrate transport in maize(Zea mays L.)", 《JOURNAL OF EXPERIMENTAL BOTANY》, vol. 55 * |
| WEN Z ET AL: "Zea mays uncharacterized LOC100279242 (LOC100279242), mRNA", 《NCBI》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN116926069A (en) * | 2023-06-28 | 2023-10-24 | 华中农业大学 | Nucleic acid sequence for detecting cotton OE-3 and detection method thereof |
Also Published As
| Publication number | Publication date |
|---|---|
| CN112662799B (en) | 2023-12-22 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| JP5769698B2 (en) | Genetically modified rice event 17314 and method of use thereof | |
| JP5762400B2 (en) | Rice genetic recombination event 17053 and methods of use thereof | |
| AU2023200906A1 (en) | Genes, constructs and maize event DP-202216-6 | |
| CN112852991B (en) | Transgenic corn event LP007-7 and detection method thereof | |
| CN112831585B (en) | Transgenic corn event LP007-4 and detection method thereof | |
| CN111206031A (en) | Nucleic acid sequence for detecting corn plant NAZ-4 and detection method thereof | |
| CN111518941B (en) | Nucleic acid sequence for detecting corn plant AN1 and detection method thereof | |
| CN113151533B (en) | Transgenic corn event LP007-6 and detection method thereof | |
| CN110881367A (en) | Corn event Ttrans-4 and methods of use thereof | |
| CN116287384B (en) | Nucleic acid molecule of insect-resistant herbicide-resistant corn transformation event LD05, detection method and application thereof | |
| CN112662799B (en) | Nucleic acid molecule for detecting corn plant ND4401 and detection method thereof | |
| CN114525277B (en) | Nucleic acid sequence for detecting 17L397-1 in cotton and detection method thereof | |
| CN112574987B (en) | Nucleic acid molecule for detecting corn plant ND6603 and detection method thereof | |
| CN112481412B (en) | Nucleic acid molecule for detecting corn plant ND4403 and detection method thereof | |
| CN112430684A (en) | Nucleic acid sequence for detecting rice plant H23 and detection method thereof | |
| CN116200529B (en) | Nucleic acid sequence of corn transformation event LG11 and detection method thereof | |
| CN116716434B (en) | Transgenic soybean event LP012-3 and detection method thereof | |
| CN116694813B (en) | Transgenic soybean event LP086-1 and detection method thereof | |
| CN116694815B (en) | Transgenic soybean event LP012-2 and detection method thereof | |
| CN116656870B (en) | Transgenic soybean event LP086-3 and detection method thereof | |
| US20230365985A1 (en) | PROTEINS AND BIOLOGICAL MATERIALS RELATED TO RICE (Oryza sativa L.) YIELD, AND USE THEREOF IN RICE YIELD INCREASE | |
| CN116694814B (en) | Transgenic soybean event LP012-1 and detection method thereof | |
| CN116694812B (en) | Transgenic soybean event LP086-2 and detection method thereof | |
| CN115820630A (en) | Exogenous insertion sequence of maize transformation event LD02 and application thereof | |
| CN116640879A (en) | Transgenic maize event p2DBEN-CP-BZ-12 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 |