CN102146383A - Plant transformation terminator DNA (deoxyribonucleic acid) sequence and application thereof - Google Patents
Plant transformation terminator DNA (deoxyribonucleic acid) sequence and application thereof Download PDFInfo
- Publication number
- CN102146383A CN102146383A CN2010101125006A CN201010112500A CN102146383A CN 102146383 A CN102146383 A CN 102146383A CN 2010101125006 A CN2010101125006 A CN 2010101125006A CN 201010112500 A CN201010112500 A CN 201010112500A CN 102146383 A CN102146383 A CN 102146383A
- Authority
- CN
- China
- Prior art keywords
- dna
- sequence
- plant
- terminator
- nucleotide sequence
- 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
- 108020004414 DNA Proteins 0.000 title claims abstract description 34
- 102000053602 DNA Human genes 0.000 title abstract description 9
- 230000009466 transformation Effects 0.000 title abstract description 8
- 241000196324 Embryophyta Species 0.000 claims abstract description 42
- 125000003729 nucleotide group Chemical group 0.000 claims abstract description 32
- 239000002773 nucleotide Substances 0.000 claims abstract description 31
- 108700007698 Genetic Terminator Regions Proteins 0.000 claims abstract description 13
- 230000009261 transgenic effect Effects 0.000 claims abstract description 9
- 240000007594 Oryza sativa Species 0.000 claims abstract description 8
- 235000007164 Oryza sativa Nutrition 0.000 claims abstract description 8
- 235000009566 rice Nutrition 0.000 claims abstract description 7
- 238000000034 method Methods 0.000 claims description 9
- 108091028043 Nucleic acid sequence Proteins 0.000 claims description 8
- 244000061176 Nicotiana tabacum Species 0.000 claims description 7
- 235000002637 Nicotiana tabacum Nutrition 0.000 claims description 7
- 241000209510 Liliopsida Species 0.000 claims description 3
- 241001233957 eudicotyledons Species 0.000 claims description 3
- 244000005700 microbiome Species 0.000 claims description 3
- 244000075850 Avena orientalis Species 0.000 claims description 2
- 235000007319 Avena orientalis Nutrition 0.000 claims description 2
- 235000007558 Avena sp Nutrition 0.000 claims description 2
- 235000016068 Berberis vulgaris Nutrition 0.000 claims description 2
- 241000335053 Beta vulgaris Species 0.000 claims description 2
- 229920000742 Cotton Polymers 0.000 claims description 2
- 244000241257 Cucumis melo Species 0.000 claims description 2
- 235000009847 Cucumis melo var cantalupensis Nutrition 0.000 claims description 2
- 240000008067 Cucumis sativus Species 0.000 claims description 2
- 235000010799 Cucumis sativus var sativus Nutrition 0.000 claims description 2
- 244000299507 Gossypium hirsutum Species 0.000 claims description 2
- 244000020551 Helianthus annuus Species 0.000 claims description 2
- 235000003222 Helianthus annuus Nutrition 0.000 claims description 2
- 240000005979 Hordeum vulgare Species 0.000 claims description 2
- 235000007340 Hordeum vulgare Nutrition 0.000 claims description 2
- 240000008415 Lactuca sativa Species 0.000 claims description 2
- 235000003228 Lactuca sativa Nutrition 0.000 claims description 2
- 235000007688 Lycopersicon esculentum Nutrition 0.000 claims description 2
- 235000010582 Pisum sativum Nutrition 0.000 claims description 2
- 240000004713 Pisum sativum Species 0.000 claims description 2
- 240000003768 Solanum lycopersicum Species 0.000 claims description 2
- 244000061456 Solanum tuberosum Species 0.000 claims description 2
- 235000002595 Solanum tuberosum Nutrition 0.000 claims description 2
- 235000021307 Triticum Nutrition 0.000 claims description 2
- 244000098338 Triticum aestivum Species 0.000 claims description 2
- 240000008042 Zea mays Species 0.000 claims description 2
- 235000005824 Zea mays ssp. parviglumis Nutrition 0.000 claims description 2
- 235000002017 Zea mays subsp mays Nutrition 0.000 claims description 2
- 235000005822 corn Nutrition 0.000 claims description 2
- 235000013311 vegetables Nutrition 0.000 claims description 2
- 230000001131 transforming effect Effects 0.000 claims 1
- 230000014509 gene expression Effects 0.000 abstract description 19
- 238000012217 deletion Methods 0.000 abstract description 4
- 230000037430 deletion Effects 0.000 abstract description 4
- 238000010276 construction Methods 0.000 abstract description 3
- 239000013598 vector Substances 0.000 abstract description 3
- 238000007792 addition Methods 0.000 abstract 1
- 230000008827 biological function Effects 0.000 abstract 1
- 238000006467 substitution reaction Methods 0.000 abstract 1
- 239000013612 plasmid Substances 0.000 description 15
- 206010020649 Hyperkeratosis Diseases 0.000 description 10
- 239000013604 expression vector Substances 0.000 description 10
- 239000001963 growth medium Substances 0.000 description 10
- 108090000623 proteins and genes Proteins 0.000 description 10
- 210000004027 cell Anatomy 0.000 description 9
- 241000589158 Agrobacterium Species 0.000 description 8
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 8
- 238000003752 polymerase chain reaction Methods 0.000 description 8
- 238000006243 chemical reaction Methods 0.000 description 7
- 238000005516 engineering process Methods 0.000 description 7
- 239000012634 fragment Substances 0.000 description 7
- 210000001519 tissue Anatomy 0.000 description 7
- 230000006870 function Effects 0.000 description 6
- 239000007788 liquid Substances 0.000 description 6
- 238000012216 screening Methods 0.000 description 6
- 241000894006 Bacteria Species 0.000 description 5
- 230000003321 amplification Effects 0.000 description 5
- 239000002585 base Substances 0.000 description 5
- 238000003199 nucleic acid amplification method Methods 0.000 description 5
- 108020004707 nucleic acids Proteins 0.000 description 5
- 102000039446 nucleic acids Human genes 0.000 description 5
- 150000007523 nucleic acids Chemical class 0.000 description 5
- 238000012545 processing Methods 0.000 description 5
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 5
- 238000004458 analytical method Methods 0.000 description 4
- 230000001580 bacterial effect Effects 0.000 description 4
- 230000008859 change Effects 0.000 description 4
- 238000013467 fragmentation Methods 0.000 description 4
- 238000006062 fragmentation reaction Methods 0.000 description 4
- 239000000499 gel Substances 0.000 description 4
- 108020004999 messenger RNA Proteins 0.000 description 4
- 239000013641 positive control Substances 0.000 description 4
- 230000008929 regeneration Effects 0.000 description 4
- 238000011069 regeneration method Methods 0.000 description 4
- 239000000126 substance Substances 0.000 description 4
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 3
- 244000025254 Cannabis sativa Species 0.000 description 3
- 241000234643 Festuca arundinacea Species 0.000 description 3
- 108700019146 Transgenes Proteins 0.000 description 3
- 230000004087 circulation Effects 0.000 description 3
- 239000012153 distilled water Substances 0.000 description 3
- 229960004756 ethanol Drugs 0.000 description 3
- 230000002068 genetic effect Effects 0.000 description 3
- 101150054900 gus gene Proteins 0.000 description 3
- 238000009396 hybridization Methods 0.000 description 3
- 239000000203 mixture Substances 0.000 description 3
- 238000012360 testing method Methods 0.000 description 3
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 description 2
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 2
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- 239000002126 C01EB10 - Adenosine Substances 0.000 description 2
- LZZYPRNAOMGNLH-UHFFFAOYSA-M Cetrimonium bromide Chemical compound [Br-].CCCCCCCCCCCCCCCC[N+](C)(C)C LZZYPRNAOMGNLH-UHFFFAOYSA-M 0.000 description 2
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 description 2
- 102000004594 DNA Polymerase I Human genes 0.000 description 2
- 108010017826 DNA Polymerase I Proteins 0.000 description 2
- 108090000790 Enzymes Proteins 0.000 description 2
- 102000004190 Enzymes Human genes 0.000 description 2
- 102000003960 Ligases Human genes 0.000 description 2
- 108090000364 Ligases Proteins 0.000 description 2
- 229920002684 Sepharose Polymers 0.000 description 2
- 229960005305 adenosine Drugs 0.000 description 2
- UDMBCSSLTHHNCD-KQYNXXCUSA-N adenosine 5'-monophosphate Chemical compound C1=NC=2C(N)=NC=NC=2N1[C@@H]1O[C@H](COP(O)(O)=O)[C@@H](O)[C@H]1O UDMBCSSLTHHNCD-KQYNXXCUSA-N 0.000 description 2
- OIRDTQYFTABQOQ-KQYNXXCUSA-N adenosine group Chemical group [C@@H]1([C@H](O)[C@H](O)[C@@H](CO)O1)N1C=NC=2C(N)=NC=NC12 OIRDTQYFTABQOQ-KQYNXXCUSA-N 0.000 description 2
- 229950006790 adenosine phosphate Drugs 0.000 description 2
- 238000010170 biological method Methods 0.000 description 2
- 238000007796 conventional method Methods 0.000 description 2
- 125000004122 cyclic group Chemical group 0.000 description 2
- 238000004043 dyeing Methods 0.000 description 2
- 239000000284 extract Substances 0.000 description 2
- 238000005286 illumination Methods 0.000 description 2
- 230000001939 inductive effect Effects 0.000 description 2
- 210000001161 mammalian embryo Anatomy 0.000 description 2
- 230000001404 mediated effect Effects 0.000 description 2
- 239000002243 precursor Substances 0.000 description 2
- 230000008569 process Effects 0.000 description 2
- 239000012879 subculture medium Substances 0.000 description 2
- 239000006228 supernatant Substances 0.000 description 2
- 230000014621 translational initiation Effects 0.000 description 2
- 238000011144 upstream manufacturing Methods 0.000 description 2
- 238000005406 washing Methods 0.000 description 2
- JXCKZXHCJOVIAV-UHFFFAOYSA-N 6-[(5-bromo-4-chloro-1h-indol-3-yl)oxy]-3,4,5-trihydroxyoxane-2-carboxylic acid;cyclohexanamine Chemical compound [NH3+]C1CCCCC1.O1C(C([O-])=O)C(O)C(O)C(O)C1OC1=CNC2=CC=C(Br)C(Cl)=C12 JXCKZXHCJOVIAV-UHFFFAOYSA-N 0.000 description 1
- 241000589155 Agrobacterium tumefaciens Species 0.000 description 1
- 241000193830 Bacillus <bacterium> Species 0.000 description 1
- 108091026890 Coding region Proteins 0.000 description 1
- 108020004705 Codon Proteins 0.000 description 1
- UDMBCSSLTHHNCD-UHFFFAOYSA-N Coenzym Q(11) Natural products C1=NC=2C(N)=NC=NC=2N1C1OC(COP(O)(O)=O)C(O)C1O UDMBCSSLTHHNCD-UHFFFAOYSA-N 0.000 description 1
- 102000004163 DNA-directed RNA polymerases Human genes 0.000 description 1
- 108090000626 DNA-directed RNA polymerases Proteins 0.000 description 1
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 1
- 241000620209 Escherichia coli DH5[alpha] Species 0.000 description 1
- 108700039691 Genetic Promoter Regions Proteins 0.000 description 1
- 102000053187 Glucuronidase Human genes 0.000 description 1
- 108010060309 Glucuronidase Proteins 0.000 description 1
- KFZMGEQAYNKOFK-UHFFFAOYSA-N Isopropanol Chemical compound CC(C)O KFZMGEQAYNKOFK-UHFFFAOYSA-N 0.000 description 1
- 108091092724 Noncoding DNA Proteins 0.000 description 1
- 208000020584 Polyploidy Diseases 0.000 description 1
- 108091034057 RNA (poly(A)) Proteins 0.000 description 1
- 108700008625 Reporter Genes Proteins 0.000 description 1
- CWHJIJJSDGEHNS-MYLFLSLOSA-N Senegenin Chemical compound C1[C@H](O)[C@H](O)[C@@](C)(C(O)=O)[C@@H]2CC[C@@]3(C)C(CC[C@]4(CCC(C[C@H]44)(C)C)C(O)=O)=C4[C@@H](CCl)C[C@@H]3[C@]21C CWHJIJJSDGEHNS-MYLFLSLOSA-N 0.000 description 1
- 108091081024 Start codon Proteins 0.000 description 1
- 108700009124 Transcription Initiation Site Proteins 0.000 description 1
- 108091023040 Transcription factor Proteins 0.000 description 1
- 102000040945 Transcription factor Human genes 0.000 description 1
- 229920004890 Triton X-100 Polymers 0.000 description 1
- 239000013504 Triton X-100 Substances 0.000 description 1
- 229960000583 acetic acid Drugs 0.000 description 1
- 206010000496 acne Diseases 0.000 description 1
- 239000003513 alkali Substances 0.000 description 1
- 125000003275 alpha amino acid group Chemical group 0.000 description 1
- 238000004113 cell culture Methods 0.000 description 1
- 210000000349 chromosome Anatomy 0.000 description 1
- 239000004927 clay Substances 0.000 description 1
- 238000003776 cleavage reaction Methods 0.000 description 1
- 230000001276 controlling effect Effects 0.000 description 1
- 238000013016 damping Methods 0.000 description 1
- 229960000935 dehydrated alcohol Drugs 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 230000004069 differentiation Effects 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 230000005611 electricity Effects 0.000 description 1
- 238000001962 electrophoresis Methods 0.000 description 1
- DNJIEGIFACGWOD-UHFFFAOYSA-N ethyl mercaptane Natural products CCS DNJIEGIFACGWOD-UHFFFAOYSA-N 0.000 description 1
- 210000003527 eukaryotic cell Anatomy 0.000 description 1
- 238000011156 evaluation Methods 0.000 description 1
- 239000013613 expression plasmid Substances 0.000 description 1
- 239000012530 fluid Substances 0.000 description 1
- 239000012362 glacial acetic acid Substances 0.000 description 1
- 238000000227 grinding Methods 0.000 description 1
- RQFCJASXJCIDSX-UUOKFMHZSA-N guanosine 5'-monophosphate Chemical compound C1=2NC(N)=NC(=O)C=2N=CN1[C@@H]1O[C@H](COP(O)(O)=O)[C@@H](O)[C@H]1O RQFCJASXJCIDSX-UUOKFMHZSA-N 0.000 description 1
- 235000013928 guanylic acid Nutrition 0.000 description 1
- 239000004226 guanylic acid Substances 0.000 description 1
- 230000001744 histochemical effect Effects 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 230000003834 intracellular effect Effects 0.000 description 1
- JEIPFZHSYJVQDO-UHFFFAOYSA-N iron(III) oxide Inorganic materials O=[Fe]O[Fe]=O JEIPFZHSYJVQDO-UHFFFAOYSA-N 0.000 description 1
- PHTQWCKDNZKARW-UHFFFAOYSA-N isoamylol Chemical compound CC(C)CCO PHTQWCKDNZKARW-UHFFFAOYSA-N 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 239000002609 medium Substances 0.000 description 1
- 238000000520 microinjection Methods 0.000 description 1
- 230000000394 mitotic effect Effects 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 238000010369 molecular cloning Methods 0.000 description 1
- 229910052757 nitrogen Inorganic materials 0.000 description 1
- 108010058731 nopaline synthase Proteins 0.000 description 1
- 210000004940 nucleus Anatomy 0.000 description 1
- 210000000056 organ Anatomy 0.000 description 1
- 230000008520 organization Effects 0.000 description 1
- 230000008488 polyadenylation Effects 0.000 description 1
- 238000006116 polymerization reaction Methods 0.000 description 1
- 229920001184 polypeptide Polymers 0.000 description 1
- 238000001556 precipitation Methods 0.000 description 1
- 238000004321 preservation Methods 0.000 description 1
- 108090000765 processed proteins & peptides Proteins 0.000 description 1
- 102000004196 processed proteins & peptides Human genes 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
- 230000009711 regulatory function Effects 0.000 description 1
- 108091008146 restriction endonucleases Proteins 0.000 description 1
- 230000010496 root system development Effects 0.000 description 1
- SUKJFIGYRHOWBL-UHFFFAOYSA-N sodium hypochlorite Chemical compound [Na+].Cl[O-] SUKJFIGYRHOWBL-UHFFFAOYSA-N 0.000 description 1
- 230000001954 sterilising effect Effects 0.000 description 1
- 238000004659 sterilization and disinfection Methods 0.000 description 1
- 238000003756 stirring Methods 0.000 description 1
- 239000009871 tenuigenin Substances 0.000 description 1
- XOGGUFAVLNCTRS-UHFFFAOYSA-N tetrapotassium;iron(2+);hexacyanide Chemical compound [K+].[K+].[K+].[K+].[Fe+2].N#[C-].N#[C-].N#[C-].N#[C-].N#[C-].N#[C-] XOGGUFAVLNCTRS-UHFFFAOYSA-N 0.000 description 1
- 238000013518 transcription Methods 0.000 description 1
- 230000035897 transcription Effects 0.000 description 1
- 230000005030 transcription termination Effects 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
- BYGOPQKDHGXNCD-UHFFFAOYSA-N tripotassium;iron(3+);hexacyanide Chemical compound [K+].[K+].[K+].[Fe+3].N#[C-].N#[C-].N#[C-].N#[C-].N#[C-].N#[C-] BYGOPQKDHGXNCD-UHFFFAOYSA-N 0.000 description 1
- 239000005418 vegetable material Substances 0.000 description 1
- 239000013603 viral vector Substances 0.000 description 1
- DGVVWUTYPXICAM-UHFFFAOYSA-N β‐Mercaptoethanol Chemical compound OCCS DGVVWUTYPXICAM-UHFFFAOYSA-N 0.000 description 1
Images
Landscapes
- Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention relates to a DNA (deoxyribonucleic acid) sequence separated from rice genome, which can be used as a terminator region in an exogenous gene expression structure in plant transformation. The invention comprises a DNA of which the nucleotide sequence is disclosed as SEQ ID NO.1, or a DNA of which the nucleotide sequence is deletion, substitution or addition of one or a plurality of bases on the basis of the nucleotide sequence disclosed as SEQ ID NO.1 and has more than 90% nucleotide sequence homology with any region composed of 1194 or more bases in the nucleotide sequence disclosed as SEQ ID NO.1. The biological functions of the DNA are equivalent to those of the DNA of which the nucleotide sequence is disclosed as SEQ ID NO.1. The invention also relates to construction of a transformation vector comprising the terminator sequence, and acquisition of a corresponding transgenic plant.
Description
Technical field
The present invention relates to from the rice genome sequence, separate the purposes of the terminator sequence that obtains, and the application in vector construction, Plant Transformation and the genetic expression in plant biotechnology field.
Background technology
In plant transgenic technology, expression of exogenous gene will be passed through series of steps such as transcribing, transcribe post-treatment, translate and translate post-treatment, and produces specific aminoacid sequence in specific time and space.Can controlling gene phraseology and expression level by the different steps of genetic expression is regulated and control.In these control methods, except that the characteristics of gene itself, the expression regulation sequence at its two ends, the especially promoter sequence of 5 ' end and gene 3 ' end non-coding sequence (terminator sequence) often have intense influence to expression of gene.
In eukaryotic cell, precursor mRNA (Pre-mRNA) needs to translate active mRNA through just being formed with after further processing after transcribing, mRNA3 ' end often adds the afterbody of being made up of 25-250 nucleotide residue poly (A) in this process, this process is determined by one section nucleotide sequence of gene 3 ' end non-coding region that mainly this zone is referred to as 3 ' end processing signal (3 '-processing signal) or terminator (Terminator).
The most important factor is the poly-adenosine signal (Polyadenylation signal) that is positioned at the poly-adenosine site upstream 15-20 Nucleotide place of mRNA in the terminator sequence, generally by six Nucleotide AAUAAA that the conservative property of height arranged (or AAUUAA) composition.The 6th Nucleotide changes sometimes to some extent in plant mRNA, and the AAUAAA sequence is necessary for the fracture of precursor mRNA3 ' end and the polymerization of adenylic acid (AMP).The sequence of uridylic (U) or uridylic guanylic acid (UC) generally is rich in AAUAAA signal downstream, and its conservative property is not as good as the AAUAAA sequence, but also is necessary for cleavage reaction.
Though terminator does not have the function of enhanser, the plant terminators of different sources has very different influences for the expression of external source.Bibliographical information shows that 3 ' terminal sequence of different sources has strong influence for the Npt-II enzyme at the intracellular expression level of transformation of tobacco, reaches as high as and differs 60 times.
The most frequently used terminator is rouge alkali synthetase (NOS) terminator (T-Nos) in the transgenic engineering at present.The present invention has comprised one section dna sequence dna ostrbcs that originates from rice genome, test-results shows: in plant transgene was used, the ostrbcs sequence had the terminator function identical with T-Nos.
Summary of the invention
The purpose of this invention is to provide a kind of terminator sequence that in Plant Transformation, can be used as in the exogenous gene expression structure.
Terminator sequence provided by the present invention derives from rice genome, has following nucleotide sequence.
1) the SEQ ID № in the sequence table: 1 dna sequence dna;
2) with SEQ ID № in the sequence table: one or more base deletions, replacement or adding are arranged in 1 the nucleotide sequence, and with shown in the nucleotide sequence by 1194 or more any regional nucleotide sequence homology formed of polybase base surpass the DNA of 90% nucleotide sequence, this DNA has the terminator function.
Described one or more base deletion, replacement or adding are meant no more than 10% base deletion, replacement or adding.
3) under the rigorous condition of height can with SEQ ID № in the sequence table: the nucleotide sequence of the 1 dna sequence dna hybridization that limits.
The rigorous condition of above-mentioned height can be 60 ℃ of hybridization down in hybridization solution, and at 0.5 * SSC, in the solution of 0.1% SDS, washes film under 60 ℃.
Contain the expression vector of dna sequence dna of the present invention, artificial transfered cell system, host bacterium and expression cassette and all belong to protection scope of the present invention.
Use contains the expression vector of dna sequence dna of the present invention, manually transfered cell system, host bacterium and expression cassette all belong to protection scope of the present invention by the existing available conversion microorganism cells of molecular biological method and transformed plant cells, tissue and plant etc.
Terminator sequence of the present invention can add any promoter sequence and target gene sequences before its sequence in being building up to plant expression vector the time.By the plant transformed host both can be monocotyledons, it also can be dicotyledons, wherein, monocotyledons can be turfgrass, wheat, barley, oat, paddy rice or corn etc., and dicotyledons can be potato, tobacco, cotton, lettuce, tomato, muskmelon, cucumber, pea, beet or Sunflower Receptacle etc.
Carry this terminator sequence expression vector can Ti-plasmids, Ri plasmid, plant viral vector, directly DNA conversion, microinjection, electricity be led, conventional biological method transformed plant cells or tissue such as agriculture bacillus mediated by using, and the tissue cultivating that transforms is become plant.
Description of drawings
Fig. 1 plant expression vector pBI121 physical map
Fig. 2 plant expression vector pBI121-ostrbcs physical map
Fig. 3 transgene tobacco
Fig. 4 transgenic tall fescue grass
Fig. 5 transfer-gen plant PCR detects
Fig. 6 transgenic plant GUS tissue chemical analysis
Embodiment
Method therefor is ordinary method if no special instructions among the following embodiment, concrete steps can be with reference to (people such as Sambrook, the molecular cloning laboratory manual, New York:Cold Spring Harbor LaboratoryPress, 1989), used term and abbreviation all are general term of those skilled in the art and abbreviation.
The term that part is used for this specification sheets and claims has following implication:
-term " nucleic acid " is meant DNA or RNA;
-term " nucleotide sequence " is meant from 5 ' end and reads to the oligomer or the polymkeric substance of the nucleotide base of 3 ' strand of holding or two strands that it comprises self-replacation type plasmid, infectivity or noninfective gene and DNA or RNA polymkeric substance and functional or non-functional DNA or RNA.In being used for the application's Nucleotide symbol, except that specifically mentioning, the left end of strand or double chain nucleotide sequence is 5 ' end;
-term " promotor " or phrase " promoter region nucleotide sequence " are meant identification and the bonded nucleic acid district that is positioned at translation initiation codon upstream, RNA polymerase and other transcription factor;
-" plant promoter " is to start the promotor of transcribing in vegetable cell;
-phrase " connects with functional mode " or " functional connection " is meant: one section nucleotide sequence (for example promotor or regulate or functional block) is connected with another section nucleotide sequence (for example another adjusting or functional block or the gene proteic to be expressed to be produced of encoding), make described sequence in the cell of introducing it, genome, carrier or expression cassette, bring into play its original function, promptly make them in such environment, function be arranged.With regard to promotor, promoter sequence is also included within the transcription sequence between transcription initiation site and the translation initiation site;
-term " expression cassette " is meant such nucleotide sequence, and described nucleotide sequence can instruct the nucleotide sequence or the expression of gene of coding polypeptide to be produced in the host living beings compatible with this sequence.Such box comprises other factor that a promotor and transcription termination signal and optional expression are needed or can be used for expressing at least;
-term " carrier " is meant expression system, for example the projectile of DNA bag quilt, based on the transport vehicle of nucleic acid be changed nucleic acid molecule and autonomous self-replacation type cyclic DNA, for example plasmid, clay, phagemid etc. to transport described nucleic acid.Described carrier can or as being incorporated into Autonomous Structure in the host genome by during mitotic division, duplicating by described cytotostatic, perhaps maintain in host's the nucleus or tenuigenin;
-term " plasmid " is meant the autonomous ring-shaped DNA molecule that can duplicate in cell, comprise so-called " expression " plasmid and so-called " non-expression " plasmid.If it is the host of " expression plasmid " that a kind of reconstitution cell culture or microorganism are described to, then this term had both comprised karyomit(e) outer ring-like dna molecular, comprised the DNA that has been incorporated in the host chromosome again.If described plasmid is kept by host cell, then described plasmid or between m period as a kind of Autonomous Structure by described cytotostatic duplicate, perhaps be incorporated in described host's the genome;
-term " frame " is meant the nucleotide sequence of being responsible for regulatory function;
-term " is arranged in " and is meant the position of discriminating element (for example " frame ", restriction site or codon with specific function) at nucleotide sequence.The position that provides with numeral is meant the zero position of described element in described nucleotide sequence, just when specifically mentioning, so that the reading direction of described nucleotide sequence promptly 5 '---3 ' direction is mentioned;
-term " transgenic plant " is meant the plant that obtains by Genetic Manipulative Technology, and comprise the whole strain plant that obtains by these technology, in its genome, integrated this generic operation or express aftergrowth, its offspring and the plant organ of this generic operation, for example root, stem and leaf.Can have different ploidy levels according to transgenic plant of the present invention, can be polyploid, diploid or monoploid.
(1) extracting of genomic dna
With CTAB method (Rogers, S.O. etc., 1985) extract oryza sativa genomic dna, concrete grammar is as follows: water intaking rice blade liquid nitrogen grinding, add 5 milliliters in the vegetable material of per 1 gram through the CTAB of 68 ℃ of preheatings solution (with before adding 0.2% mercaptoethanol), 60 ℃ were heated 30 minutes.Add the equal-volume chloroform then: primary isoamyl alcohol, centrifugal 15 minutes of 15000g gets supernatant.The Virahol that in supernatant liquor, adds 2/3 times of volume, mixing stirs out the DNA silk with the entry needle needle point and to place new pipe, adds 75% ethanol again, and washing precipitation and centrifugal tube wall blot ethanol then, and air is dried remaining ethanol, adding TE dissolving DNA.And in-20 ℃ of preservations.
(3) clone of ostrbcs sequence and analysis
With PCR (polymerase chain reaction) method amplification ostrbcs terminator dna fragmentation.The PCR reaction solution contains 1 μ g genomic dna, 1.5mM MgCl
2, 20mM Tris-HCl (pH8.4), 50mM KCl, 0.8mM dNTP mixture, 1 μ M, 5 ' primer and 1 μ M, 3 ' primer, and the Taq polysaccharase of 1 unit (Shanghai Shen You biotech company).Primer sequence is as follows:
5 ' primer: 5 '-ggcaactaagccgtcatcgtca-3 '
3 ' primer: 5 '-ctcgctttgtctccacttccgg-3 '
In PCR-thermal cycler (Eppendorf company, Germany), carry out PCR circulation with amplification ostrbcsDNA fragment according to following scheme: 94 ℃ 5 minutes; 94 ℃ 1 minute; 60 ℃ 1 minute; 72 ℃ 2 minutes, totally 30 circulations and 72 ℃ are 10 minutes.About 1194bp dna fragmentation that amplification is obtained reclaims this fragment with the scheme that gel reclaims test kit (sky, Beijing latitude Time Technology company limited) and provides by supplier, with (Promega company of pGEM-Teasy vector system, the U.S.) scheme that provides by supplier is cloned this fragment, order-checking.
(1) selection of positive control plasmid
The structure of plant expression vector carries out according to a conventional method.With plasmid pBI121 (Genebank:AF485783) as positive control, β-glucuronidase (Jefferson etc. encode in this plasmid, 1987) gus gene is under the control of the nopaline synthase gene terminator of CaMV 35S promoter and root Agrobacterium (Agrobacterium tumefaciens), as shown in Figure 1.
(2) structure of conversion carrier pBI121-ostrbcs
The structure of plant expression vector carries out according to a conventional method.The 1194bp dna fragmentation that obtains in embodiment 1, (precious biotechnology (Dalian) company limited) carries out flush end processing (37 ℃, 30 minutes) with dna polymerase i (the big fragment of Klenow).The scheme that reclaims test kit (sky, Beijing latitude Time Technology company limited) and provide by supplier with dna gel is separated on 0.8% sepharose and is reclaimed.
Getting 5 μ g plasmid pBI121 digested 1 hour in 37 ℃ with restriction enzyme SacI (precious biotechnology (Dalian) company limited) and EcoRI (precious biotechnology (Dalian) company limited).(precious biotechnology (Dalian) company limited) carries out flush end processing (37 ℃, 30 minutes) with dna polymerase i (the big fragment of Klenow).The scheme that reclaims test kit (sky, Beijing latitude Time Technology company limited) and provide by supplier with dna gel is separated on 0.8% sepharose and is reclaimed the long dna fragmentation of 1194bp that be, and this fragment is the skeleton structure of having removed the Nos terminator, having contained the former pBI121 plasmid of CaMV 35S promoter and gus gene.
Get above-mentioned two kinds of gels and reclaim each about 0.2 μ g of fragment, in 16 ℃ of connections of spending the night, transformed into escherichia coli DH5 α cuts evaluation through colony screening and enzyme, obtains plasmid pBI121-ostrbcs, as shown in Figure 2 with the T4 ligase enzyme.
(1) cultivation of root Agrobacterium
The plant expression vector pBI121-ostrbcs that makes up among positive control matter pBI121 and the embodiment 2 is transformed Agrobacterium respectively, and the Agrobacterium bacterial classification is LBA4404.The Agrobacterium-mediated Transformation bacterial strain that obtains was cultivated 2 days on the YEB substratum, with little spoon bacterium is scraped at liquid and break up in the culture medium altogether, left standstill 2 hours, shake up the back and adjust bacterial concentration to OD
600Be 0.1.
(2) acquisition of transgene tobacco
Utilize improvement Ye Panfa, by the agrobacterium mediation converted tobacco.Get complete unfolded tobacco aseptic seedling blade, (diameter 0.9cm) makes the leaf dish with punch tool, soaks 10min in bacterium liquid.Take out the leaf dish, after blotting with aseptic filter paper, change in the common culture medium that is coated with one deck aseptic filter paper in 25 ℃ dark cultivate 2～3 days after, the leaf dish is changed in the subculture medium.Illumination cultivation in subculture medium (light/dark cycle is 16/8hrs) changed the leaf dish over to screening culture medium after 3 days.Continue to cultivate after 7～10 days, change the leaf dish over to regeneration culture medium.Promptly can be observed that blade edge grows callus or the regeneration bud of growing thickly in 15～20 days, can grow to 1～2cm about one month, change in the new regeneration culture medium, treat that it is long during to 3～4cm, with scalper regeneration bud is downcut, and change in the root media.Regrowth is taken root on the root media that contains kantlex (50mg/L), treat after one month that root system development fully after, seedling is transferred to the greenhouse growth.As shown in Figure 3.
(2) acquisition of transgenic tall fescue grass
Choose tall grass (the Festuca arundinacea Schreb.) seed of mature and plump, in distilled water, soak after 4 hours under the room temperature and peel off kind of a skin, put into distilled water again and continue soaked overnight.Soaked peeling seed crosses twice with 70% alcohol earlier, again with 30% clorox sterilization 30 minutes, aseptic water washing three times.Under aseptic condition,, callus inducing medium will be inserted again after the embryo crosscut into two respectively with embryo complete peeling from the seed that disinfects.
Use above-mentioned callus of induce substratum, per two to three weeks are changeed ware once with callus.Select for use through state adjust 10 days white of succeeding transfer culture or yellow-white, translucent, dry, surface has the callus of pimple to infect.
Placing callus through the resuspended bacterial concentration that leaves standstill is OD
600Infected 10 minutes in=0.1 the LBA4404 root Agrobacterium bacterium liquid, remove bacterium liquid, on aseptic filter paper, callus is dried (about 5 minutes), place the common culture medium that is covered with one deck aseptic filter paper, 25 ℃ of dark cultivations 3 days.
Cultivate the back altogether with inducing screening culture medium that callus is screened.To be connected to through the callus of cultivating altogether and induce screening culture medium at first to screen for 2 weeks, the resistant calli that as seen is fresh ivory buff grows.
To reach the above resistant calli of 0.5cm through the diameter of screening and be put on the differentiation screening culture medium and break up, and in 25 ℃ of following illumination cultivation (16h light/8h is dark).Have green budlet to occur after 10 days, an about afterwards week can be grown to seedling.
The seedling that will break up before taking root is removed the unnecessary callus of leaf and bottom on top, be placed on the division culture medium in the triangular flask slow seedling and carry out root culture about 10 days again, after being inoculated in root media, approximately visible white young root grows after 3-5 days, about afterwards two weeks can form more flourishing sophisticated root system, as shown in Figure 4.
The PCR of embodiment 4, transformed plant identifies
Extract the genomic dna of transformed plant as method as described in the embodiment 1 (1), and be that template is carried out the PCR reaction, the part of amplification gus gene 3 ' end and ostrbcs terminator 5 ' end with the 100ng genomic dna.Reaction solution contains 100ng genomic dna, 1.5mM MgCl
2, 20mM Tris-HCl (pH8.4), 50mM KCl, 0.8mM dNTP mixture, 1 μ M, 5 ' primer and 1 μ M, 3 ' primer, and the Taq polysaccharase of 1 unit (Shanghai Shen You biotech company).Primer sequence is as follows:
5 ' primer: 5 '-tgcatcagccgattatcatc-3 '
3 ' primer: 5 '-ggtcgtcgtacagatttgct-3 '
In PCR-thermal cycler (Eppendorf company, Germany), carry out the PCR cyclic amplification according to following scheme: 94 ℃ 5 minutes; 94 ℃ 1 minute; 58 ℃ 1 minute; 72 ℃ 1 minute, totally 30 circulations and 72 ℃ are 10 minutes.Amplified production is carried out electrophoretic analysis as seen band is clearly arranged at the 1060bp place that estimates.As shown in Figure 5.
The GUS histochemical stain is with reference to the detection method of (1987) such as Jefferson.The fresh blade of getting transfer-gen plant be soaked in the GUS reaction solution (0.1M NaPO4 damping fluid, pH 7.0,10mM EDTA, pH7.0, the 5mM Tripotassium iron hexacyanide, 5mM yellow prussiate of potash, 1.0mM X-Gluc, 0.1% Triton X-100), the tissue that soaks is vacuumized, spend the night in 37 ℃ of dark.Clean the dyeing tissue with distilled water, fix 1 hour, decolour, dewater, observe with stationary liquid (dehydrated alcohol/Glacial acetic acid, 3/1).The result shows: the plant (left three strains) that changes pBI121-ostrbcs is the same with the positive control plant (middle three strains) that changes pBI121, obviously display organization dyeing, but not transfer-gen plant (right two strains) is a white.As shown in Figure 6.
Claims (7)
1. the terminator that can bring into play function in vegetable cell is characterized by the DNA that includes nucleotide sequence shown in the SEQ ID NO:1.
2. a carrier is characterized by the terminator dna sequence dna that comprises claim 1.
3. produce the method for transformant, it is characterized by the step that comprises the importing of the carrier in the claim 2 host cell.
4. microorganism transformed cell is characterized by terminator sequence in the claim 1 that contains importing or the carrier in the claim 2.
5. plant transformed cell and tissue is characterized by terminator sequence in the claim 1 that contains importing or the carrier in the claim 2.
6. transfer-gen plant, it is characterized by this plant and be application rights and require terminator sequence or the carrier in the claim 2 in 1 to obtain, contain in its final transformant or removed terminator sequence in the claim 1 or the carrier in the claim 2 through transforming.
7. according to described in the claim 6, transgenic plant comprise monocotyledons and dicotyledons, as turfgrass, wheat, barley, oat, paddy rice, corn, potato, tobacco, cotton, lettuce, tomato, muskmelon, cucumber, pea, beet or Sunflower Receptacle etc.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2010101125006A CN102146383B (en) | 2010-02-05 | 2010-02-05 | Plant transformation terminator DNA (deoxyribonucleic acid) sequence and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2010101125006A CN102146383B (en) | 2010-02-05 | 2010-02-05 | Plant transformation terminator DNA (deoxyribonucleic acid) sequence and application thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN102146383A true CN102146383A (en) | 2011-08-10 |
| CN102146383B CN102146383B (en) | 2012-11-28 |
Family
ID=44420854
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN2010101125006A Expired - Fee Related CN102146383B (en) | 2010-02-05 | 2010-02-05 | Plant transformation terminator DNA (deoxyribonucleic acid) sequence and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN102146383B (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN118147165A (en) * | 2024-05-09 | 2024-06-07 | 中国热带农业科学院三亚研究院 | Application of FTO gene in improving total solid content of latex |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US7294504B1 (en) * | 2001-12-27 | 2007-11-13 | Allele Biotechnology & Pharmaceuticals, Inc. | Methods and compositions for DNA mediated gene silencing |
-
2010
- 2010-02-05 CN CN2010101125006A patent/CN102146383B/en not_active Expired - Fee Related
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN118147165A (en) * | 2024-05-09 | 2024-06-07 | 中国热带农业科学院三亚研究院 | Application of FTO gene in improving total solid content of latex |
| CN118147165B (en) * | 2024-05-09 | 2024-07-02 | 中国热带农业科学院三亚研究院 | Application of FTO gene in improving total solid content of latex |
Also Published As
| Publication number | Publication date |
|---|---|
| CN102146383B (en) | 2012-11-28 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Terada et al. | A large-scale Agrobacterium-mediated transformation procedure with a strong positive-negative selection for gene targeting in rice (Oryza sativa L.) | |
| CN105647925B (en) | Rice anther strong expression promoter OsAnth4 and application thereof | |
| CN105755021A (en) | Rice cadmium-tolerant gene OsGSTU37 and application thereof | |
| CN106957355A (en) | It is a kind of to the resistance to low light of plant and low temperature resistant related PPR albumen and its encoding gene and application | |
| CN102242119B (en) | Rice root-specific promoter Os03g01700 and application thereof | |
| CN102146383B (en) | Plant transformation terminator DNA (deoxyribonucleic acid) sequence and application thereof | |
| CN101880664B (en) | Monocotyledon promoter as well as preparation method and application thereof | |
| CN104651359A (en) | Bidirectional promoters separated out of corn and application thereof | |
| CN103789312A (en) | Corn endosperm tissue specificity promoter and application thereof | |
| CN102191243B (en) | DNA sequence of plant transformation terminator derived from rice and use thereof | |
| CN103667291B (en) | Derive from endosperm specificity expression promoter and the application thereof of paddy rice | |
| CN106434692A (en) | Applications of rice OsPCF7 gene in culturing high-tillering rice varieties | |
| CN102181444A (en) | Rice callus-specific promoter and application thereof | |
| CN102146384B (en) | Promoter BgIosP573, preparation method and application | |
| CN106399312B (en) | Inducible promoter NtPCS1P and application thereof | |
| CN100392081C (en) | Wheat WRAB 17 gene promotor and application thereof | |
| CN100485037C (en) | Wheat WRAB19 gene promotor and application thereof | |
| JP3782013B2 (en) | Improved promoter and use thereof | |
| CN102146399B (en) | Promoter BgIosP581 as well as preparation method and application thereof | |
| CN102146385B (en) | Promoter BgIosP576, and preparation method and use thereof | |
| CN100469881C (en) | Wheat WCOR726 gene promotor and application thereof | |
| KR101825960B1 (en) | Root―specific promoter derived from Oryza sativa and use thereof | |
| CN104328124A (en) | Promoter capable of driving or regulating expression of gene in plant stamen | |
| CN102146386B (en) | Promoter BgIosP557, preparation method and application | |
| CN102146398B (en) | Promoter BgIosP537, and preparation method and use thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| DD01 | Delivery of document by public notice |
Addressee: Beijing Guardian Technology Co., Ltd. Document name: Notification that Application Deemed not to be Proposed |
|
| DD01 | Delivery of document by public notice |
Addressee: BEIJING NORTH ELITE BIOTECHNOLOGY Co.,Ltd. Document name: Notification to Pay the Fees |
|
| DD01 | Delivery of document by public notice | ||
| DD01 | Delivery of document by public notice |
Addressee: Chen Lei Document name: Notice of termination of patent |
|
| DD01 | Delivery of document by public notice | ||
| DD01 | Delivery of document by public notice |
Addressee: Chen Lei Document name: Notice of correction of right of resumption |
|
| DD01 | Delivery of document by public notice | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20121128 Termination date: 20200205 |
|
| CF01 | Termination of patent right due to non-payment of annual fee | ||
| DD01 | Delivery of document by public notice |
Addressee: Patent of Beijing North Jieshi Biotechnology Co.,Ltd. The person in charge Document name: Notice of resumption of claim approval |
|
| DD01 | Delivery of document by public notice |