CN102250905A - Rice seed 16kD gliadin gene terminator and application thereof - Google Patents
Rice seed 16kD gliadin gene terminator and application thereof Download PDFInfo
- Publication number
- CN102250905A CN102250905A CN 201110197633 CN201110197633A CN102250905A CN 102250905 A CN102250905 A CN 102250905A CN 201110197633 CN201110197633 CN 201110197633 CN 201110197633 A CN201110197633 A CN 201110197633A CN 102250905 A CN102250905 A CN 102250905A
- Authority
- CN
- China
- Prior art keywords
- gene
- plant
- sequence
- expression
- prolamine
- 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
- 235000007164 Oryza sativa Nutrition 0.000 title claims abstract description 36
- 235000009566 rice Nutrition 0.000 title claims abstract description 35
- 240000007594 Oryza sativa Species 0.000 title claims description 37
- 108010061711 Gliadin Proteins 0.000 title abstract 3
- 241000196324 Embryophyta Species 0.000 claims abstract description 56
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 54
- 230000014509 gene expression Effects 0.000 claims abstract description 37
- 108020004414 DNA Proteins 0.000 claims abstract description 32
- 239000002773 nucleotide Substances 0.000 claims abstract description 5
- 125000003729 nucleotide group Chemical group 0.000 claims abstract description 5
- 108091028043 Nucleic acid sequence Proteins 0.000 claims abstract description 4
- 102000053602 DNA Human genes 0.000 claims abstract 3
- 230000009261 transgenic effect Effects 0.000 claims description 25
- 238000000034 method Methods 0.000 claims description 19
- 102000004169 proteins and genes Human genes 0.000 claims description 10
- 239000013598 vector Substances 0.000 claims description 10
- 241000894006 Bacteria Species 0.000 claims description 6
- 241000209510 Liliopsida Species 0.000 claims description 4
- 241001233957 eudicotyledons Species 0.000 claims description 4
- 238000009396 hybridization Methods 0.000 claims description 4
- 244000068988 Glycine max Species 0.000 claims description 3
- 235000010469 Glycine max Nutrition 0.000 claims description 3
- 240000008042 Zea mays Species 0.000 claims description 3
- 235000005824 Zea mays ssp. parviglumis Nutrition 0.000 claims description 3
- 235000002017 Zea mays subsp mays Nutrition 0.000 claims description 3
- 235000005822 corn Nutrition 0.000 claims description 3
- 229920000742 Cotton Polymers 0.000 claims description 2
- 244000299507 Gossypium hirsutum Species 0.000 claims description 2
- 240000005979 Hordeum vulgare Species 0.000 claims description 2
- 235000007340 Hordeum vulgare Nutrition 0.000 claims description 2
- 244000017020 Ipomoea batatas Species 0.000 claims description 2
- 235000002678 Ipomoea batatas Nutrition 0.000 claims description 2
- 244000061176 Nicotiana tabacum Species 0.000 claims description 2
- 235000002637 Nicotiana tabacum Nutrition 0.000 claims description 2
- 244000061456 Solanum tuberosum Species 0.000 claims description 2
- 235000002595 Solanum tuberosum Nutrition 0.000 claims description 2
- 244000046109 Sorghum vulgare var. nervosum Species 0.000 claims description 2
- 235000021307 Triticum Nutrition 0.000 claims description 2
- 244000098338 Triticum aestivum Species 0.000 claims description 2
- 240000001866 Vernicia fordii Species 0.000 claims description 2
- 241000700605 Viruses Species 0.000 claims description 2
- 108091027963 non-coding RNA Proteins 0.000 claims description 2
- 102000042567 non-coding RNA Human genes 0.000 claims description 2
- 230000008521 reorganization Effects 0.000 claims description 2
- 238000011160 research Methods 0.000 abstract description 5
- 230000006872 improvement Effects 0.000 abstract description 4
- 238000009825 accumulation Methods 0.000 abstract description 3
- 239000003814 drug Substances 0.000 abstract description 3
- 241000209094 Oryza Species 0.000 abstract 2
- RWRDLPDLKQPQOW-UHFFFAOYSA-N Pyrrolidine Chemical compound C1CCNC1 RWRDLPDLKQPQOW-UHFFFAOYSA-N 0.000 description 115
- DBMJMQXJHONAFJ-UHFFFAOYSA-M Sodium laurylsulphate Chemical compound [Na+].CCCCCCCCCCCCOS([O-])(=O)=O DBMJMQXJHONAFJ-UHFFFAOYSA-M 0.000 description 15
- 239000004141 Sodium laurylsulphate Substances 0.000 description 15
- 235000019333 sodium laurylsulphate Nutrition 0.000 description 15
- 239000013604 expression vector Substances 0.000 description 11
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 10
- 101000702488 Rattus norvegicus High affinity cationic amino acid transporter 1 Proteins 0.000 description 9
- 230000008859 change Effects 0.000 description 8
- 239000011734 sodium Substances 0.000 description 8
- 239000000243 solution Substances 0.000 description 7
- 238000012408 PCR amplification Methods 0.000 description 6
- 230000029087 digestion Effects 0.000 description 6
- 230000000694 effects Effects 0.000 description 6
- 238000002156 mixing Methods 0.000 description 6
- 241000589158 Agrobacterium Species 0.000 description 5
- 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 5
- 108010050181 aleurone Proteins 0.000 description 4
- 238000006243 chemical reaction Methods 0.000 description 4
- 101150054900 gus gene Proteins 0.000 description 4
- 108010005054 Deoxyribonuclease BamHI Proteins 0.000 description 3
- YQYJSBFKSSDGFO-UHFFFAOYSA-N Epihygromycin Natural products OC1C(O)C(C(=O)C)OC1OC(C(=C1)O)=CC=C1C=C(C)C(=O)NC1C(O)C(O)C2OCOC2C1O YQYJSBFKSSDGFO-UHFFFAOYSA-N 0.000 description 3
- 108091081024 Start codon Proteins 0.000 description 3
- 230000003321 amplification Effects 0.000 description 3
- 238000010276 construction Methods 0.000 description 3
- 238000001514 detection method Methods 0.000 description 3
- 238000011161 development Methods 0.000 description 3
- 230000018109 developmental process Effects 0.000 description 3
- 238000005516 engineering process Methods 0.000 description 3
- 239000012634 fragment Substances 0.000 description 3
- 230000002068 genetic effect Effects 0.000 description 3
- 230000008676 import Effects 0.000 description 3
- 238000004519 manufacturing process Methods 0.000 description 3
- 238000003199 nucleic acid amplification method Methods 0.000 description 3
- 239000000047 product Substances 0.000 description 3
- 229960005486 vaccine Drugs 0.000 description 3
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- 241000701489 Cauliflower mosaic virus Species 0.000 description 2
- 102000004190 Enzymes Human genes 0.000 description 2
- 108090000790 Enzymes Proteins 0.000 description 2
- 102000008857 Ferritin Human genes 0.000 description 2
- 238000008416 Ferritin Methods 0.000 description 2
- 108050000784 Ferritin Proteins 0.000 description 2
- 206010020649 Hyperkeratosis Diseases 0.000 description 2
- 108700019146 Transgenes Proteins 0.000 description 2
- 229920004890 Triton X-100 Polymers 0.000 description 2
- 239000013504 Triton X-100 Substances 0.000 description 2
- 108090000848 Ubiquitin Proteins 0.000 description 2
- 102000044159 Ubiquitin Human genes 0.000 description 2
- 230000003115 biocidal effect Effects 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- 230000004087 circulation Effects 0.000 description 2
- 238000001962 electrophoresis Methods 0.000 description 2
- 239000003623 enhancer Substances 0.000 description 2
- 108010083391 glycinin Proteins 0.000 description 2
- 230000036541 health Effects 0.000 description 2
- 239000007788 liquid Substances 0.000 description 2
- 239000003550 marker Substances 0.000 description 2
- 238000005259 measurement Methods 0.000 description 2
- 239000013612 plasmid Substances 0.000 description 2
- 230000001124 posttranscriptional effect Effects 0.000 description 2
- 230000008929 regeneration Effects 0.000 description 2
- 238000011069 regeneration method Methods 0.000 description 2
- 230000004044 response Effects 0.000 description 2
- 238000012216 screening Methods 0.000 description 2
- 241000894007 species Species 0.000 description 2
- 239000006228 supernatant Substances 0.000 description 2
- 238000012360 testing method Methods 0.000 description 2
- 230000002103 transcriptional effect Effects 0.000 description 2
- 230000009466 transformation Effects 0.000 description 2
- 238000013519 translation Methods 0.000 description 2
- 238000012795 verification Methods 0.000 description 2
- FPIPGXGPPPQFEQ-UHFFFAOYSA-N 13-cis retinol Natural products OCC=C(C)C=CC=C(C)C=CC1=C(C)CCCC1(C)C FPIPGXGPPPQFEQ-UHFFFAOYSA-N 0.000 description 1
- ARQXEQLMMNGFDU-JHZZJYKESA-N 4-methylumbelliferone beta-D-glucuronide Chemical compound C1=CC=2C(C)=CC(=O)OC=2C=C1O[C@@H]1O[C@H](C(O)=O)[C@@H](O)[C@H](O)[C@H]1O ARQXEQLMMNGFDU-JHZZJYKESA-N 0.000 description 1
- ARQXEQLMMNGFDU-UHFFFAOYSA-N 4MUG Natural products C1=CC=2C(C)=CC(=O)OC=2C=C1OC1OC(C(O)=O)C(O)C(O)C1O ARQXEQLMMNGFDU-UHFFFAOYSA-N 0.000 description 1
- 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
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 1
- LZZYPRNAOMGNLH-UHFFFAOYSA-M Cetrimonium bromide Chemical compound [Br-].CCCCCCCCCCCCCCCC[N+](C)(C)C LZZYPRNAOMGNLH-UHFFFAOYSA-M 0.000 description 1
- 101150074155 DHFR gene Proteins 0.000 description 1
- 101150111720 EPSPS gene Proteins 0.000 description 1
- 239000005562 Glyphosate Substances 0.000 description 1
- 229940124872 Hepatitis B virus vaccine Drugs 0.000 description 1
- 206010020751 Hypersensitivity Diseases 0.000 description 1
- 206010061217 Infestation Diseases 0.000 description 1
- 101100288095 Klebsiella pneumoniae neo gene Proteins 0.000 description 1
- 102000003960 Ligases Human genes 0.000 description 1
- 108090000364 Ligases Proteins 0.000 description 1
- 108060001084 Luciferase Proteins 0.000 description 1
- 108091022912 Mannose-6-Phosphate Isomerase Proteins 0.000 description 1
- IAJOBQBIJHVGMQ-UHFFFAOYSA-N Phosphinothricin Natural products CP(O)(=O)CCC(N)C(O)=O IAJOBQBIJHVGMQ-UHFFFAOYSA-N 0.000 description 1
- 238000009009 Protein Assay Kit II Methods 0.000 description 1
- 108700008625 Reporter Genes Proteins 0.000 description 1
- 108020005038 Terminator Codon Proteins 0.000 description 1
- 241000218636 Thuja Species 0.000 description 1
- FPIPGXGPPPQFEQ-BOOMUCAASA-N Vitamin A Natural products OC/C=C(/C)\C=C\C=C(\C)/C=C/C1=C(C)CCCC1(C)C FPIPGXGPPPQFEQ-BOOMUCAASA-N 0.000 description 1
- 241000607479 Yersinia pestis Species 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 230000009418 agronomic effect Effects 0.000 description 1
- 239000003513 alkali Substances 0.000 description 1
- FPIPGXGPPPQFEQ-OVSJKPMPSA-N all-trans-retinol Chemical compound OC\C=C(/C)\C=C\C=C(/C)\C=C\C1=C(C)CCCC1(C)C FPIPGXGPPPQFEQ-OVSJKPMPSA-N 0.000 description 1
- 208000026935 allergic disease Diseases 0.000 description 1
- 230000000735 allogeneic effect Effects 0.000 description 1
- WQZGKKKJIJFFOK-PQMKYFCFSA-N alpha-D-mannose Chemical compound OC[C@H]1O[C@H](O)[C@@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-PQMKYFCFSA-N 0.000 description 1
- 208000007502 anemia Diseases 0.000 description 1
- 230000001363 autoimmune Effects 0.000 description 1
- 101150103518 bar gene Proteins 0.000 description 1
- 239000002585 base Substances 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 229940098773 bovine serum albumin Drugs 0.000 description 1
- 238000009395 breeding Methods 0.000 description 1
- 239000000872 buffer Substances 0.000 description 1
- 239000007853 buffer solution Substances 0.000 description 1
- 229940027138 cambia Drugs 0.000 description 1
- 230000030570 cellular localization Effects 0.000 description 1
- 239000012295 chemical reaction liquid Substances 0.000 description 1
- 239000002299 complementary DNA Substances 0.000 description 1
- 230000001276 controlling effect Effects 0.000 description 1
- 230000002596 correlated effect Effects 0.000 description 1
- 238000013461 design Methods 0.000 description 1
- 206010012601 diabetes mellitus Diseases 0.000 description 1
- KXZOIWWTXOCYKR-UHFFFAOYSA-M diclofenac potassium Chemical compound [K+].[O-]C(=O)CC1=CC=CC=C1NC1=C(Cl)C=CC=C1Cl KXZOIWWTXOCYKR-UHFFFAOYSA-M 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 230000005284 excitation Effects 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 238000001917 fluorescence detection Methods 0.000 description 1
- 230000008014 freezing Effects 0.000 description 1
- 238000007710 freezing Methods 0.000 description 1
- 230000030279 gene silencing Effects 0.000 description 1
- IAJOBQBIJHVGMQ-BYPYZUCNSA-N glufosinate-P Chemical compound CP(O)(=O)CC[C@H](N)C(O)=O IAJOBQBIJHVGMQ-BYPYZUCNSA-N 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
- 239000004009 herbicide Substances 0.000 description 1
- 230000001744 histochemical effect Effects 0.000 description 1
- 101150029559 hph gene Proteins 0.000 description 1
- 230000009610 hypersensitivity Effects 0.000 description 1
- 230000003914 insulin secretion 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
- 229930027917 kanamycin Natural products 0.000 description 1
- SBUJHOSQTJFQJX-NOAMYHISSA-N kanamycin Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CN)O[C@@H]1O[C@H]1[C@H](O)[C@@H](O[C@@H]2[C@@H]([C@@H](N)[C@H](O)[C@@H](CO)O2)O)[C@H](N)C[C@@H]1N SBUJHOSQTJFQJX-NOAMYHISSA-N 0.000 description 1
- 229960000318 kanamycin Drugs 0.000 description 1
- 229930182823 kanamycin A Natural products 0.000 description 1
- 238000009630 liquid culture Methods 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 230000004060 metabolic process Effects 0.000 description 1
- 239000013642 negative control Substances 0.000 description 1
- 230000012666 negative regulation of transcription by glucose Effects 0.000 description 1
- 229910052757 nitrogen Inorganic materials 0.000 description 1
- 230000035479 physiological effects, processes and functions Effects 0.000 description 1
- 239000013641 positive control Substances 0.000 description 1
- 230000004481 post-translational protein modification Effects 0.000 description 1
- 230000002265 prevention Effects 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 108090000765 processed proteins & peptides Proteins 0.000 description 1
- 238000004445 quantitative analysis Methods 0.000 description 1
- 238000003259 recombinant expression Methods 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
- 108091008146 restriction endonucleases Proteins 0.000 description 1
- JQXXHWHPUNPDRT-WLSIYKJHSA-N rifampicin Chemical compound O([C@](C1=O)(C)O/C=C/[C@@H]([C@H]([C@@H](OC(C)=O)[C@H](C)[C@H](O)[C@H](C)[C@@H](O)[C@@H](C)\C=C\C=C(C)/C(=O)NC=2C(O)=C3C([O-])=C4C)C)OC)C4=C1C3=C(O)C=2\C=N\N1CC[NH+](C)CC1 JQXXHWHPUNPDRT-WLSIYKJHSA-N 0.000 description 1
- 229960001225 rifampicin Drugs 0.000 description 1
- 230000024053 secondary metabolic process Effects 0.000 description 1
- 238000010187 selection method Methods 0.000 description 1
- 239000001488 sodium phosphate Substances 0.000 description 1
- 229910000162 sodium phosphate Inorganic materials 0.000 description 1
- 239000012064 sodium phosphate buffer Substances 0.000 description 1
- 238000010186 staining Methods 0.000 description 1
- 238000003860 storage Methods 0.000 description 1
- 238000005728 strengthening Methods 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
- 230000005026 transcription initiation Effects 0.000 description 1
- 230000014621 translational initiation Effects 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
- RYFMWSXOAZQYPI-UHFFFAOYSA-K trisodium phosphate Chemical compound [Na+].[Na+].[Na+].[O-]P([O-])([O-])=O RYFMWSXOAZQYPI-UHFFFAOYSA-K 0.000 description 1
- 238000011144 upstream manufacturing Methods 0.000 description 1
- 238000003805 vibration mixing Methods 0.000 description 1
- 235000019155 vitamin A Nutrition 0.000 description 1
- 239000011719 vitamin A Substances 0.000 description 1
- 229940045997 vitamin a Drugs 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 discloses a rice seed 16kD gliadin gene terminator and application thereof. The terminator is a DNA molecule which can be the following 1) or 2) or 3): 1) the DNA molecule composed of a nucleotide sequence represented by a sequence 1 in a sequence table; 2) molecules at least having 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% of homology with the sequence of the DNA limited by 1); 3) a molecule in hybrid with the DNA sequence limited by 1) or 2). The expression and accumulation level of an exogenous gene in a target plant can be improved by matching the terminator with different promotors, thereby laying a foundation for researches on improvement of the seed quality and molecular medicine farms by using the biotechnology; and the rice seed 16kD gliadin gene terminator has great application prospect.
Description
Technical field
The present invention relates to a kind of rice paddy seed 16kD alcohol soluble protein gene terminator and application thereof.
Background technology
The recent development of Plant Biotechnology has not only realized the improvement (as improving crop yield, strengthening disease-resistant, pest-resistant, antiweed characteristic, improvement quality etc.) of traditional economical character, and makes plant become the bio-reactor of biological medicine and Industrial products.Characteristics such as most gramineous crops have that output height, production cost are low, storage endurance, industrial scale are controlled easily, direct-edible, and it possesses the ability of posttranslational modification in the body, thereby become the ideal carrier of s-generation transgene product.Utilize rice paddy seed production foreign protein with health role and the development of edibility vaccine research very fast in recent years, and obtained immense success.All successfully in paddy rice, express and the high level accumulation as vitamin A, glycinin (Glycinin), soybean ferritin (Ferritin), GLP, Hepatitis B virus vaccine and the pollen hypersensitivity vaccine etc. that have prevention and treatment hypoferric anemia and improve autoimmune function with the prevent diabetes generation function that stimulates insulin secretion with lowering blood glucose effect.Yet, realize that further efficiently expressing of foreign gene must improve genetic expression simultaneously transcribing with post-transcriptional level.Promotor is mainly in transcriptional level control genetic expression, and terminator then plays regulating and controlling effect simultaneously transcribing with post-transcriptional level.
Although the terminator of widespread use at present as Nos, Ocs, and can start the high expression level of reporter gene in plant after the allogeneic promoter combination, can satisfy the research demand of biological chemistry, physiology and cellular localization aspect.Yet, less for other terminator researchs that can improve genetic expression.Because some Main Agronomic Characters and Secondary Metabolism of Plant product all are by controlled by multiple genes, the expression that improves individual gene is not obvious for the correlated character improving effect.By traditional method for transformation, as repeat to transform or hybridization realizes that polygene transforms and but wastes time and energy.The polygene conversion system that development in recent years is got up can insert a plurality of genes simultaneously in an expression vector, for fear of the too high transgene silencing that causes of transgenosis homology, these genes need be expressed by different promoters driven, and different terminators stops transcribing.
Summary of the invention
An object of the present invention is to provide a terminator, name is called t16kD prolamine, and this terminator is compared with the no terminator, can improve the expression of exogenous gene level.
Terminator provided by the present invention is following 1) or 2) or 3) dna molecular:
1) dna molecular of forming by the nucleotide sequence shown in the sequence in the sequence table 1;
2) with 1) sequence of the DNA that limits has 70% at least, have 75% at least, have 80% at least, have 85% at least, have 90% at least, have 95% at least, have 96% at least, have 97% at least, have 98% or the molecule that has 99% homology at least at least;
3) under stringent condition with 1) or 2) molecule of the dna sequence dna hybridization that limits.
Described stringent condition can be as follows: 50 ℃, and at 7% sodium lauryl sulphate (SDS), 0.5M Na
3PO
4With hybridize in the mixing solutions of 1mM EDTA, at 50 ℃, 2 * SSC, rinsing among the 0.1%SDS; Also can be: 50 ℃, at 7%SDS, 0.5M Na
3PO
4With hybridize in the mixing solutions of 1mM EDTA, at 50 ℃, 1 * SSC, rinsing among the 0.1%SDS; Also can be: 50 ℃, at 7%SDS, 0.5M Na
3PO
4With hybridize in the mixing solutions of 1mM EDTA, at 50 ℃, 0.5 * SSC, rinsing among the 0.1%SDS; Also can be: 50 ℃, at 7%SDS, 0.5M Na
3PO
4With hybridize in the mixing solutions of 1mM EDTA, at 50 ℃, 0.1 * SSC, rinsing among the 0.1%SDS; Also can be: 50 ℃, at 7%SDS, 0.5M Na
3PO
4With hybridize in the mixing solutions of 1mM EDTA, at 65 ℃, 0.1 * SSC, rinsing among the 0.1%SDS; Also can be: at 6 * SSC, in the solution of 0.5%SDS, 65 ℃ of hybridization down, use 2 * SSC then, 0.1%SDS and 1 * SSC, 0.1%SDS respectively wash film once.
Wherein, sequence 1 is made up of 531 Nucleotide in the sequence table.
The recombinant vectors, expression cassette, transgenic cell line, reorganization bacterium or the recombinant virus that contain described dna molecular also belong to protection scope of the present invention.
Available existing plant expression vector construction contains the recombinant expression vector of described dna molecular.Described plant expression vector comprises the double base agrobacterium vector and can be used for the carrier etc. of plant micropellet bombardment.As pROKII, pBin438, pCAMBIA1302, pCAMBIA2301, pCAMBIA1301, pCAMBIA1300, pBI121, pCAMBIA1391-Xa or pCAMBIA1391-Xb (CAMBIA company) etc.When using described dna molecular to make up the recombinant plant expression vector, can add any enhancement type promotor (as the ubiquitin promoter (Ubiquitin) of cauliflower mosaic virus (CAMV) 35S promoter, corn), constitutive promoter or organizing specific expression promotor (as the promotor of seed specific expression) before transcription of foreign genes nuclei originis thuja acid, they can use separately or be used in combination with other plant promoter; In addition, when using dna molecular of the present invention to make up plant expression vector, also can use enhanser, comprise translational enhancer or transcriptional enhancer, these enhanser zones can be ATG initiator codon or neighboring region initiator codon etc., but must be identical with the reading frame of encoding sequence, to guarantee the correct translation of whole exogenous gene sequence.The source of described translation control signal and initiator codon is widely, can be natural, also can be synthetic.Translation initiation region can be from transcription initiation zone or structure gene.For the ease of transgenic plant cells or plant being identified and screening, can process used plant expression vector, can produce the enzyme of colour-change or the gene (gus gene of luminophor as adding the coding that in plant, to express, luciferase genes etc.), antibiotic marker gene (as is given nptII gene to kantlex and associated antibiotic resistance, give bar gene to weedicide phosphinothricin resistance, give hph gene to the microbiotic hygromycin resistance, with the dhfr gene of giving the methatrexate resistance, give EPSPS gene to the glyphosate resistance) or anti-chemical reagent marker gene etc. (as anti-weedkiller gene), the mannose-6-phosphate isomerase gene of metabolism seminose ability is provided.
Described recombinant vectors specifically can be pGluB-3-t16kD prolamine.Described pGluB-3-t16kD prolamine replaces with the recombinant vectors that the t16kD prolamine terminator shown in the sequence 1 obtains in the sequence table with the no terminator among the pGluB-3-nos.
Described expression cassette is meant and starts by promotor, by described promotor that the goal gene of transcribing and the described dna molecular that is positioned at described goal gene downstream form.Wherein, described promotor can be constitutive promoter or organizing specific expression promotor (as endosperm specificity promoter).Described goal gene can be protein coding gene and/or non-protein coding gene; Described protein coding gene is preferably the quality-improving gene; Described non-protein coding gene is just rna gene and/or sense-rna gene.
Another object of the present invention provides a kind of method of cultivating transgenic plant.
The method of cultivation transgenic plant provided by the present invention, be that the expression cassette that will contain described dna molecular imports in the purpose plant, obtain described destination gene expression level and be higher than the transgenic plant that following expression cassette imported described purpose plant: the described dna molecular in the described expression cassette is replaced with the expression cassette that the no terminator of rouge alkali synthetase obtains.
Described purpose plant specifically can be monocotyledons or dicotyledons.
Described monocotyledons specifically can be paddy rice, wheat, and corn, Chinese sorghum or barley, described dicotyledons specifically can be soybean, rape, cotton, tobacco, potato, sweet potato or tung oil tree.
Described transgenic plant are interpreted as not only to comprise the described expression cassette that contains described dna molecular are transformed the first-generation transgenic plant that the purpose plant obtains, also comprise its filial generation.For transgenic plant, can in these species, breed this and contain the expression cassette of described dna molecular, also available traditional breeding method shifts other kind that enters same species with the expression cassette that this contains described dna molecular, in commercial variety.
Described dna molecular in cultivating transgenic plant application or as the application of terminator also in protection scope of the present invention.
Utilize terminator of the present invention and different promotor cooperations, can improve expression and the accumulation level of foreign gene in the purpose plant, but improved seed quality, the albumen that will have physiologically active or small peptide import initiative health function new variety in the seed, utilize seed to produce useful foreign protein or edibility vaccine as bio-reactor, increase agricultural-food science and technology added value etc.Terminator of the present invention and being applied as utilizes researchs such as bio-technology improvement seed quality, molecule medicine farm to lay a good foundation, and has great application prospect.
Description of drawings
Fig. 1 is the electrophoretogram of pcr amplification 16kD prolamine terminator (t16kD prolamine) from oryza sativa genomic dna.Wherein 1 is dna molecular amount standard, and 2 is the fragment of 16kD prolamine terminator.Wherein, the standard molecular weight size is followed successively by from the bottom to top: 0.1kb, 0.2kb, 0.3kb, 0.4kb, 0.5kb, 0.6kb, 0.7kb, 0.8kb, 0.9kb, 1.0kb, 1.2kb, 1.5kb, 2.0kb, 3.0kb, 4.0kb, 5.0kb, 6.0kb, 8.0kb, 10.0kb.
Fig. 2 is that carrier pMD-t16kD prolamine identifies collection of illustrative plates through the double digestion of Sac I and EcoR I.Wherein 1 is dna molecular amount standard, and 2 is the endonuclease bamhi of pMD-t16kD prolamine.Wherein, the standard molecular weight size is followed successively by from the bottom to top: 0.1kb, 0.2kb, 0.3kb, 0.4kb, 0.5kb, 0.6kb, 0.7kb, 0.8kb, 0.9kb, 1.0kb, 1.2kb, 1.5kb, 2.0kb, 3.0kb, 4.0kb, 5.0kb, 6.0kb, 8.0kb, 10.0kb endonuclease bamhi is followed successively by from the bottom to top: t16kD prolamine, 531bp; PMD-t16kD prolamine carrier frame, 2700bp.
Fig. 3 is the structural representation of carrier pGluB-3-t16kD prolamine.Wherein, 16kD proT represents t16kD prolamine.
Fig. 4 is for changeing pGluB-3-t16kD prolamine carrier T
0For rice plant chimeric primers pcr amplification electrophoretogram.1 is dna molecular amount standard, and 2 for being the positive control of template with pGluB-3-t16kD prolamine, and 3 for being the negative control of template with unconverted strain, and 4～13 for changeing the regeneration plant of pGluB-3-t16kD prolamine plant expression vector.
Fig. 5 is not genetically modified wild-type paddy rice Kitaake plant GUS coloration result.Wherein, 1,2,3,4 coloration results that are respectively not genetically modified wild-type paddy rice Kitaake plant root, stem, leaf and seed.
Fig. 6 is T
0For transgenic paddy rice Kitaake/pGluB-3-nos plant GUS coloration result.Wherein, 1,2,3,4 be respectively the coloration result that changes pGluB-3-nos plant root, stem, leaf and seed.
Fig. 7 is T
0For transgenic paddy rice Kitaake/pGluB-3-t16kD prolamine plant GUS coloration result.Wherein, 1,2,3,4 be respectively the coloration result that changes pGluB-3-t16kD prolamine plant root, stem, leaf and seed.
Fig. 8 is T
0For the seed bearing GUS fluorescence activity measurement result of transgenic paddy rice Kitaake/pGluB-3-t16kD prolamine.
Fig. 9 is T
1For the seed bearing GUS fluorescence activity measurement result of transgenic paddy rice Kitaake/pGluB-3-t16kD prolamine.
Embodiment
Employed experimental technique is ordinary method if no special instructions among the following embodiment.
Used material, reagent etc. if no special instructions, all can obtain from commercial channels among the following embodiment.
The acquisition of embodiment 1, rice paddy seed 16kD alcohol soluble protein gene terminator (t16kD prolamine)
According to paddy rice 16kD alcohol soluble protein gene cDNA sequence (GenBank number is AK107785), from GenBank, search the genomic dna sequence of 16kD alcohol soluble protein gene, the sequence of 531bp is rice paddy seed 16kD alcohol soluble protein gene terminator of the present invention (t16kD prolamine) after the terminator codon, design primer amplification t16kD prolamine.For ease of vector construction, on primer, add restriction enzyme site (shown in the underscore) respectively.The forward primer of t16kD prolamine is 16kD prolamine SacF:5 '-AA
GAGCTCTAAAATTTGGAATAGTCTTTGG-3 ' (Sac I), reverse primer are 16kD prolamine EcoR:5 '-AAA
GAATTCACTTCTTCTATGTTTATAG-3 ' (EcoR I).
The CTAB method is from wild-type paddy rice Kitaake (document Qu et al., J.Exp.Bot.2008,59:2417-2424) extract genomic dna in the blade in a small amount, with it is template, with 16kD prolamine SacF and 16kD prolamine EcoR is primer, pcr amplification t16kD prolamine sequence.Response procedures is: 94 ℃ of pre-sex change 5min, 94 ℃ of 30sec then, 55 ℃ of 30sec, 72 ℃ of 1min, 30 circulations, last 72 ℃ of 10min.Pcr amplification obtains the purpose band of 531bp, as shown in Figure 1.Reclaim amplified production, be directly connected on the pMD18-T carrier (available from TaKaRa company), check order, the result shows that the t16kD prolamine sequence size that amplification obtains is 531bp, has the nucleotide sequence of sequence 1 in the sequence table.To check order to detect and show and contain the segmental recombinant vectors called after of t16kD prolamine pMD-t16kD prolamine.PMD-t16kD prolamine identifies collection of illustrative plates as shown in Figure 2 through the double digestion of Sac I and EcoR I.
The stably express vector construction of embodiment 2, rice paddy seed 16kD alcohol soluble protein gene terminator (t16kD prolamine)
1, t16kD prolamine merges the plant stability expression vector establishment of gus gene
With Sac I and EcoR I double digestion pMD-t16kD prolamine plasmid, reclaim t16kD prolamine 531bp endonuclease bamhi, this fragment is inserted between the SacI of the pGluB-3-nos that contains the GluB-3 promotor and the EcoR I double digestion recognition site makes up stable conversion carrier pGluB-3-t16kD prolamine (its structural representation as shown in Figure 3).PGluB-3-nos is according to document Qu et al., J.Exp.Bot.2008, and the described method of 59:2417-2424 makes up: with 65 genomic dna in the paddy rice platform is template, with GluB-3 forward primer 5 '-CCC
AAGCTTATTTTACTTGTACTGTTTAACC-3 ' (HindIII) and GluB-3 reverse primer 5 '-AAA
CCCGGGAGCTTTCTGTATATGCTAATG-3 ' is a primer (SmaI), pcr amplification obtains the GluB-3 promotor, with GluB-3 promotor HindIII and SmaI double digestion, be inserted into pGPTV-35S-HPT (Qu et al., J.Exp.Bot.2008, the HindIII of GUS upstream 59:2417-2424) and SmaI site, the recombinant vectors that obtains is pGluB-3-nos.PGPTV-35S-HPT is foreign gene with GUS, and no is a terminator.
Utilizing Sac I and EcoR I to carry out double digestion on the basis that PCR identifies the recombinant vectors pGluB-3-t16kD prolamine that obtains identifies, obtains containing the fragment of 531bp t16kD prolamine.
2, change the acquisition of pGluB-3-t16kD prolamine and pGluB-3-nos rice regeneration plant
The pGluB-3-t16kD prolamine expression vector that pGluB-3-nos and make up finishes through enzyme cut with its exactness of sequence verification after, utilize freeze-thaw method to import respectively among the Agrobacterium EHA105, concrete grammar is as follows: draw 7 μ l expression vector plasmids and add in the 100 μ l EHA105 Agrobacterium competent cells, flick mixing and be placed on freezing 7min in the liquid nitrogen, change over to afterwards in 37 ℃ of water-baths and leave standstill 3min, add the 800ulYEB liquid culture then and leave standstill recovery cultivation 3h～5h based on 28 ℃, get 400 μ l bacterium liquid and coat (kantlex 50mg/L on the YEB resistant panel, Rifampin Rif 50mg/L), be inverted cultivation 2～3 days in 28 ℃.After the bacterium colony PCR that the single bacterium colony of a little Agrobacterium of picking carries out target gene detects positive bacteria dropped on the YEB resistant panel line and expand numerous cultivation, utilize the callus of Agrobacterium infestation method rice transformation kind kitaake, use the hygromycin selection kanamycin-resistant callus tissue, and further cultivate and be divided into intermediate house behind the seedling.
Utilize the hygromycin selection method (according to document Hiei et al., Plant J.1994, the described method of 6:271-282 is carried out), obtain 10 strain T
0In generation, changeed pGluB-3-t16kD prolamine rice plant.The part of utilizing PCR method that above-mentioned screening is obtained is changeed pGluB-3-t16kD prolamine paddy rice and is carried out the PCR Molecular Detection, the PCR primer is the chimeric primers of t16kD prolamine and gus gene sequence, that is: t16kD prolamine sequence reverse primer 16kD prolamine EcoR:5 '-AAA
GAATTCThe forward primer GUSF185:5 '-TCGTCGGTGAACAGGTATGG-3 ' of ACTTCTTCTATGTTTATAG-3 ' and GUS sequence inside.The PCR response procedures is: 94 ℃ of pre-sex change 5min, 94 ℃ of 30sec then, 55 ℃ of 30sec, 72 ℃ of 1min, 30 circulations, last 72 ℃ of 10min.The purpose band that pcr amplification obtains 0.7kb is the detection positive.The result shows that 10 strains that PCR detects change pGluB-3-t16kD prolamine plant (being expressed as Kitaake/pGluB-3-t16kD prolamine) and all be positive (as shown in Figure 4).
According to the method described above, change pGluB-3-nos over to wild-type paddy rice Kitaake, obtain the positive pGluB-3-nos plant (being expressed as Kitaake/pGluB-3-nos) that pGluB-3-nos is changeed in 9 strains.Change the PCR detection method of pGluB-3-nos rice plant: the forward primer GUSF185:5 '-TCGTCGGTGAACAGGTATGG-3 ' with tnos sequence reverse primer tnosR:5 '-GATCTAGTAACATAGATGAC-3 ' and GUS sequence inside is a primer, the chimeric sequences of amplification tnos and gus gene, the purpose band that obtains 500bp is positive transformant.
T
0The seed of tying for transfer-gen plant and be T by the plant that this seed grows up to
1In generation, the rest may be inferred, T
2, T
3Represent the 2nd generation of transfer-gen plant and the 3rd generation respectively.
The functional verification of embodiment 3, rice paddy seed 16kD alcohol soluble protein gene terminator (t16kD prolamine)
1, T
0GUS histological chemistry for transgenic paddy rice is detected
To the transgenic paddy rice Kitaake/pGluB-3-nos of PCR test positive and the T of Kitaake/pGluB-3-t16kD prolamine
0Carrying out histochemical stain for plant, is contrast with not genetically modified wild-type paddy rice Kitaake plant.Concrete steps are: with transgenic paddy rice Kitaake/pGluB-3-nos and Kitaake/pGluB-3-t16kD prolamineT
0Partial blade, root, cane tissue for plant and not genetically modified wild-type paddy rice Kitaake plant are cut into small pieces; The filling stage seed that to bloom back 17 days with scalper from the middle part longitudinal incision.The sample of handling well is soaked in GUS staining reaction liquid (0.1M sodium phosphate buffer (pH 7.0), 10mM Na
2-EDTA (pH7.0), the 5mM Tripotassium iron hexacyanide, 5mM yellow prussiate of potash, 1.0mM X-Gluc, 0.1%Triton X-100), 37 ℃ were reacted 0.5-4 hour.Preserve in 70% ethanol, observe.Observe under the stereoscope and take pictures.Result such as Fig. 5, Fig. 6, shown in Figure 7: root, blade, the cane of not genetically modified wild-type paddy rice Kitaake plant and the aleurone layer of the back 17 days seed of blooming and inferior aleurone layer are not all observed GUS and are expressed; T
0All do not observe GUS for root, blade, the cane of transgenic paddy rice Kitaake/pGluB-3-nos and Kitaake/pGluB-3-t16kD prolamine plant and express, and the aleurone layer of the back 17 days seed of blooming and inferior aleurone layer all there are GUS to express.The result shows that pGluB-3-t16kD prolamine is similar to pGluB-3-nos expression intensity in endosperm, and t16kD prolamine and no terminator all do not change endosperm specificity expression.
2, T
0And T
1GUS fluorescence activity for transgenic paddy rice is measured
To the transgenic paddy rice Kitaake/pGluB-3-nos of PCR test positive and the T of Partial K itaake/pGluB-3-t16kD prolamine
0Generation and T
1Carry out the GUS quantitative analysis for 17 days filling stage seed behind the plant blossom.Method according to the fluorescence detection method of Jefferson etc. (Jefferson, Plant Mol.Biol.Report, 1987,5:387-405) carry out.Be specially: 3 seeds are got in every strain, place 3 1.5ml eppendorf pipes respectively, with pestle seed are pulverized, and add 200 μ l extract (50mM buffer solution of sodium phosphate (pH7.0), 10mM beta-mercaptoethanol, 10mM Na
2EDTA (pH 8.0), 0.1%SDS, 0.1%Triton X-100), the vibration mixing.4 ℃ 13, the centrifugal 5min of 000rpm gets supernatant in new pipe.10 μ l supernatants add 90 μ l reaction solutions (1mM 4-MUG is dissolved in the GUS extract), and 37 ℃ were reacted 60 minutes, and added 900 μ l stop buffer (0.2M Na
2CO
3), the room temperature termination reaction.Do typical curve with 4-MU, under 360nm excitation wavelength and 460nm absorbing wavelength, detect relative 4-MU content with F-4500 (Hitachi) type spectrophotofluorometer.With the bovine serum albumin is contrast, utilizes BIO-Rad Protein Assay Kit II (available from Bio Rad Laboratories, numbering GPR6611) to measure protein content.
To transgenic line T
0The Dai Suojie seed carries out GUS determination of activity result as shown in Figure 8.T at 9 Kitaake/pGluB-3-t16kD prolamine
0In the strain system, the active maximum of GUS is 34.34pmol 4-MU min
-1μ g
-1Albumen, Schwellenwert are 5.36pmol 4-MU min
-1μ g
-1Albumen, mean value are 13.2 ± 10.2pmol 4-MU min
-1μ g
-1Albumen.And the contrast T of 9 Kitaake/pGluB-3-nos
0In the strain system, the active maximum of GUS is 17.38pmol 4-MU min
-1μ g
-1Albumen, Schwellenwert are 2.39pmol 4-MU min
-1μ g
-1Albumen, mean value are 11.2 ± 5.0pmol 4-MU min
-1μ g
-1Albumen.Changeing pGluB-3-t16kD prolamine carrier maximum value is peaked 1.97 times of the latter, is 14.34 times of its minimum value, and mean value is 1.17 times of contrast mean value.
T to the transgenic line part
1The Dai Suojie seed carries out GUS determination of activity result as shown in Figure 9.The T of 8 Kitaake/pGluB-3-t16kD prolamine
1Strain is that the active maximum of GUS is 29.24pmol 4-MU min
-1μ g
-1Albumen, Schwellenwert are 6.38pmol 4-MU min
-1μ g
-1Albumen, mean value are 15.97 ± 8.3pmol 4-MU min
-1μ g
-1Albumen.And the T of 7 contrast Kitaake/pGluB-3-nos
1In the strain system, then the active maximum of GUS is 21.52pmol 4-MU min
-1μ g
-1Albumen, Schwellenwert are 2.82pmol 4-MU min
-1μ g
-1Albumen, mean value are 9.9 ± 5.8pmol 4-MU min
-1μ g
-1Albumen.The former mean value is 1.60 times of contrast.
The result shows that t16kD prolamine compares with the no terminator and strengthened the expression level of foreign gene in paddy endosperm.
Claims (10)
1.DNA molecule is characterized in that: described dna molecular is following 1) or 2) or 3) molecule:
1) dna molecular of forming by the nucleotide sequence shown in the sequence in the sequence table 1;
2) with 1) sequence of the DNA that limits has 70% at least, have 75% at least, have 80% at least, have 85% at least, have 90% at least, have 95% at least, have 96% at least, have 97% at least, have 98% or the molecule that has 99% homology at least at least;
3) under stringent condition with 1) or 2) molecule of the dna sequence dna hybridization that limits.
2. the recombinant vectors, expression cassette, transgenic cell line, reorganization bacterium or the recombinant virus that contain the described dna molecular of claim 1.
3. expression cassette according to claim 2 is characterized in that: described expression cassette is by promotor, start the goal gene of transcribing and the described dna molecular of claim 1 that is positioned at described goal gene downstream is formed by described promotor.
4. expression cassette according to claim 3 is characterized in that: described promotor is constitutive promoter or organizing specific expression promotor, and described organizing specific expression promotor specifically can be endosperm specificity expression promoter.
5. according to claim 3 or 4 described expression cassettes, it is characterized in that: described goal gene is protein coding gene and/or non-protein coding gene; Described protein coding gene is preferably the quality-improving gene; Described non-protein coding gene is just rna gene and/or sense-rna gene.
6. method of cultivating transgenic plant, be that arbitrary described expression cassette among the claim 3-5 is imported in the purpose plant, obtain described destination gene expression level and be higher than the transgenic plant that following expression cassette imported described purpose plant: the described dna molecular of claim 1 in the described expression cassette of claim 3 is replaced with the expression cassette that the no terminator obtains.
7. method according to claim 6 is characterized in that: described purpose plant is monocotyledons or dicotyledons.
8. method according to claim 7 is characterized in that: described monocotyledons is a paddy rice, wheat, corn, Chinese sorghum or barley; Described dicotyledons is soybean, rape, cotton, tobacco, potato, sweet potato or tung oil tree.
9. the application of the described dna molecular of claim 1 in cultivating transgenic plant.
10. the described dna molecular of claim 1 is as the application of terminator.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 201110197633 CN102250905B (en) | 2011-07-15 | 2011-07-15 | Rice seed 16kD gliadin gene terminator and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 201110197633 CN102250905B (en) | 2011-07-15 | 2011-07-15 | Rice seed 16kD gliadin gene terminator and application thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN102250905A true CN102250905A (en) | 2011-11-23 |
| CN102250905B CN102250905B (en) | 2013-04-03 |
Family
ID=44978474
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN 201110197633 Expired - Fee Related CN102250905B (en) | 2011-07-15 | 2011-07-15 | Rice seed 16kD gliadin gene terminator and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN102250905B (en) |
-
2011
- 2011-07-15 CN CN 201110197633 patent/CN102250905B/en not_active Expired - Fee Related
Non-Patent Citations (3)
| Title |
|---|
| 《Journal of the Science of Food and Agriculture》 20051231 Hanaa A S Oraby 等 Screening food products for the presence of CaMV 35S promoter and NOS 3'terminator 1974-1980 1-10 第85卷, * |
| 《PLANT BIOTECHNOLOGY JOURNAL》 20040331 Qu, LQ 等 Evaluation of tissue specificity and expression strength of rice seed component gene promoters in transgenic rice 113-125 1-10 第2卷, 第2期 * |
| 《Plant Cell Physiol. 》 19981231 Chuan-Yin Wu 等 Promoters of Rice Seed Storage Protein Genes Direct Endosperm-Specific Gene Expression in Transgenic Rice 885-889 1-10 第39卷, 第8期 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN102250905B (en) | 2013-04-03 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN100562574C (en) | Plant endosperm specificity promoter and application thereof | |
| CN103820445A (en) | Identification and application of plant anther specific expression promoter | |
| CN111154756B (en) | Plant anther pollen late development specific expression promoter and application thereof | |
| CN102712929B (en) | Identification and use of plant root-specific expression promoter | |
| WO2021141082A1 (en) | Enhancer | |
| CN103923196B (en) | Derive from the disease resistance associated protein TaPK-R1 of Semen Tritici aestivi and relevant biological material thereof and application | |
| CN103740717B (en) | A kind of EMBRYO IN RICE specific expressing promoter and application thereof | |
| CN103275202A (en) | Disease resistance-related protein RCR1 derived from wheat, related biomaterials thereof, and application for same | |
| CN106148344B (en) | One kind having the active 5 ' UTR sequence of enhancing gene expression in plants and its application | |
| CN102250908B (en) | Rice seed 26kD globulin Glb-1 gene terminator and application thereof | |
| CN102250907B (en) | Rice glutelin gene GluA-2 terminator and use thereof | |
| CN102250892B (en) | Plant endosperm specific expression promoter and use thereof | |
| CN102260675B (en) | Rice seed glutelin GluB-5 gene terminator and application thereof | |
| CN103667291B (en) | Derive from endosperm specificity expression promoter and the application thereof of paddy rice | |
| CN102250905B (en) | Rice seed 16kD gliadin gene terminator and application thereof | |
| CN102250906B (en) | Rice seed glutelin GluD-1 gene terminator and application thereof | |
| CN102260676B (en) | Glutelin GluA-3 gene terminator of rice seed and application thereof | |
| CN102260674B (en) | Glutelin GluB-4 gene terminator of rice seed and application thereof | |
| CN102260677B (en) | Rice seed glutelin GluC gene terminator and application thereof | |
| CN102517284A (en) | Promoter miR390 and applications thereof | |
| CN102250893B (en) | Plant endosperm specific expression promoter and application thereof | |
| CN105316333A (en) | Identification and application of plant anther specific expression promoter pTaASG005 | |
| CN102277355A (en) | Rice seed glutelin GluA-2 gene terminator and application thereof | |
| CN102250895B (en) | Specific expression promoter for plant embryo and application thereof | |
| CN102250894B (en) | Specific expression promoter for plant embryo and application 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 | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20130403 Termination date: 20140715 |
|
| EXPY | Termination of patent right or utility model |