CN108060167B - 一种长牡蛎tyrosinase1启动子及其重组表达载体及应用 - Google Patents
一种长牡蛎tyrosinase1启动子及其重组表达载体及应用 Download PDFInfo
- Publication number
- CN108060167B CN108060167B CN201711401175.3A CN201711401175A CN108060167B CN 108060167 B CN108060167 B CN 108060167B CN 201711401175 A CN201711401175 A CN 201711401175A CN 108060167 B CN108060167 B CN 108060167B
- Authority
- CN
- China
- Prior art keywords
- promoter
- expression vector
- recombinant expression
- application
- crassostrea
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
- 101001091417 Agaricus bisporus Polyphenol oxidase 1 Proteins 0.000 title claims abstract description 21
- 101001125487 Bombyx mori Phenoloxidase subunit 1 Proteins 0.000 title claims abstract description 21
- 101000606125 Margaritifera margaritifera Tyrosinase-like protein 1 Proteins 0.000 title claims abstract description 21
- 239000013604 expression vector Substances 0.000 title claims abstract description 19
- 241000548230 Crassostrea angulata Species 0.000 title claims abstract description 18
- 238000003259 recombinant expression Methods 0.000 title claims abstract description 17
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 17
- 235000015170 shellfish Nutrition 0.000 claims abstract description 9
- 108091028043 Nucleic acid sequence Proteins 0.000 claims description 2
- 238000002360 preparation method Methods 0.000 claims description 2
- 241000237501 Crassostrea Species 0.000 claims 4
- 238000005516 engineering process Methods 0.000 abstract description 2
- 239000013598 vector Substances 0.000 description 15
- 238000002347 injection Methods 0.000 description 7
- 239000007924 injection Substances 0.000 description 7
- 108020004414 DNA Proteins 0.000 description 5
- 239000013612 plasmid Substances 0.000 description 5
- 239000000047 product Substances 0.000 description 5
- 241000588724 Escherichia coli Species 0.000 description 4
- 235000013601 eggs Nutrition 0.000 description 4
- 239000013535 sea water Substances 0.000 description 4
- 241000237852 Mollusca Species 0.000 description 3
- 239000002609 medium Substances 0.000 description 3
- 239000000203 mixture Substances 0.000 description 3
- 238000011144 upstream manufacturing Methods 0.000 description 3
- 235000017166 Bambusa arundinacea Nutrition 0.000 description 2
- 235000017491 Bambusa tulda Nutrition 0.000 description 2
- 241001330002 Bambuseae Species 0.000 description 2
- 240000006808 Chimaphila maculata Species 0.000 description 2
- 238000012408 PCR amplification Methods 0.000 description 2
- BELBBZDIHDAJOR-UHFFFAOYSA-N Phenolsulfonephthalein Chemical compound C1=CC(O)=CC=C1C1(C=2C=CC(O)=CC=2)C2=CC=CC=C2S(=O)(=O)O1 BELBBZDIHDAJOR-UHFFFAOYSA-N 0.000 description 2
- 235000015334 Phyllostachys viridis Nutrition 0.000 description 2
- 230000003321 amplification Effects 0.000 description 2
- 239000011425 bamboo Substances 0.000 description 2
- 230000015572 biosynthetic process Effects 0.000 description 2
- 238000010367 cloning Methods 0.000 description 2
- 238000012258 culturing Methods 0.000 description 2
- 108010048367 enhanced green fluorescent protein Proteins 0.000 description 2
- 238000003199 nucleic acid amplification method Methods 0.000 description 2
- 229960003531 phenolsulfonphthalein Drugs 0.000 description 2
- 238000012163 sequencing technique Methods 0.000 description 2
- 239000000243 solution Substances 0.000 description 2
- 229910021642 ultra pure water Inorganic materials 0.000 description 2
- 239000012498 ultrapure water Substances 0.000 description 2
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 1
- FWMNVWWHGCHHJJ-SKKKGAJSSA-N 4-amino-1-[(2r)-6-amino-2-[[(2r)-2-[[(2r)-2-[[(2r)-2-amino-3-phenylpropanoyl]amino]-3-phenylpropanoyl]amino]-4-methylpentanoyl]amino]hexanoyl]piperidine-4-carboxylic acid Chemical compound C([C@H](C(=O)N[C@H](CC(C)C)C(=O)N[C@H](CCCCN)C(=O)N1CCC(N)(CC1)C(O)=O)NC(=O)[C@H](N)CC=1C=CC=CC=1)C1=CC=CC=C1 FWMNVWWHGCHHJJ-SKKKGAJSSA-N 0.000 description 1
- 108020005544 Antisense RNA Proteins 0.000 description 1
- RYGMFSIKBFXOCR-UHFFFAOYSA-N Copper Chemical compound [Cu] RYGMFSIKBFXOCR-UHFFFAOYSA-N 0.000 description 1
- 238000007400 DNA extraction Methods 0.000 description 1
- 108091092584 GDNA Proteins 0.000 description 1
- 239000012880 LB liquid culture medium Substances 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 108090000854 Oxidoreductases Proteins 0.000 description 1
- 102000004316 Oxidoreductases Human genes 0.000 description 1
- 108700008625 Reporter Genes Proteins 0.000 description 1
- 108060008724 Tyrosinase Proteins 0.000 description 1
- 102000003425 Tyrosinase Human genes 0.000 description 1
- 238000000246 agarose gel electrophoresis Methods 0.000 description 1
- 238000000137 annealing Methods 0.000 description 1
- 230000003851 biochemical process Effects 0.000 description 1
- 230000003115 biocidal effect Effects 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 239000003184 complementary RNA Substances 0.000 description 1
- 238000010276 construction Methods 0.000 description 1
- 229910052802 copper Inorganic materials 0.000 description 1
- 239000010949 copper Substances 0.000 description 1
- 238000004925 denaturation Methods 0.000 description 1
- 230000036425 denaturation Effects 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 230000029087 digestion Effects 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 230000004720 fertilization Effects 0.000 description 1
- 239000012634 fragment Substances 0.000 description 1
- 230000006870 function Effects 0.000 description 1
- 239000000499 gel Substances 0.000 description 1
- 230000008105 immune reaction Effects 0.000 description 1
- 238000001802 infusion Methods 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
- 239000007788 liquid Substances 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 239000000049 pigment Substances 0.000 description 1
- 238000003752 polymerase chain reaction Methods 0.000 description 1
- 238000012257 pre-denaturation Methods 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- 230000009711 regulatory function Effects 0.000 description 1
- 230000001568 sexual effect Effects 0.000 description 1
- 230000035939 shock Effects 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/43504—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
- C12N15/8509—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells for producing genetically modified animals, e.g. transgenic
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K2267/00—Animals characterised by purpose
- A01K2267/01—Animal expressing industrially exogenous proteins
Landscapes
- Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Engineering & Computer Science (AREA)
- Zoology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Biotechnology (AREA)
- General Engineering & Computer Science (AREA)
- Molecular Biology (AREA)
- General Health & Medical Sciences (AREA)
- Biophysics (AREA)
- Biochemistry (AREA)
- Wood Science & Technology (AREA)
- Biomedical Technology (AREA)
- Physics & Mathematics (AREA)
- Microbiology (AREA)
- Plant Pathology (AREA)
- Veterinary Medicine (AREA)
- Tropical Medicine & Parasitology (AREA)
- Toxicology (AREA)
- Gastroenterology & Hepatology (AREA)
- Medicinal Chemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Peptides Or Proteins (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
本发明涉及分子生物学技术，具体的说是一种长牡蛎tyrosinase 1启动子及其重组表达载体及应用。启动子为SEQ ID No.1所示的碱基序列。所述启动子为与SEQ ID No.1所示的碱基序列具有80％或80％以上同源性的碱基序列，且具有启动子功能。通过注射重组表达载体，启动目的基因的表达，实现目的基因在贝类幼虫中的表达。
Description
技术领域
本发明涉及分子生物学技术，具体的说是一种长牡蛎tyrosinase 1 启动子及其重组表达载体及应用。
背景技术
在软体动物早期发育研究中，目前尚无可用的启动子。Tyrosinase 是一种含铜氧化酶，参与众多生化过程，包括贝壳形成、色素合成及免疫反应等。tyrosinase 1启动子可以在贝类幼虫中驱动报告基因的表达，为软体动物的发育研究提供便利。
发明内容
本发明的目的在于提供一种长牡蛎tyrosinase 1启动子及其重组表达载体及应用。
为实现上述目的，本发明采用技术方案为：
一种长牡蛎tyrosinase 1启动子，启动子为SEQ ID No.1所示的碱基序列。
所述启动子为与SEQ ID No.1所示的碱基序列具有80％或80％以上同源性的碱基序列，且具有启动子功能。
一种长牡蛎tyrosinase 1启动子的应用，所述启动子在制备重组表达载体中的应用。
一种长牡蛎tyrosinase 1启动子的应用，所述启动子在贝类幼虫中启动目的基因表达。
所述目的基因为可在细胞中转录或翻译的功能基因或RNA序列，如编码蛋白的各种功能基因(如EGFP)、反义RNA或其它具有调节功能的长 RNA序列(如LnRNA)，在本例应用中是EGFP。
含所述的长牡蛎tyrosinase 1启动子的重组表达载体。
一种所述的含长牡蛎tyrosinase 1启动子的重组表达载体的应用，其特征在于：所述重组表达载体在贝类幼虫中启动目的基因表达中的应用。
本发明所具有的优点：
通过注射重组表达载体，启动目的基因的表达，实现目的基因在贝类幼虫中的表达。
附图说明
图1为本发明实施例提供的pEGFP-1质粒示意图。
图2为本发明实施例提供的pEGFP-1质粒中的多克隆位点图。
图3为本发明实施例提供的注射重组载体的笠贝早期幼虫中tyrosinase 1启动子驱动GFP表达的结果图，其中，注射重组载体的幼虫出现GFP的绿色荧光。
具体实施方式
下面结合附图和实施例对本发明做进一步解释说明。
实施例1
启动子扩增：
使用海洋动物组织基因组DNA提取试剂盒(TIANGEN，目录号：DP324)提取长牡蛎(Crassostrea gigas)基因组DNA，根据长牡蛎基因组数据库 (http://www.oysterdb.com)设计长牡蛎基因启动子特异性引物。
(上游引物Cgtyr1PmF，加16碱基的载体pEGFP-1同源末端1，下游引物Cgtyr1PmR，加16碱基的载体pEGFP-1同源末端2)
以上述提取的长牡蛎的gDNA为模板，使用Premix TaqTM(LA TaqTM Version 2.0)(Takara，目录号：RR900Q)聚合酶进行PCR扩增。如表 1所示。
表1基因启动子扩增的PCR体系
| 组成 | 体积 |
| Premix Taq(LA TaqVersion 2.0) | 25μl |
| DNA模板 | 2μl |
| 上游引物Cgtyr1PmF(10μM) | 2μl |
| 下游引物Cgtyr1PmR(10μM) | 2μl |
| 超纯水 | 19μl |
PCR扩增程序为：98℃预变性2min；98℃变性20s，58℃退火20s，72 ℃延伸2min30s，进行35个反应循环；72℃延伸7min。
上游引物Cgtyr1PmF：CGCGGGCCCGGGATCCTACAGTAAAGGTCAGCCCAACTAT，其中下划线代表16碱基的载体pEGFP-1同源末端1；下游引物Cgtyr1PmR：GGCGACCGGTGGATCCCTTGAAAGAGAATTTCAGAAATTCA，其中下划线代表16碱基的载体pEGFP-1同源末端2。
PCR扩增产物经1.0％琼脂糖凝胶电泳分离，得到大小为2300bp左右的条带，使用Thermo Scientific的GeneJET Gel Extraction Kit(目录号：K0692)进行纯化回收，即得cgtyr1启动子碱基序列如序列表1 所示。
实施例2
cgtyr1::GFP重组载体构建：
将pEGFP-1质粒(中国科学院海洋研究所张晓军提供，还可以从优宝生物(http://www.youbio.cn/)订购，货号：VT1106，其碱基序列如序列 2中所示)用BamHI酶切线性化，将如上述得到的PCR回收产物cgtyr1 启动与线性化的pEGFP-1进行In-Fusion连接(In-Fusion HD Cloning Kit，TaKaRa，目录号：638909)，转化大肠杆菌，挑取阳性转化子测序(如cgtyr1启动子序列所示)，证明准确，具体为：
In-Fusion连接体系：
| 组成 | 体积 |
| PCR回收产物(cgtyr1启动子) | 1.5μl |
| 超纯水 | 3.5μl |
| 线性化pEGFP-1 | 3μl |
| Infusion Mix | 2μl |
| 总体积 | 10μl |
将上述记载的两片段按照上述表中记载的连接体系混合，50℃水浴中连接15min，经过连接后用感受态细胞DH5α进行转化。取出-80℃保存感大肠杆菌受态，立即静置于冰中；将适量体积的待转化DNA(每100μL感受态加入50ng DNA，体积不要超过感受态细胞体积的5％)与感受态轻轻混匀，冰上静置20min；42℃热激90s，然后冰上放置2-3min。加入900 μL的LB液体培养基(不含抗生素)，37℃150rpm振荡培养1-1.5h； 5000rpm离心3min，弃上清，用100μL LB液体培养基重悬浮菌体。将重悬液涂于卡那霉素抗性LB平板上，37℃倒置培养12-20h。选取单菌落，进行菌落PCR鉴定。空载质粒的产物大小约为100bp，重组载体产物长度约为2400bp。鉴定用引物：pEGFP-1-F,CGAGCTCAAGCTTCGAATTCTG； pEGFP-1-R,CGCCGTCCAGCTCGACCAG。获得含有pEGFP-1+cgtyr1表达载体的重组大肠杆菌，命名为cgtyr1::GFP(参见图1)。对cgtyr1::GFP表达载体中的cgtyr1启动子进行测序，测序结果如cgtyr1启动子所示。测序结果表明，获得的cgtyr1::GFP重组表达载体中cgtyr1启动子序列正确。
用无内毒素质粒小提中量试剂盒(目录号：DP118-02)从大肠杆菌 cgtyr1::GFP中提取重组载体cgtyr1::GFP。
实施例3
软体动物早期幼虫中GFP的表达
将提取的重组载体cgtyr1::GFP配置成注射液：重组载体50ng/uL,酚红 0.05％。
将性成熟的笠贝放入70ml新鲜海水的培养杯中(每杯一只)，待其产卵排精后，分别收集精子和卵细胞。
将收集的卵细胞转入盛有5ml新鲜海水的培养皿中，放置于解剖镜下。将显微注射仪(WARNER，目录号：PLI-100A)输入气压维持在200pa。用 Microloader(eppendorf，目录号：5242956003)吸取0.5ul注射液注入注射针Femtotip II(eppendorf，目录号：5242957000)，将注射针连接于注射仪。设置注射参数：输入气压200pa，平衡气压3pa，注射压力 15pa，注射时间0.08s。注射体积依据酚红颜色指示，不超过卵体积的 5％。
将注射后的卵转移至12孔培养板，加入5ml新鲜海水，按体积5％的比例加入备好的精子，室温受精一小时后将精子洗脱，重新加入5ml新鲜海水。培养20hpf后检测重组载体的的表达情况，拍照记录(参见图3)。由图3可见注射重组载体的笠贝早期幼虫中GFP表达(绿色荧光)，证明该重组载体可以在贝类幼虫中启动目的基因表达。
tyrosinase 1启动子序列：该启动子名称cgi tyrosinase 1,或简称cgtyr1启动子
TACAGTAAAGGTCAGCCCAACTATGATCAATTCAGTACTTGCAAAAATAAATACTACGAA ACTTACAACTTAAGGTATTGTTGTTTCTTTATTATTTATCGAAAATCATTTGATTTAATT GAAAACAATTGTTAATTGATGCTTTGGATTATTAAATAAAACCAAGATAAAAAAAATCGA CTAAAAAAATTAATCAACCAAAATTGCATAATGTAAAATATCAGCACATCCAAATTTTCA TGAGATTTCGGATGTATCGTCAAAAAATATATATTGTTAGAATCATTTCATGACTTACAG AAAACGATCAATTGATAATAAAAGTAAACAGGATACATTGTATTTCTGTAAGAATAAATT ATTCAGAGTTACTGATAGATTTTGGAGCCACCTGTGATCGTTCAATGTACCACCTACCTT AATCACAGTAGAACCTGGTGAATGGGGTTGATTATACAGAGCCGTCAGAATTTTAAATAC TCATTTTTCATATGAATTTCATTTTTTAAGTACAATAAAAAAACAATCGCAATCAATCAA TCTCTCGCGTGCTTTTTAGTGATTTTAATGGGTAATTTATGAAAATTAAATTAAACTGAT CAGGTCTGAACATATGATTTATATCTCTCTCTCCATAACCACAATTTCTAATGGATTAAA CAGAAGAATTTTCTATACTCATCAAAAAAACTTTTTACAATGCGTTCACATAACCGAGAC GTCACAGCCGTTGGAAAATTCATTTAAAAAATTAATTTAAAGAATAGGGAGAACTTGTTC GGCACTAAAGGCCATTGCACAATACACCCCTGAAAATCAGCTTCATCTTATAGTGTGATG TTCAAATTTTATATGAAGGTTTTACTCCGCAACAAAATAATAAGTTCATACAATCAATTA GTACCATACTTCGTTCAAAAATCGTGTTAACTTCCATAATAAACGATGACTTTAATTTCC TTATGATGTTGAATAAGCGATGTAAAGAAACTCGCTCTCATATATTGAACATATCAATGT TTACAATTTTTCGGAACAAATGGTTAAAGATTTTATTACATCTGAGATTTGATCAGATAG CTTGCAACGCATGGTCATAATCAAGTTCAATTTAGAACGAAAATACGTTCCAAATGATTA CACTCTGCACATTGCAGAATACAAAAAATTTAAAATACAAATTATTAAATTTTTTATCAA GTTATATCTATGCCTTTGTCTTAATTCAATGACTGATCGCTAACAAAATTCACAAAAGAA CATAGAAATAAAATCATTTAGTTAAATTGATCCTTCTATGAAAAGATTTCAAATACGAGA CGTGAATTCCATGCATTTACTTGTGCTCTAAAAAGCAAAGAAATGAATGAAAGAAACTAT CCTAATGAACTCCTTCTGGTTAGAATTTCTAAAGTTCAGACTTGTCTCAAATCAAAAAAC CGTGTAAACGGATCTATTAATAAATACTAAGTAGGCAGGAATCTCTTAATTCAGTTAGAA AATCAAACCCTTTTTGACACATAAAAAGACACATCTTGTATTCAAATTTTACCTTAGTAA TTTTTAATAATTGTTAGTCATGTCAGAAACTCCTAATGATTATTTTGAATGAATGATGGA TTGTTTTCCCAGGGCATTACTAACAGAATGGTGAGCAGTCTGTCACTCTAGGCGTTACAC AGAGGTTTCCATTTCCCACCAATCACCGAGCCCGCACGTACACAAGCTGTATATAAAGCT CCCGAACCCGAGACCAAAGACACGGCCTCCGGACAATCAAGAATCCAAGGTGCGTTACCA AAAAAGCTTATTCTATTCTCGCTTCTCTTTTTTATGACATTTTTATTAAGAAGCGAGACA TAAAGTTTGATTATTAGTATGCTGAGGTTTAAAGTTAATTTACTGTTTTTGCTTAAAAAA AAAAAATCCTATAAAAAATAAACTTTTGTTAGCCCTAGTTATTTTTTAAGGGTTGGCTAG GGTGTTCTCTTGCTTTTCAAAAATGACATTTATAGTGCATAATCAAATAAATAGACATAA AGTTTGATTATTAATATGCTGAGGTTTAAAGTTAATCTACTGTTTTTGCTTTAAAAAAAG AAAATACCTATTAAAAAAAATAAACTTTAGATAGTCCTAACTATTTTATAACAGTTTGGCTGGGATGTTCTCTTTATTTTTCAAAAATTACATTTAGTGCATAATAATTTCAAAATTTAC ACTTAATCCCTGTAGTTCATATATTCAAATGTTTCCATTAATGAATTTCTGAAATTCTCTTTCAAG
pEGFP-1载体序列：
TAGTTATTACTAGCGCTACCGGACTCAGATCTCGAGCTCAAGCTTCGAATTCTGCAGTCGACGGTACCGCGGG CCCGGGATCCACCGGTCGCCACCATGGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGTCGAGCTGGACGGCGACGTAAACGGCCACAAGTTCAGCGTGTCCGGCGAGGGCGAGGGCGATGCCACCT ACGGCAAGCTGACCCTGAAGTTCATCTGCACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACC ACCCTGACCTACGGCGTGCAGTGCTTCAGCCGCTACCCCGACCACATGAAGCAGCACGACTTCTTCAAGTCC GCCATGCCCGAAGGCTACGTCCAGGAGCGCACCATCTTCTTCAAGGACGACGGCAACTACAAGACCCGCGC CGAGGTGAAGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGAC GGCAACATCCTGGGGCACAAGCTGGAGTACAACTACAACAGCCACAACGTCTATATCATGGCCGACAAGCA GAAGAACGGCATCAAGGTGAACTTCAAGATCCGCCACAACATCGAGGACGGCAGCGTGCAGCTCGCCGAC CACTACCAGCAGAACACCCCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCACTACCTGAGCACCCA GTCCGCCCTGAGCAAAGACCCCAACGAGAAGCGCGATCACATGGTCCTGCTGGAGTTCGTGACCGCCGCCG GGATCACTCTCGGCATGGACGAGCTGTACAAGTAAAGCGGCCGCGACTCTAGATCATAATCAGCCATACCAC ATTTGTAGAGGTTTTACTTGCTTTAAAAAACCTCCCACACCTCCCCCTGAACCTGAAACATAAAATGAATGCA ATTGTTGTTGTTAACTTGTTTATTGCAGCTTATAATGGTTACAAATAAAGCAATAGCATCACAAATTTCACAAA TAAAGCATTTTTTTCACTGCATTCTAGTTGTGGTTTGTCCAAACTCATCAATGTATCTTAAGGCGTAAATTGTA AGCGTTAATATTTTGTTAAAATTCGCGTTAAATTTTTGTTAAATCAGCTCATTTTTTAACCAATAGGCCGAAATC GGCAAAATCCCTTATAAATCAAAAGAATAGACCGAGATAGGGTTGAGTGTTGTTCCAGTTTGGAACAAGAGT CCACTATTAAAGAACGTGGACTCCAACGTCAAAGGGCGAAAAACCGTCTATCAGGGCGATGGCCCACTACG TGAACCATCACCCTAATCAAGTTTTTTGGGGTCGAGGTGCCGTAAAGCACTAAATCGGAACCCTAAAGGGAG CCCCCGATTTAGAGCTTGACGGGGAAAGCCGGCGAACGTGGCGAGAAAGGAAGGGAAGAAAGCGAAAGG AGCGGGCGCTAGGGCGCTGGCAAGTGTAGCGGTCACGCTGCGCGTAACCACCACACCCGCCGCGCTTAATG CGCCGCTACAGGGCGCGTCAGGTGGCACTTTTCGGGGAAATGTGCGCGGAACCCCTATTTGTTTATTTTTCTA AATACATTCAAATATGTATCCGCTCATGAGACAATAACCCTGATAAATGCTTCAATAATATTGAAAAAGGAAGA GTCCTGAGGCGGAAAGAACCAGCTGTGGAATGTGTGTCAGTTAGGGTGTGGAAAGTCCCCAGGCTCCCCAG CAGGCAGAAGTATGCAAAGCATGCATCTCAATTAGTCAGCAACCAGGTGTGGAAAGTCCCCAGGCTCCCCAGCAGGCAGAAGTATGCAAAGCATGCATCTCAATTAGTCAGCAACCATAGTCCCGCCCCTAACTCCGCCCATC CCGCCCCTAACTCCGCCCAGTTCCGCCCATTCTCCGCCCCATGGCTGACTAATTTTTTTTATTTATGCAGAGGC CGAGGCCGCCTCGGCCTCTGAGCTATTCCAGAAGTAGTGAGGAGGCTTTTTTGGAGGCCTAGGCTTTTGCAA AGATCGATCAAGAGACAGGATGAGGATCGTTTCGCATGATTGAACAAGATGGATTGCACGCAGGTTCTCCGG CCGCTTGGGTGGAGAGGCTATTCGGCTATGACTGGGCACAACAGACAATCGGCTGCTCTGATGCCGCCGTGT TCCGGCTGTCAGCGCAGGGGCGCCCGGTTCTTTTTGTCAAGACCGACCTGTCCGGTGCCCTGAATGAACTGC AAGACGAGGCAGCGCGGCTATCGTGGCTGGCCACGACGGGCGTTCCTTGCGCAGCTGTGCTCGACGTTGTC ACTGAAGCGGGAAGGGACTGGCTGCTATTGGGCGAAGTGCCGGGGCAGGATCTCCTGTCATCTCACCTTGCT CCTGCCGAGAAAGTATCCATCATGGCTGATGCAATGCGGCGGCTGCATACGCTTGATCCGGCTACCTGCCCAT TCGACCACCAAGCGAAACATCGCATCGAGCGAGCACGTACTCGGATGGAAGCCGGTCTTGTCGATCAGGAT GATCTGGACGAAGAGCATCAGGGGCTCGCGCCAGCCGAACTGTTCGCCAGGCTCAAGGCGAGCATGCCCGA CGGCGAGGATCTCGTCGTGACCCATGGCGATGCCTGCTTGCCGAATATCATGGTGGAAAATGGCCGCTTTTCT GGATTCATCGACTGTGGCCGGCTGGGTGTGGCGGACCGCTATCAGGACATAGCGTTGGCTACCCGTGATATTG CTGAAGAGCTTGGCGGCGAATGGGCTGACCGCTTCCTCGTGCTTTACGGTATCGCCGCTCCCGATTCGCAGC GCATCGCCTTCTATCGCCTTCTTGACGAGTTCTTCTGAGCGGGACTCTGGGGTTCGAAATGACCGACCAAGC GACGCCCAACCTGCCATCACGAGATTTCGATTCCACCGCCGCCTTCTATGAAAGGTTGGGCTTCGGAATCGTT TTCCGGGACGCCGGCTGGATGATCCTCCAGCGCGGGGATCTCATGCTGGAGTTCTTCGCCCACCCTAGGGGG AGGCTAACTGAAACACGGAAGGAGACAATACCGGAAGGAACCCGCGCTATGACGGCAATAAAAAGACAGA ATAAAACGCACGGTGTTGGGTCGTTTGTTCATAAACGCGGGGTTCGGTCCCAGGGCTGGCACTCTGTCGATA CCCCACCGAGACCCCATTGGGGCCAATACGCCCGCGTTTCTTCCTTTTCCCCACCCCACCCCCCAAGTTCGG GTGAAGGCCCAGGGCTCGCAGCCAACGTCGGGGCGGCAGGCCCTGCCATAGCCTCAGGTTACTCATATATAC TTTAGATTGATTTAAAACTTCATTTTTAATTTAAAAGGATCTAGGTGAAGATCCTTTTTGATAATCTCATGACCA AAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGATCTTCTTGAG ATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCC GGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTCCT TCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATC CTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACA CCGAACTGAGATACCTACAGCGTGAGCTATGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAG GTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTAT CTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGA GCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCACATGTT CTTTCCTGCGTTATCCCCTGATTCTGTGGATAACCGTATTACCGCCATGCAT。
序列表
<110> 中国科学院海洋研究所
<120> 一种长牡蛎 tyrosinase 1启动子及其重组表达载体及应用
<140> 2017114011753
<141> 2017-12-22
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 2286
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 1
tacagtaaag gtcagcccaa ctatgatcaa ttcagtactt gcaaaaataa atactacgaa 60
acttacaact taaggtattg ttgtttcttt attatttatc gaaaatcatt tgatttaatt 120
gaaaacaatt gttaattgat gctttggatt attaaataaa accaagataa aaaaaatcga 180
ctaaaaaaat taatcaacca aaattgcata atgtaaaata tcagcacatc caaattttca 240
tgagatttcg gatgtatcgt caaaaaatat atattgttag aatcatttca tgacttacag 300
aaaacgatca attgataata aaagtaaaca ggatacattg tatttctgta agaataaatt 360
attcagagtt actgatagat tttggagcca cctgtgatcg ttcaatgtac cacctacctt 420
aatcacagta gaacctggtg aatggggttg attatacaga gccgtcagaa ttttaaatac 480
tcatttttca tatgaatttc attttttaag tacaataaaa aaacaatcgc aatcaatcaa 540
tctctcgcgt gctttttagt gattttaatg ggtaatttat gaaaattaaa ttaaactgat 600
caggtctgaa catatgattt atatctctct ctccataacc acaatttcta atggattaaa 660
cagaagaatt ttctatactc atcaaaaaaa ctttttacaa tgcgttcaca taaccgagac 720
gtcacagccg ttggaaaatt catttaaaaa attaatttaa agaataggga gaacttgttc 780
ggcactaaag gccattgcac aatacacccc tgaaaatcag cttcatctta tagtgtgatg 840
ttcaaatttt atatgaaggt tttactccgc aacaaaataa taagttcata caatcaatta 900
gtaccatact tcgttcaaaa atcgtgttaa cttccataat aaacgatgac tttaatttcc 960
ttatgatgtt gaataagcga tgtaaagaaa ctcgctctca tatattgaac atatcaatgt 1020
ttacaatttt tcggaacaaa tggttaaaga ttttattaca tctgagattt gatcagatag 1080
cttgcaacgc atggtcataa tcaagttcaa tttagaacga aaatacgttc caaatgatta 1140
cactctgcac attgcagaat acaaaaaatt taaaatacaa attattaaat tttttatcaa 1200
gttatatcta tgcctttgtc ttaattcaat gactgatcgc taacaaaatt cacaaaagaa 1260
catagaaata aaatcattta gttaaattga tccttctatg aaaagatttc aaatacgaga 1320
cgtgaattcc atgcatttac ttgtgctcta aaaagcaaag aaatgaatga aagaaactat 1380
cctaatgaac tccttctggt tagaatttct aaagttcaga cttgtctcaa atcaaaaaac 1440
cgtgtaaacg gatctattaa taaatactaa gtaggcagga atctcttaat tcagttagaa 1500
aatcaaaccc tttttgacac ataaaaagac acatcttgta ttcaaatttt accttagtaa 1560
tttttaataa ttgttagtca tgtcagaaac tcctaatgat tattttgaat gaatgatgga 1620
ttgttttccc agggcattac taacagaatg gtgagcagtc tgtcactcta ggcgttacac 1680
agaggtttcc atttcccacc aatcaccgag cccgcacgta cacaagctgt atataaagct 1740
cccgaacccg agaccaaaga cacggcctcc ggacaatcaa gaatccaagg tgcgttacca 1800
aaaaagctta ttctattctc gcttctcttt tttatgacat ttttattaag aagcgagaca 1860
taaagtttga ttattagtat gctgaggttt aaagttaatt tactgttttt gcttaaaaaa 1920
aaaaaatcct ataaaaaata aacttttgtt agccctagtt attttttaag ggttggctag 1980
ggtgttctct tgcttttcaa aaatgacatt tatagtgcat aatcaaataa atagacataa 2040
agtttgatta ttaatatgct gaggtttaaa gttaatctac tgtttttgct ttaaaaaaag 2100
aaaataccta ttaaaaaaaa taaactttag atagtcctaa ctattttata acagtttggc 2160
tgggatgttc tctttatttt tcaaaaatta catttagtgc ataataattt caaaatttac 2220
acttaatccc tgtagttcat atattcaaat gtttccatta atgaatttct gaaattctct 2280
ttcaag 2286
Claims (6)
1.一种长牡蛎 tyrosinase 1启动子，其特征在于：启动子为SEQ ID No.1所示的碱基序列。
2.一种按权利要求1所述的长牡蛎 tyrosinase 1启动子的应用，其特征在于：所述启动子在制备重组表达载体中的应用。
3.一种按权利要求1所述的长牡蛎 tyrosinase 1启动子的应用，其特征在于：所述启动子在贝类幼虫中启动目的基因中的应用。
4.按权利要求3所述的长牡蛎 tyrosinase 1启动子的应用，其特征在于：所述目的基因为在细胞中转录或翻译的功能基因或RNA序列。
5.含权利要求1所述的长牡蛎 tyrosinase 1启动子的重组表达载体。
6.一种权利要求5所述的含长牡蛎 tyrosinase 1启动子的重组表达载体的应用，其特征在于：所述重组表达载体在贝类幼虫中启动目的基因表达中的应用。
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201711401175.3A CN108060167B (zh) | 2017-12-22 | 2017-12-22 | 一种长牡蛎tyrosinase1启动子及其重组表达载体及应用 |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201711401175.3A CN108060167B (zh) | 2017-12-22 | 2017-12-22 | 一种长牡蛎tyrosinase1启动子及其重组表达载体及应用 |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN108060167A CN108060167A (zh) | 2018-05-22 |
| CN108060167B true CN108060167B (zh) | 2020-12-08 |
Family
ID=62139963
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201711401175.3A Active CN108060167B (zh) | 2017-12-22 | 2017-12-22 | 一种长牡蛎tyrosinase1启动子及其重组表达载体及应用 |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN108060167B (zh) |
-
2017
- 2017-12-22 CN CN201711401175.3A patent/CN108060167B/zh active Active
Non-Patent Citations (2)
| Title |
|---|
| Crassostrea gigas genome assembly, linkage group: LG3;Genbank;《Genbank》;20200215;全文 * |
| Crassostrea gigas tyrosinase (tyr) mRNA, complete cds;Genbank;《Genbank》;20131105;全文 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN108060167A (zh) | 2018-05-22 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN1495200A (zh) | 作为载体的新的融合蛋白质 | |
| CN108396030B (zh) | 凡纳滨对虾抗菌肽基因Lv-BigPEN及其重组蛋白和应用 | |
| Zhang et al. | Characterization of four heat-shock protein genes from Nile tilapia (Oreochromis niloticus) and demonstration of the inducible transcriptional activity of Hsp70 promoter | |
| CN104694547A (zh) | 斑节对虾热休克蛋白40基因及其编码的蛋白 | |
| CN108060167B (zh) | 一种长牡蛎tyrosinase1启动子及其重组表达载体及应用 | |
| CN113583102A (zh) | 一种牙鲆肽聚糖识别蛋白的重组蛋白及其制备方法和应用 | |
| CN110747199B (zh) | 蜜蜂抗逆相关基因nf-y及其应用 | |
| CN113005069A (zh) | 一种减毒鰤鱼诺卡氏菌及其制备方法和应用 | |
| RU2020113297A (ru) | ДНК-вакцина против вируса SARS-CoV-2 на основе генотерапевтического ДНК-вектора GDTT1.8NAS12, способ ее получения, штаммы, несущие генотерапевтические ДНК-вектора, способ их получения, способ производства в промышленных масштабах генотерапевтических ДНК-векторов | |
| CN112575006B (zh) | 一种生防腐霉菌中诱导HR和活性氧积累的Elicitin类基因及其表达载体和应用 | |
| CN108060166B (zh) | 一种长牡蛎gata2/3启动子及其重组表达载体及应用 | |
| CN112538490B (zh) | 一种生防腐霉菌中诱导坏死和活性氧积累的nlp类基因及其表达载体和应用 | |
| Mahardika et al. | The effects of crude recombinant viral protein vaccines against grouper sleepy disease iridovirus (GSDIV) on humpback grouper (Cromileptes altivelis) | |
| CN113337523A (zh) | Crn类效应蛋白基因在诱导植物抗性中的应用 | |
| CN108004243B (zh) | 一种长牡蛎engrailed2启动子及其重组表达载体及应用 | |
| CN111662370A (zh) | 南极鱼hepcidin抗菌肽及其制备方法和应用 | |
| CN107904241B (zh) | 一种长牡蛎brachyury启动子及其重组表达载体及应用 | |
| CN116144667B (zh) | 卵形鲳鲹胰岛素样生长因子结合蛋白1基因、蛋白及应用 | |
| RU2018145694A (ru) | Генотерапевтический ДНК-вектор на основе генотерапевтического ДНК-вектора VTvaf17, несущий целевой ген, выбранный из группы генов NOS2, NOS3, VIP, KCNMA1, CGRP, для повышения уровня экспрессии этих целевых генов, способ его получения и применения, штамм Escherichia coli SCS110-AF/VTvaf17-NOS2, или Escherichia coli SCS110-AF/VTvaf17-NOS3, или Escherichia coli SCS110-AF/VTvaf17-VIP, или Escherichia coli SCS110-AF/VTvaf17-KCNMA1, или Escherichia coli SCS110-AF/VTvaf17-CGRP, несущий генотерапевтический ДНК-вектор, способ его получения, способ производства в промышленных масштабах генотерапевтического ДНК-вектора | |
| CN116004640B (zh) | 卵形鲳鲹b细胞淋巴瘤-2的基因、蛋白、质粒及其应用 | |
| CN114164159B (zh) | 一种具有防治鱼类杀鲑气和迟缓爱德华氏菌感染的二联疫苗及其制备方法与应用 | |
| CN112941104B (zh) | 一种携带斑马鱼bid序列的表达载体、构建方法及其用途 | |
| CN112646822B (zh) | 一种双胞旋沟藻细胞核增殖抗原基因及其应用 | |
| CN112043836B (zh) | 海水青鳉HSP90ab1基因及其编码蛋白的应用 | |
| CN110563828B (zh) | 二化螟雄性特异性致死相关蛋白MSL3及其编码基因、dsRNA干扰序列和应用 |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |