CN116425852A - 鱼类促摄食成熟肽adropin及其应用和鱼类诱食剂 - Google Patents
鱼类促摄食成熟肽adropin及其应用和鱼类诱食剂 Download PDFInfo
- Publication number
- CN116425852A CN116425852A CN202310475344.7A CN202310475344A CN116425852A CN 116425852 A CN116425852 A CN 116425852A CN 202310475344 A CN202310475344 A CN 202310475344A CN 116425852 A CN116425852 A CN 116425852A
- Authority
- CN
- China
- Prior art keywords
- fish
- mature peptide
- adropin
- gene
- aropin
- 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
- 241000251468 Actinopterygii Species 0.000 title claims abstract description 64
- 108090000765 processed proteins & peptides Proteins 0.000 title claims abstract description 36
- 230000037406 food intake Effects 0.000 title claims abstract description 29
- 230000001737 promoting effect Effects 0.000 title claims abstract description 17
- 101710161788 Adropin Proteins 0.000 title claims description 41
- 102100030805 Adropin Human genes 0.000 title claims description 29
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 28
- 125000003275 alpha amino acid group Chemical group 0.000 claims abstract description 7
- 235000021050 feed intake Nutrition 0.000 claims abstract description 5
- 241000276707 Tilapia Species 0.000 claims description 46
- 108090000189 Neuropeptides Proteins 0.000 claims description 12
- 239000002299 complementary DNA Substances 0.000 claims description 12
- 210000004556 brain Anatomy 0.000 claims description 10
- 102000004169 proteins and genes Human genes 0.000 claims description 9
- 102000002512 Orexin Human genes 0.000 claims description 8
- 108060005714 orexin Proteins 0.000 claims description 8
- 210000001519 tissue Anatomy 0.000 claims description 8
- 230000033228 biological regulation Effects 0.000 claims description 7
- 230000000694 effects Effects 0.000 claims description 7
- 238000003753 real-time PCR Methods 0.000 claims description 6
- 230000008859 change Effects 0.000 claims description 5
- 210000003016 hypothalamus Anatomy 0.000 claims description 5
- 239000007928 intraperitoneal injection Substances 0.000 claims description 5
- 239000002773 nucleotide Substances 0.000 claims description 5
- 125000003729 nucleotide group Chemical group 0.000 claims description 5
- 230000001817 pituitary effect Effects 0.000 claims description 5
- 108700026244 Open Reading Frames Proteins 0.000 claims description 4
- 108010076504 Protein Sorting Signals Proteins 0.000 claims description 4
- 150000001413 amino acids Chemical class 0.000 claims description 4
- 210000003734 kidney Anatomy 0.000 claims description 4
- 210000004185 liver Anatomy 0.000 claims description 4
- 238000000034 method Methods 0.000 claims description 4
- 239000002243 precursor Substances 0.000 claims description 4
- 210000003205 muscle Anatomy 0.000 claims description 3
- 210000001672 ovary Anatomy 0.000 claims description 3
- 241000287828 Gallus gallus Species 0.000 claims description 2
- 241000282414 Homo sapiens Species 0.000 claims description 2
- 238000010367 cloning Methods 0.000 claims description 2
- 238000005516 engineering process Methods 0.000 claims description 2
- 230000001939 inductive effect Effects 0.000 claims description 2
- 210000000936 intestine Anatomy 0.000 claims description 2
- 238000010369 molecular cloning Methods 0.000 claims description 2
- 210000000952 spleen Anatomy 0.000 claims description 2
- 238000011144 upstream manufacturing Methods 0.000 claims description 2
- 238000001514 detection method Methods 0.000 claims 1
- 210000005036 nerve Anatomy 0.000 claims 1
- 230000001105 regulatory effect Effects 0.000 abstract description 10
- 238000002474 experimental method Methods 0.000 abstract description 4
- 238000011160 research Methods 0.000 abstract description 4
- 230000008844 regulatory mechanism Effects 0.000 abstract description 3
- 238000010353 genetic engineering Methods 0.000 abstract description 2
- 210000000653 nervous system Anatomy 0.000 abstract description 2
- 230000037396 body weight Effects 0.000 description 10
- 235000013305 food Nutrition 0.000 description 7
- 230000002267 hypothalamic effect Effects 0.000 description 5
- 108020004999 messenger RNA Proteins 0.000 description 5
- 238000010839 reverse transcription Methods 0.000 description 5
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 4
- 238000012408 PCR amplification Methods 0.000 description 4
- 230000037149 energy metabolism Effects 0.000 description 4
- 239000012634 fragment Substances 0.000 description 4
- 241000276701 Oreochromis mossambicus Species 0.000 description 3
- 238000003786 synthesis reaction Methods 0.000 description 3
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- 241000588724 Escherichia coli Species 0.000 description 2
- 241000124008 Mammalia Species 0.000 description 2
- 241000699670 Mus sp. Species 0.000 description 2
- 102000003797 Neuropeptides Human genes 0.000 description 2
- 230000003321 amplification Effects 0.000 description 2
- 238000004458 analytical method Methods 0.000 description 2
- 239000003674 animal food additive Substances 0.000 description 2
- 230000036528 appetite Effects 0.000 description 2
- 235000019789 appetite Nutrition 0.000 description 2
- 238000011161 development Methods 0.000 description 2
- 238000010586 diagram Methods 0.000 description 2
- 238000001962 electrophoresis Methods 0.000 description 2
- 230000019439 energy homeostasis Effects 0.000 description 2
- 239000011521 glass Substances 0.000 description 2
- 239000007924 injection Substances 0.000 description 2
- 238000002347 injection Methods 0.000 description 2
- 230000007246 mechanism Effects 0.000 description 2
- 238000003199 nucleic acid amplification method Methods 0.000 description 2
- 239000002504 physiological saline solution Substances 0.000 description 2
- 238000012163 sequencing technique Methods 0.000 description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 2
- 238000005303 weighing Methods 0.000 description 2
- 108020004463 18S ribosomal RNA 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
- 102000016911 Deoxyribonucleases Human genes 0.000 description 1
- 108010053770 Deoxyribonucleases Proteins 0.000 description 1
- ISWSIDIOOBJBQZ-UHFFFAOYSA-N Phenol Chemical compound OC1=CC=CC=C1 ISWSIDIOOBJBQZ-UHFFFAOYSA-N 0.000 description 1
- 239000013614 RNA sample Substances 0.000 description 1
- 241000700159 Rattus Species 0.000 description 1
- 210000000683 abdominal cavity Anatomy 0.000 description 1
- 230000005856 abnormality Effects 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 210000000577 adipose tissue Anatomy 0.000 description 1
- 239000011543 agarose gel Substances 0.000 description 1
- 208000022531 anorexia Diseases 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 210000005013 brain tissue Anatomy 0.000 description 1
- 210000001638 cerebellum Anatomy 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 206010061428 decreased appetite Diseases 0.000 description 1
- 238000013461 design Methods 0.000 description 1
- 230000006866 deterioration Effects 0.000 description 1
- 230000037213 diet Effects 0.000 description 1
- 235000005911 diet Nutrition 0.000 description 1
- 238000002224 dissection Methods 0.000 description 1
- 230000002124 endocrine Effects 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 238000013467 fragmentation Methods 0.000 description 1
- 238000006062 fragmentation reaction Methods 0.000 description 1
- 239000000499 gel Substances 0.000 description 1
- 210000002816 gill Anatomy 0.000 description 1
- 210000004907 gland Anatomy 0.000 description 1
- 230000002710 gonadal effect Effects 0.000 description 1
- 210000005003 heart tissue Anatomy 0.000 description 1
- 235000003642 hunger Nutrition 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 230000002503 metabolic effect Effects 0.000 description 1
- 238000006241 metabolic reaction Methods 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 210000004165 myocardium Anatomy 0.000 description 1
- 229910052757 nitrogen Inorganic materials 0.000 description 1
- 231100000252 nontoxic Toxicity 0.000 description 1
- 230000003000 nontoxic effect Effects 0.000 description 1
- 235000003715 nutritional status Nutrition 0.000 description 1
- 210000000496 pancreas Anatomy 0.000 description 1
- 230000001766 physiological effect Effects 0.000 description 1
- 238000009372 pisciculture Methods 0.000 description 1
- 230000029556 regulation of feeding behavior Effects 0.000 description 1
- 239000000243 solution Substances 0.000 description 1
- 230000037351 starvation Effects 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 230000002195 synergetic effect Effects 0.000 description 1
- 210000001587 telencephalon Anatomy 0.000 description 1
- 210000001550 testis Anatomy 0.000 description 1
- 231100000331 toxic Toxicity 0.000 description 1
- 230000002588 toxic effect Effects 0.000 description 1
- 230000001131 transforming effect Effects 0.000 description 1
- 230000002861 ventricular Effects 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
- C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
- C07K14/461—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from fish
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K20/00—Accessory food factors for animal feeding-stuffs
- A23K20/10—Organic substances
- A23K20/142—Amino acids; Derivatives thereof
- A23K20/147—Polymeric derivatives, e.g. peptides or proteins
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K50/00—Feeding-stuffs specially adapted for particular animals
- A23K50/80—Feeding-stuffs specially adapted for particular animals for aquatic animals, e.g. fish, crustaceans or molluscs
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/124—Animal traits, i.e. production traits, including athletic performance or the like
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/158—Expression markers
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
- Y02A40/80—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in fisheries management
- Y02A40/81—Aquaculture, e.g. of fish
Landscapes
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Zoology (AREA)
- Health & Medical Sciences (AREA)
- Polymers & Plastics (AREA)
- Organic Chemistry (AREA)
- Engineering & Computer Science (AREA)
- Analytical Chemistry (AREA)
- Biophysics (AREA)
- Food Science & Technology (AREA)
- Molecular Biology (AREA)
- Genetics & Genomics (AREA)
- General Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- Animal Husbandry (AREA)
- Wood Science & Technology (AREA)
- Toxicology (AREA)
- Immunology (AREA)
- Microbiology (AREA)
- Gastroenterology & Hepatology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- Biotechnology (AREA)
- Physics & Mathematics (AREA)
- Medicinal Chemistry (AREA)
- Insects & Arthropods (AREA)
- Marine Sciences & Fisheries (AREA)
- Birds (AREA)
- Peptides Or Proteins (AREA)
Abstract
本发明公开了一种鱼类促摄食成熟肽adropin及其应用和鱼类诱食剂，涉及基因工程领域。本发明提供了adropin成熟肽在提高鱼类摄食量中的应用，adropin成熟肽的氨基酸序列如SEQ ID NO:2所示。该鱼类促摄食成熟肽及其应用发明鱼类能量动态平衡相关基因编码基因在鱼类的神经系统高表达，并通过分子调控机制研究及生理实验证实该基因产生的成熟肽adropin具有调节鱼类摄食的功能，能够用于调节鱼类摄食以及用于制备促进鱼类摄食的饲料。
Description
本申请是申请日为2018年04月27日、申请号为201810418188.X、发明名称为《鱼类促摄食成熟肽及其应用》的分案申请。
技术领域
本发明涉及基因工程领域，具体来说，本发明涉及一种鱼类促摄食成熟肽adropin及其应用和鱼类诱食剂。
背景技术
鱼类的摄食和能量代谢调节主要受下丘脑-靶腺轴的控制，其中下丘脑-肝脏轴能够感应机体的营养状况，并做出适当的食欲调节行为和代谢反应，以维持机体的能量稳态，而能量自我平衡机制的损坏将会导致食欲和能量代谢异常。近年来鱼类内分泌代谢调控机制的研究表明，神经肽是参与调控摄食和能量代谢的一类重要物质。因此，研究鱼类神经肽调控摄食和能量代谢的作用机制将为鱼类养殖业持续健康发展提供技术支持。
Adropin是由能量动态平衡相关基因编码的神经肽，主要表达于脑部、肝脏、肌肉、肾脏、心肌、胰腺等组织中，提示adropin可能具有广泛的生理作用。近期研究表明adropin在哺乳动物中参与了摄食行为的调控。腹腔注射adropin可使饮食诱导肥胖型(DIO)小鼠的摄食量降低和体重减轻，但脑室注射adropin对大鼠摄食无明显影响；然而，敲除adropin基因的小鼠则表现出食欲减退。由此可见，Adropin在哺乳动物中参与调控摄食的研究结论并不一致，具体调控机制亦未阐明。通过搜索EST数据库与鱼类的基因组数据库，并进行基因共线性分析，首次证实了鱼类能量动态平衡相关基因的存在，并证实了鱼类能量动态平衡相关基因编码的成熟肽adropin能增加罗非鱼的饵料摄入量，显示出了较强的促摄食功能，从而为开发一种新型诱食剂奠定了基础。现有诱食剂活性低，用量大，生产成本较高并且有一定的毒副作用。
发明内容
本发明所要解决的技术问题是提供一种鱼类促摄食成熟肽adropin及其应用和鱼类诱食剂。基于罗非鱼的理论研究，鉴定了鱼类adripin神经肽基因编码的成熟肽adropin具有调节鱼类摄食的功能，并且可以和摄食调控关键因子orexin协同调控摄食。这为开发出高活性、无毒和成本低廉的诱食剂，作为饲料添加剂，促进养殖鱼类等的发展具有广泛的应用价值。
为实现上述目的，本发明提供以下的技术方案：
本发明提供了adropin蛋白在提高鱼类摄食量中的应用，adropin蛋白的氨基酸序列如SEQ ID NO:2所示。
本发明提供了adropin蛋白的编码基因adropin在提高鱼类摄食量中的应用，所述编码基因adropin的核苷酸序列如SEQ ID NO:1所示。
优选的，所述鱼类包括罗非鱼。
优选的，adropin成熟肽的有效作用剂量为100ng/gBW、250ng/gBW或500ng/gBW。
本发明提供了一种鱼类促摄食成熟肽，所述鱼类促摄食成熟肽的核苷酸序列如SEQ ID NO:1所示。
本发明提供了一种鱼类诱食剂，所述诱食剂包括adropin成熟肽，所述adropin成熟肽的氨基酸序列如SEQ ID NO:2所示。
本发明图1中的adropin基因的核苷酸序列如SEQ ID NO：1所示，图1中的adropin蛋白氨基酸序列如SEQ ID NO：2所示。
SEQ ID NO：1序列：ACGCGGGACGCCCCCCGGGCTCGCAGTTTGGT GACCTACTTCGGGACAAAAGTTCCCCCTTTTTTTTCTTTTTTAGAAACGTCGTGGTGAACTTGCTGAAGATTTGGAAGTCATTCCTTATAATCCGTTTTGCTCTTGGGGTGCGCGCCAAGACAACAAATGCGTGGACCCAAAGCTTCCGATTGGAGTGTCAGGGATAATCTGTGGAGCTGGGGTATCTAACAGACCTCAGGCTGACCACCAGGATCTTGGAGACCACTGTCCCACCTACCATGGAGCATGAACTGAGAAATGGCCTGTCTATATGGAGCATCGTGGCCATTGTTTGCAACTCGGTGGTAGGCTTTCTCATTGTCATCCTCTTTGTCATCCTCTACAAGGCCTGCAAGGTACCTTCACACCAAGAGAAAGTGCCTGCTTTCGCAGCAAAGCTGGAGCAGAAGAAGGATGAACAGAGGTATATGCTGACTGCAGCCTAATCGAGACCCATAGATATGAACACTAATGGAGCTGGACATCATATATGCATGCCATTACATTCCCTCGGTATCTCCTTTTGTGTTACATAGATGACTGTTTACATCTCTGCATTTTGACTGTGCCAGTGAGTGAAGCTCAGAGTAAGCTAACTGCATTAAGAGTTGTGGCAAGACCAACAACAACAAGCGGGCCCAATCCTTTTATTTTTGCTGTGCGTCGCGGGGACTGTCCATCTGTCAGTCAGTTTGGTTTTGCTCGGCACAGATACACCATGATCTCCTCCTTTGCTCCGTTCCTTTTCAACCCTCCATCTGAAGCCACAGAGTGGATGTGGAGGACAAACAGCAAAGTTAACTGGAAGAGAGGA ACTGAGGTGACAAGGTTGGTCGAGAGGCACAAAGACATAAAATCAAATTTTTAGTGGTAAAGAGAAAAAGCAACGGCAGCTGGGATTTTTTTTTCCCCATAGCTGCACACTAATGATGCCAGCAACAGAGCGGCAGAAACACGACAGGTAGACACTAAAAACCCAAACGAGGCAGAAAAAAAAAAAAAAAAAAAAAAAA。
SEQ ID NO：2序列：MEHELRNGLSIWSIVAIVCNSVVGFLIVILFVILYKACKVPSHQEKVPAFAAKLEQKKDEQRYMLTAA。
SEQ ID NO：1中下划线代表加尾信号，SEQ ID NO：2中下划线代表信号肽，SEQ IDNO：2中除去下划线的其余部分代表成熟肽。成熟肽和信号肽组成adropin蛋白。
本发明提供了鱼类促摄食成熟肽及其应用包括如下步骤：
(1)通过分析人、鸡及鱼类基因组和EST数据库，在鱼类中发现adropin基因的存在，根据这些序列设计引物，结合分子克隆技术，在罗非鱼中克隆得到adropin cDNA序列1046bp，其中开放阅读框(ORF)序列204bp，编码的adropin蛋白前体为68个氨基酸。蛋白前体等电点为8.65，分子量为7.69千道尔顿，其中N-端信号肽由25个氨基酸组成。
(2)采用实时荧光定量PCR检测罗非鱼adropin基因在各种组织中的表达，结果显示在脑、肝脏、脾脏、卵巢、垂体和肌肉中都有较高的表达；另外在心脏和肠的表达量较弱；在肾脏和鳃中检测不到表达信号。从中显示出adropin具有明显的组织表达特异性，如图2所示。Adropin基因在脑和垂体中的高表达预示着adropin在上游神经中枢系统中的调控作用；
(3)用实时荧光定量PCR检测罗非鱼基因在进食状态下的变化，结果表明：在进食1h后，下丘脑中adropin基因的表达量显著高于对照组(P＜0.05)，表明adropin与摄食调控相关，如图3所示；
(4)鱼类adropin神经肽基因可以用于鱼类摄食调控。采用腹腔注射化学合成的adropin成熟肽，实际应用表明，adropin能够显著性增加罗非鱼的摄食量，如图4所示，并且可以和orexin一起叠加刺激摄食。因此，该鱼类adropin神经肽基因编码的成熟肽adropin可以用于诱导鱼类摄食。
采用以上技术方案的有益效果是：该鱼类促摄食成熟肽及其应用可以让鱼类adropin神经肽基因在鱼类下丘脑中大量表达，该基因产生的成熟肽具有调节鱼类摄食的功能，可以通过化学合成的方法进行开发利用，使用方便，效果显著，具有广泛的应用价值。
附图说明
下面结合附图对本发明的具体实施方式作进一步详细的描述。
图1是本发明鱼类adropin神经肽的序列图；
图2是本发明鱼类adropin神经肽基因在各个组织的表达分析图；
图3是本发明鱼类adropin神经肽基因在进食过程的表达变化图；
图4是本发明鱼类adropin神经肽腹腔注射对罗非鱼摄食的影响图。
具体实施方式
下面结合附图详细说明本发明鱼类促摄食成熟肽及其应用的优选实施方式。
图1至图4出示本发明鱼类促摄食成熟肽及其应用的具体实施方式：
实施例1：
1.罗非鱼脑总RNA的提取
取健康罗非鱼，解剖后立即分离出脑组织。采用Trizol试剂法提取获得罗非鱼脑总RNA，其OD260/280＝1.9，电泳结果显示，28S rRNA，18S rRNA条带清晰，28S条带亮度为18S的两倍，表明所得总RNA未被蛋白质、酚和基因组DNA污染，纯度高。
2.cDNA第一链的合成
取1μg罗非鱼鱼脑总RNA样品进行DNA酶处理以去除基因组DNA的污染应用iScriptTM反转录试剂盒进行反转录，所得产物置于-20℃保存备用。
3.引物设计
通过分析罗非鱼基因组，我们在罗非鱼adropin基因保守区设计引物，扩增片段大小约为1046bp。
引物序列为：F：5’-CTCAC(T/C)TCCAT(T/C)GC(A/C)GGAGG-3’(SEQ ID NO：3)；
R：5’-GCTTTCG(A/C)TTTGG(C/G)AGGAAG-3’(SEQ ID NO：4)。
4.罗非鱼adropin基因cDNA全序列的克隆
以合成的第一链cDNA作为模板，以引物进行PCR扩增，所得PCR产物上样至1.5％琼脂糖凝胶，以低电压电泳分离DNA片段，从凝胶中纯化回收目的产物。将纯化后的目的产物连接至
Easy载体，转化DH5α大肠杆菌，挑选阳性克隆测序。
根据已得到的cDNA片段序列设计特异性引物，利用cDNA末端快速扩增技术(RapidAmplification ofcDNAends，RACE)对目的基因的3’-和5’-末端进行PCR扩增。按照RACE试剂盒GeneRacerTM Kit说明书对罗非鱼脑总RNA进行去磷酸，去mRNA 5’帽子结构，与RNAOligo连接，最终通过反转录合成cDNA第一链。用罗非鱼adropin cDNA的3’-和5’-RACE特异引物(AD-F2，AD-F3和AD-R2，AD-R3)和GeneRacerTM Kit的通用引物，以GeneRacerTMKit反转录合成的第一链cDNA为第一轮PCR模板，以稀释的第一轮PCR产物为第二轮PCR模板，扩增罗非鱼adropin cDNA的3’端和5’端片段，对所得到的PCR产物的回收纯化、连接T载体、转化大肠杆菌、阳性克隆鉴定及DNA测序。先以GeneRacerTMKit反转录合成的第一链cDNA为第一轮PCR模板，以稀释的第一轮PCR产物为第二轮PCR模板采用特异性引物AD-F2和AD-R2进行PCR扩增，再利用特异性引物AD-F3和AD-R3对PCR扩增引物进行验证。
实施例2：
罗非鱼adropin的组织表达分布。
采用Real-time PCR检测罗非鱼adropin在各种组织中的表达，结果显示adropin在下丘脑、视顶盖-丘脑及精巢中表达最高，在端脑、小脑、延脑、肾脏、肝脏、垂体和心脏等组织也有表达，而在卵巢和脂肪组织中没有表达，如图2所示。下丘脑adropin表达为99.9792％、性腺adropin表达为4.599478％。
实施例3：
罗非鱼adropin在饥饿过程的表达变化。
将体重30±5g的罗非鱼随机分配到四个玻璃缸中(分别为对照组和投喂组)，平均每个缸8条罗非鱼。罗非鱼每天定时(上午10点；下午4点左右)投喂两次，食物投喂量约为体重的3％。驯养两周后，实验正式开始。对照组的罗非鱼停止投喂，实验组的罗非鱼在实验开始后正常投喂。在进食1h，2h和3h后，分别将实验组和对照组的罗非鱼用MS-222麻醉，经处死收集下丘脑样品，液氮速冻处理，存放于-80℃超低温冰箱备用。通过荧光定量PCR检测罗非鱼下丘脑中adropin基因在对照组和进食组的表达量变化，结果表明：下丘脑adropinmRNA表达水平在进食1h后被显著抑制，如图3所示。未进食前2h下丘脑adropinmRNA表达水平为99.04794286％，进食后2h下丘脑adropinmRNA表达水平为57.04232857％；未进食前1h下丘脑adropinmRNA表达水平为105.5845714％，进食后1h下丘脑adropinmRNA表达水平为82.21737143％。
实施例4：
采用腹腔注射方式研究化学合成的氨基酸序列如SEQ ID NO：2所示的罗非鱼adropin对罗非鱼摄食的影响。
将体重30±5g的罗非鱼随机分配到六个玻璃缸中，平均每个缸8条罗非鱼。罗非鱼每天定时(上午10点；下午4点左右)投喂两次，食物投喂量约为体重的3％。驯养两周后开始正式实验。罗非鱼用MS-222麻醉后，称量每组中个体体重(bodyweight，BW)，根据体重分别由腹腔注射0.7％鱼类生理盐水(对照组)、50ng/gBWadropin、100ng/gBWadropin、250ng/gBWadropin和500ng/gBW adropin，另外单独注射orexin(100ng/gBW)、共同注射adropin(500ng/gBW)与Orexin(100ng/gBW)。注射15min后，分别向每组罗非鱼投喂固定重量的食物，让每组罗非鱼自由摄食2h。同时将等量的食物放入水中，以计算食物在水中2h中的重量变化。将各组剩余的食物分别收集并烘干，根据剩余的食物的重量计算各组中单位体重罗非鱼的摄食量，如图4所示。根据图4可知，注射100ng/gBWadropin、250ng/gBWadropin和500ng/gBWadropin的罗非鱼的摄食量显著增加。Adropin和Orexin存在协同作用，共同应用可以显著提高罗非鱼的摄食量。注射0.7％鱼类生理盐水的罗非鱼的摄食量为14.988075mg/g BW/120min，500ng/gBW adropin的罗非鱼的摄食量为26.8549125mg/g BW/120min，同时注射500ng/gBW adropin和Orexin(100ng/gBW)的罗非鱼摄食量为41.00621429mg/g BW/120min。
以上的仅是本发明的优选实施方式，应当指出，对于本领域的普通技术人员来说，在不脱离本发明创造构思的前提下，还可以做出若干变形和改进，这些都属于本发明的保护范围。
Claims (7)
1.adropin成熟肽在提高鱼类摄食量中的应用，adropin成熟肽的氨基酸序列如SEQ IDNO:2所示。
2.adropin成熟肽的编码基因adropin在提高鱼类摄食量中的应用，所述编码基因adropin的核苷酸序列如SEQ ID NO:1所示。
3.根据权利要求1或2所述的应用，其特征在于，所述鱼类包括罗非鱼。
4.根据权利要求1或2所述的应用，其特征在于，adropin成熟肽的有效作用量为100ng/gBW、250ng/gBW或500ng/gBW。
5.一种鱼类促摄食成熟肽，其特征在于：所述鱼类促摄食成熟肽的核苷酸序列如SEQID NO:1所示。
6.一种鱼类诱食剂，其特征在于，所述诱食剂包括adropin成熟肽，所述adropin成熟肽的氨基酸序列如SEQ ID NO:2所示。
7.一种检测确定鱼类促摄食成熟肽的方法，其特征在于：所述检测的方法包括如下步骤：
(1)通过分析人、鸡及鱼类基因组和EST数据库，在鱼类中发现adropin基因的存在，根据这些序列设计引物，结合分子克隆技术，在罗非鱼中克隆得到adropincDNA序列1046bp，其中开放阅读框(ORF)序列204bp，编码的adropin蛋白前体为68个氨基酸，蛋白前体等电点为8.65，分子量为7.69千道尔顿，其中N-端信号肽由25个氨基酸组成；
(2)采用实时荧光定量PCR检测罗非鱼adropin基因在各种组织中的表达，结果显示在脑、肝脏、脾脏、卵巢、垂体和肌肉中都有较高的表达；另外在心脏和肠的表达量较弱；在肾脏和鳃中检测不到表达信号，从中显示出adropin具有明显的组织表达特异性，Adropin基因在脑和垂体中的高表达预示着adropin在上游神经中枢系统中的调控作用；
(3)用实时荧光定量PCR检测罗非鱼基因在进食状态下的变化，结果表明：在进食1小时后，下丘脑中adropin基因的表达量显著高于对照组(P＜0.05)，表明adropin与摄食调控相关；
(4)鱼类adropin神经肽基因可以用于鱼类摄食调控，采用腹腔注射化学合成的adropin成熟肽，实际应用表明，adropin能够显著性增加罗非鱼的摄食量，并且可以和orexin一起叠加刺激摄食，鱼类adropin神经肽基因编码的成熟肽adropin可以用于诱导鱼类摄食。
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202310475344.7A CN116425852B (zh) | 2018-04-27 | 2018-04-27 | 鱼类促摄食成熟肽及其应用 |
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202310475344.7A CN116425852B (zh) | 2018-04-27 | 2018-04-27 | 鱼类促摄食成熟肽及其应用 |
| CN201810418188.XA CN108586597A (zh) | 2018-04-27 | 2018-04-27 | 鱼类促摄食成熟肽及其应用 |
Related Parent Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201810418188.XA Division CN108586597A (zh) | 2018-04-27 | 2018-04-27 | 鱼类促摄食成熟肽及其应用 |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN116425852A true CN116425852A (zh) | 2023-07-14 |
| CN116425852B CN116425852B (zh) | 2024-05-24 |
Family
ID=63619826
Family Applications (2)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201810418188.XA Pending CN108586597A (zh) | 2018-04-27 | 2018-04-27 | 鱼类促摄食成熟肽及其应用 |
| CN202310475344.7A Active CN116425852B (zh) | 2018-04-27 | 2018-04-27 | 鱼类促摄食成熟肽及其应用 |
Family Applications Before (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201810418188.XA Pending CN108586597A (zh) | 2018-04-27 | 2018-04-27 | 鱼类促摄食成熟肽及其应用 |
Country Status (1)
| Country | Link |
|---|---|
| CN (2) | CN108586597A (zh) |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114671943B (zh) * | 2022-04-29 | 2023-05-05 | 四川大学 | 一种鱼类摄食调控蛋白的制备和应用 |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| TW200743444A (en) * | 2006-05-16 | 2007-12-01 | Academia Sinica | Peptide used to increase ingestion quantity of animals |
| CN101948838A (zh) * | 2010-08-24 | 2011-01-19 | 中山大学 | 一种鱼类rf神经肽基因、编码的蛋白质及应用 |
| CN103319587A (zh) * | 2013-06-24 | 2013-09-25 | 广东医学院附属医院 | 一种鱼类促摄食成熟肽及其应用 |
| CN105296500A (zh) * | 2015-11-11 | 2016-02-03 | 中山大学 | 鞍带石斑鱼神经肽b基因、蛋白及其应用 |
Family Cites Families (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101438761A (zh) * | 2008-12-23 | 2009-05-27 | 大连英斯特生物技术有限公司 | 鸡蛋蛋黄中高附加值蛋白质的分离提纯工艺 |
| CN104327184A (zh) * | 2014-09-28 | 2015-02-04 | 浙江省长兴艾格生物制品有限公司 | 一种蛋黄球蛋白粉的制备方法 |
| CN107383187B (zh) * | 2017-07-10 | 2020-08-11 | 华中农业大学 | 一种从鸡蛋黄中联合提取IgY、卵黄高磷蛋白、卵磷脂、蛋黄油及脱脂蛋黄粉的方法 |
-
2018
- 2018-04-27 CN CN201810418188.XA patent/CN108586597A/zh active Pending
- 2018-04-27 CN CN202310475344.7A patent/CN116425852B/zh active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| TW200743444A (en) * | 2006-05-16 | 2007-12-01 | Academia Sinica | Peptide used to increase ingestion quantity of animals |
| CN101948838A (zh) * | 2010-08-24 | 2011-01-19 | 中山大学 | 一种鱼类rf神经肽基因、编码的蛋白质及应用 |
| CN103319587A (zh) * | 2013-06-24 | 2013-09-25 | 广东医学院附属医院 | 一种鱼类促摄食成熟肽及其应用 |
| CN105296500A (zh) * | 2015-11-11 | 2016-02-03 | 中山大学 | 鞍带石斑鱼神经肽b基因、蛋白及其应用 |
Non-Patent Citations (4)
| Title |
|---|
| CHAOYI ZHANG: "Molecular cloning and characterization of the novel adropin from tilapia (Oreochromis niloticus): Involvement in the control of food intake", 《NEUROPEPTIDES》, 8 June 2021 (2021-06-08), pages 1 - 11 * |
| GENBANK: "Effects of neuropeptide Y as a feed additive on stimulating the growth of tilapia (Oreochromis niloticus) fed low fish meal diets", 《GENBANK》, 1 December 2021 (2021-12-01), pages 909784 * |
| GENBANK: "Oreochromis niloticus adropin (ENHO) mRNA, complete cds", 《GENBANK》, 1 October 2021 (2021-10-01), pages 909784 * |
| 王国文: "神经肽Y 的促摄食作用及其调控机制", 《动物营养学报》, vol. 28, no. 2, 31 December 2016 (2016-12-31), pages 385 - 391 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN108586597A (zh) | 2018-09-28 |
| CN116425852B (zh) | 2024-05-24 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Murashita et al. | Production of recombinant leptin and its effects on food intake in rainbow trout (Oncorhynchus mykiss) | |
| Shike et al. | Bass hepcidin is a novel antimicrobial peptide induced by bacterial challenge | |
| MacDonald et al. | Cloning, distribution and effects of season and nutritional status on the expression of neuropeptide Y (NPY), cocaine and amphetamine regulated transcript (CART) and cholecystokinin (CCK) in winter flounder (Pseudopleuronectes americanus) | |
| Shpilman et al. | Production, gene structure and characterization of two orthologs of leptin and a leptin receptor in tilapia | |
| CN104250646B (zh) | 一种与猪饲料转化效率性状相关的分子标记及检测方法和应用 | |
| Su et al. | Genomic organization and expression analysis of Toll-like receptor 3 in grass carp (Ctenopharyngodon idella) | |
| Wan et al. | Characterization of the foxl2 gene involved in the vtg expression in mud crab (Scylla paramamosain) | |
| Gong et al. | Molecular characterization, phylogeny and expression of a hepcidin gene in the blotched snakehead Channa maculata | |
| Feng et al. | Molecular characterization and expression of three preprosomatostatin genes and their association with growth in common carp (Cyprinus carpio) | |
| Wan et al. | Dynamic expression of HSP90B1 mRNA in the hypothalamus of two Chinese chicken breeds under heat stress and association analysis with a SNP in Huainan chickens | |
| CN104450763B (zh) | 斜带石斑鱼瘦素Leptin A蛋白表达纯化的方法及其应用 | |
| CN116425852B (zh) | 鱼类促摄食成熟肽及其应用 | |
| Li et al. | Molecular cloning, promoter analysis and induced expression of the complement component C9 gene in the grass carp Ctenopharyngodon idella | |
| CN103319587A (zh) | 一种鱼类促摄食成熟肽及其应用 | |
| CN113368218A (zh) | 包含花鲈干扰素IFNd及其受体的抗病毒组合物及应用 | |
| Suda et al. | Identification and gene expression analyses of ghrelin in the stomach of Pacific bluefin tuna (Thunnus orientalis) | |
| Liang et al. | Molecular characterization of neuropeptide Y gene in Chinese perch, an acanthomorph fish | |
| Khattiya et al. | Cloning, expression and functional analysis of a novel-chemokine gene of Japanese flounder, Paralichthys olivaceus, containing two additional cysteines and an extra fourth exon | |
| CN101948838A (zh) | 一种鱼类rf神经肽基因、编码的蛋白质及应用 | |
| Lee et al. | An avian αB-crystallin: non-lens expression and sequence similarities with both small (HSP27) and large (HSP70) heat shock proteins | |
| Cai et al. | Sequence, genomic organization and expression of ghrelin receptor in grass carp, Ctenopharyngodon idellus | |
| Xu et al. | Molecular characterization and expression analysis of IL-10 and IL-20L genes in swamp eel (Monopterus albus) against Aeromonas veronii infection | |
| Li et al. | Cloning, tissue distribution and effects of fasting on pituitary adenylate cyclase-activating polypeptide in largemouth bass | |
| Shi et al. | Genomic organization, promoter characterization and expression analysis of the leukocyte cell-derived chemotaxin-2 gene in Epinephelus akaraa | |
| McDevitt et al. | Tumour-associated hypoglycaemia in a murine cachexia model |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |