CN111849997B - Macrobrachium carinicauda compound eye development regulation gene and guide RNA as well as acquisition and application - Google Patents
Macrobrachium carinicauda compound eye development regulation gene and guide RNA as well as acquisition and application Download PDFInfo
- Publication number
- CN111849997B CN111849997B CN202010633645.4A CN202010633645A CN111849997B CN 111849997 B CN111849997 B CN 111849997B CN 202010633645 A CN202010633645 A CN 202010633645A CN 111849997 B CN111849997 B CN 111849997B
- Authority
- CN
- China
- Prior art keywords
- gene
- grna
- ecey
- sequence
- carinicauda
- 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
- 108090000623 proteins and genes Proteins 0.000 title claims abstract description 86
- 108020005004 Guide RNA Proteins 0.000 title claims abstract description 67
- 230000024415 compound eye development Effects 0.000 title claims abstract description 29
- 230000033228 biological regulation Effects 0.000 title description 7
- 241000238559 Macrobrachium Species 0.000 title description 3
- 241000851180 Palaemon carinicauda Species 0.000 claims abstract description 55
- 238000010362 genome editing Methods 0.000 claims abstract description 40
- 235000013601 eggs Nutrition 0.000 claims abstract description 11
- 239000002773 nucleotide Substances 0.000 claims abstract description 9
- 125000003729 nucleotide group Chemical group 0.000 claims abstract description 9
- 230000007547 defect Effects 0.000 claims abstract description 4
- 230000001105 regulatory effect Effects 0.000 claims abstract description 4
- 108091033409 CRISPR Proteins 0.000 claims description 13
- 108700005075 Regulator Genes Proteins 0.000 claims description 12
- 238000005516 engineering process Methods 0.000 claims description 11
- 238000000034 method Methods 0.000 claims description 11
- 230000015572 biosynthetic process Effects 0.000 claims description 10
- 239000002299 complementary DNA Substances 0.000 claims description 10
- 238000000338 in vitro Methods 0.000 claims description 10
- 239000003550 marker Substances 0.000 claims description 9
- 238000012408 PCR amplification Methods 0.000 claims description 8
- 238000013518 transcription Methods 0.000 claims description 7
- 230000035897 transcription Effects 0.000 claims description 7
- 239000013612 plasmid Substances 0.000 claims description 6
- 108091032973 (ribonucleotides)n+m Proteins 0.000 claims description 5
- 238000003786 synthesis reaction Methods 0.000 claims description 5
- 238000010839 reverse transcription Methods 0.000 claims description 4
- 238000010354 CRISPR gene editing Methods 0.000 claims description 3
- 230000008143 early embryonic development Effects 0.000 claims description 3
- 238000010276 construction Methods 0.000 claims description 2
- 230000001276 controlling effect Effects 0.000 claims description 2
- 238000000746 purification Methods 0.000 claims description 2
- 230000008685 targeting Effects 0.000 claims description 2
- 108091028664 Ribonucleotide Proteins 0.000 claims 1
- 239000002336 ribonucleotide Substances 0.000 claims 1
- 125000002652 ribonucleotide group Chemical group 0.000 claims 1
- 150000001875 compounds Chemical class 0.000 abstract description 21
- 241000238424 Crustacea Species 0.000 abstract description 10
- 238000011160 research Methods 0.000 abstract description 7
- 230000013020 embryo development Effects 0.000 abstract description 2
- 229920002477 rna polymer Polymers 0.000 abstract description 2
- 238000012163 sequencing technique Methods 0.000 description 15
- 241001465754 Metazoa Species 0.000 description 9
- 241000238557 Decapoda Species 0.000 description 8
- 238000011161 development Methods 0.000 description 8
- 230000018109 developmental process Effects 0.000 description 8
- 210000002257 embryonic structure Anatomy 0.000 description 8
- 230000035772 mutation Effects 0.000 description 8
- 125000003275 alpha amino acid group Chemical group 0.000 description 7
- 241000255601 Drosophila melanogaster Species 0.000 description 6
- 108020004414 DNA Proteins 0.000 description 5
- 238000002474 experimental method Methods 0.000 description 5
- 238000002347 injection Methods 0.000 description 5
- 239000007924 injection Substances 0.000 description 5
- 241000238421 Arthropoda Species 0.000 description 4
- 208000032170 Congenital Abnormalities Diseases 0.000 description 4
- 241000255581 Drosophila <fruit fly, genus> Species 0.000 description 4
- 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 4
- 238000000246 agarose gel electrophoresis Methods 0.000 description 4
- 239000002609 medium Substances 0.000 description 4
- 102000004169 proteins and genes Human genes 0.000 description 4
- 239000013535 sea water Substances 0.000 description 4
- 241000238578 Daphnia Species 0.000 description 3
- 238000002123 RNA extraction Methods 0.000 description 3
- 125000000539 amino acid group Chemical group 0.000 description 3
- 238000010367 cloning Methods 0.000 description 3
- 238000001514 detection method Methods 0.000 description 3
- 230000000694 effects Effects 0.000 description 3
- 210000001161 mammalian embryo Anatomy 0.000 description 3
- 238000012986 modification Methods 0.000 description 3
- 230000004048 modification Effects 0.000 description 3
- 230000008569 process Effects 0.000 description 3
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 3
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 description 2
- 241000243968 Cypria Species 0.000 description 2
- 102000007354 PAX6 Transcription Factor Human genes 0.000 description 2
- 101150081664 PAX6 gene Proteins 0.000 description 2
- 241000256248 Spodoptera Species 0.000 description 2
- 241000606278 Triplostegia Species 0.000 description 2
- 241000251539 Vertebrata <Metazoa> Species 0.000 description 2
- 150000001413 amino acids Chemical class 0.000 description 2
- AVKUERGKIZMTKX-NJBDSQKTSA-N ampicillin Chemical compound C1([C@@H](N)C(=O)N[C@H]2[C@H]3SC([C@@H](N3C2=O)C(O)=O)(C)C)=CC=CC=C1 AVKUERGKIZMTKX-NJBDSQKTSA-N 0.000 description 2
- 229960000723 ampicillin Drugs 0.000 description 2
- 230000003321 amplification Effects 0.000 description 2
- 238000000137 annealing Methods 0.000 description 2
- 230000001580 bacterial effect Effects 0.000 description 2
- 238000010804 cDNA synthesis Methods 0.000 description 2
- 238000012512 characterization method Methods 0.000 description 2
- 238000006243 chemical reaction Methods 0.000 description 2
- 238000012258 culturing Methods 0.000 description 2
- 238000012217 deletion Methods 0.000 description 2
- 230000037430 deletion Effects 0.000 description 2
- 238000004925 denaturation Methods 0.000 description 2
- 230000036425 denaturation Effects 0.000 description 2
- 230000003203 everyday effect Effects 0.000 description 2
- BPHPUYQFMNQIOC-NXRLNHOXSA-N isopropyl beta-D-thiogalactopyranoside Chemical compound CC(C)S[C@@H]1O[C@H](CO)[C@H](O)[C@H](O)[C@H]1O BPHPUYQFMNQIOC-NXRLNHOXSA-N 0.000 description 2
- 101150044508 key gene Proteins 0.000 description 2
- 239000007788 liquid Substances 0.000 description 2
- 108020004999 messenger RNA Proteins 0.000 description 2
- 238000000520 microinjection Methods 0.000 description 2
- 238000002156 mixing Methods 0.000 description 2
- 238000002887 multiple sequence alignment Methods 0.000 description 2
- 238000003199 nucleic acid amplification method Methods 0.000 description 2
- 150000007523 nucleic acids Chemical class 0.000 description 2
- 229960003531 phenolsulfonphthalein Drugs 0.000 description 2
- 238000012257 pre-denaturation Methods 0.000 description 2
- 238000002360 preparation method Methods 0.000 description 2
- 238000004153 renaturation Methods 0.000 description 2
- 238000007480 sanger sequencing Methods 0.000 description 2
- 241000894007 species Species 0.000 description 2
- 230000004083 survival effect Effects 0.000 description 2
- 238000012795 verification Methods 0.000 description 2
- 241000238017 Astacoidea Species 0.000 description 1
- 238000010453 CRISPR/Cas method Methods 0.000 description 1
- 241001390275 Carinata Species 0.000 description 1
- 108010008532 Deoxyribonuclease I Proteins 0.000 description 1
- 102000007260 Deoxyribonuclease I Human genes 0.000 description 1
- 108010053770 Deoxyribonucleases Proteins 0.000 description 1
- 102000016911 Deoxyribonucleases Human genes 0.000 description 1
- 206010058314 Dysplasia Diseases 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 241000620209 Escherichia coli DH5[alpha] Species 0.000 description 1
- 241000238631 Hexapoda Species 0.000 description 1
- 101000601647 Homo sapiens Paired box protein Pax-6 Proteins 0.000 description 1
- 241000530454 Litopenaeus schmitti Species 0.000 description 1
- 241000238553 Litopenaeus vannamei Species 0.000 description 1
- 241000124008 Mammalia Species 0.000 description 1
- 108091028043 Nucleic acid sequence Proteins 0.000 description 1
- 108700026244 Open Reading Frames Proteins 0.000 description 1
- 102100037506 Paired box protein Pax-6 Human genes 0.000 description 1
- 241001174556 Palatinus Species 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
- 238000012300 Sequence Analysis Methods 0.000 description 1
- 238000010459 TALEN Methods 0.000 description 1
- 108010043645 Transcription Activator-Like Effector Nucleases Proteins 0.000 description 1
- 108091023040 Transcription factor Proteins 0.000 description 1
- 102000040945 Transcription factor Human genes 0.000 description 1
- 241000212749 Zesius chrysomallus Species 0.000 description 1
- 230000005856 abnormality Effects 0.000 description 1
- GFFGJBXGBJISGV-UHFFFAOYSA-N adenyl group Chemical class N1=CN=C2N=CNC2=C1N GFFGJBXGBJISGV-UHFFFAOYSA-N 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 208000008303 aniridia Diseases 0.000 description 1
- 230000006399 behavior Effects 0.000 description 1
- 238000010805 cDNA synthesis kit Methods 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 108091036078 conserved sequence Proteins 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 230000001351 cycling effect Effects 0.000 description 1
- UHDGCWIWMRVCDJ-ZAKLUEHWSA-N cytidine Chemical class O=C1N=C(N)C=CN1[C@H]1[C@H](O)[C@@H](O)[C@H](CO)O1 UHDGCWIWMRVCDJ-ZAKLUEHWSA-N 0.000 description 1
- 230000002950 deficient Effects 0.000 description 1
- 230000029087 digestion Effects 0.000 description 1
- 208000008204 ectopia pupillae Diseases 0.000 description 1
- 238000001962 electrophoresis Methods 0.000 description 1
- 230000007613 environmental effect Effects 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 230000004373 eye development Effects 0.000 description 1
- 239000012634 fragment Substances 0.000 description 1
- 239000000499 gel Substances 0.000 description 1
- 238000001502 gel electrophoresis Methods 0.000 description 1
- 210000002149 gonad Anatomy 0.000 description 1
- 230000012010 growth Effects 0.000 description 1
- 239000001963 growth medium Substances 0.000 description 1
- 230000004060 metabolic process Effects 0.000 description 1
- 239000011259 mixed solution Substances 0.000 description 1
- 238000001823 molecular biology technique Methods 0.000 description 1
- 108020004707 nucleic acids Proteins 0.000 description 1
- 102000039446 nucleic acids Human genes 0.000 description 1
- 210000000158 ommatidium Anatomy 0.000 description 1
- 210000001328 optic nerve Anatomy 0.000 description 1
- 230000017448 oviposition Effects 0.000 description 1
- 108091008695 photoreceptors Proteins 0.000 description 1
- 230000035479 physiological effects, processes and functions Effects 0.000 description 1
- 238000007747 plating Methods 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- 230000033458 reproduction Effects 0.000 description 1
- 230000001850 reproductive effect Effects 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 230000035939 shock Effects 0.000 description 1
- 239000008223 sterile water Substances 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
- 208000024891 symptom Diseases 0.000 description 1
- 230000009466 transformation Effects 0.000 description 1
- 230000001131 transforming effect 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
- C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
-
- 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
- A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
- A01K67/033—Rearing or breeding invertebrates; New breeds of invertebrates
- A01K67/0333—Genetically modified invertebrates, e.g. transgenic, polyploid
- A01K67/0337—Genetically modified Arthropods
- A01K67/0338—Genetically modified Crustaceans
-
- 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
- C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
- C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
-
- 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
- C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
- C12N15/90—Stable introduction of foreign DNA into chromosome
- C12N15/902—Stable introduction of foreign DNA into chromosome using homologous recombination
-
- 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
- A01K2227/00—Animals characterised by species
- A01K2227/70—Invertebrates
-
- 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
- A01K2267/03—Animal model, e.g. for test or diseases
-
- 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
- C12N2310/00—Structure or type of the nucleic acid
- C12N2310/10—Type of nucleic acid
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Genetics & Genomics (AREA)
- Biotechnology (AREA)
- Chemical & Material Sciences (AREA)
- Zoology (AREA)
- Organic Chemistry (AREA)
- Biomedical Technology (AREA)
- Molecular Biology (AREA)
- General Engineering & Computer Science (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Wood Science & Technology (AREA)
- Biophysics (AREA)
- General Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- Microbiology (AREA)
- Environmental Sciences (AREA)
- Physics & Mathematics (AREA)
- Plant Pathology (AREA)
- Gastroenterology & Hepatology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Toxicology (AREA)
- Mycology (AREA)
- Animal Behavior & Ethology (AREA)
- Animal Husbandry (AREA)
- Biodiversity & Conservation Biology (AREA)
- Medicinal Chemistry (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention relates to a gene EcEy for regulating the compound eye development of palaemon carinicauda, which can show obvious phenotypic change in the embryonic period after being successfully edited so as to indicate the success rate of gene editing operation, and can also be applied to the establishment of other gene editing platforms of decapod crustaceans. The EcEy gene of palaemon carinicauda related by the invention has a nucleotide sequence shown as SEQ ID No.1 in a sequence list; guide RNA (gRNA) designed and synthesized based on the sequence has a ribonucleic acid sequence shown as SEQ ID No.2 in a sequence list, and the EcEy-gRNA and the commercialized Cas9mRNA are mixed and injected into fertilized eggs of palaemon carinicauda, so that the PAX structural domain of the EcEy gene can be specifically identified and cut, and an obvious compound eye defect phenotype is observed at the early stage of embryonic development. The EcEy gene can be used as a target gene for gene editing research of palaemon carinicauda, is used for verifying whether the gene editing operation is successful or not, and can also be used for establishing other decapod crustacean gene editing platforms.
Description
Technical Field
The invention relates to the technical field of exopalaemon carinicauda gene engineering, in particular to acquisition of exopalaemon carinicauda compound eye development regulation gene and guide RNA and application thereof in gene editing research of decapod crustaceans.
Background
Exopalaemon carinicauda belongs to the decapod crustacean subgenus (Decapoda), is a marine shrimp with higher economic value, is also one of decapod animal research models, is the only reported decapod animal capable of carrying out gene editing operation at present, and has the advantages of strong environmental adaptability, strong reproductive capacity, easy artificial feeding, continuous oviposition of female shrimps, large embryo, transparency and the like. The study on gene editing of palaemon carinicauda can be used for revealing the life activity rules of development, growth, metabolism, reproduction and the like of the decapod animals. However, the current gene editing research of palaemon carinicauda lacks an effective marker gene, and the marker gene should have an obvious mutation phenotype and be observed in early development stage and can be used as a mark for the success or failure of gene editing operation.
The compound eye is one of important characteristic traits of crustacean, is an important photoreceptor of the crustacean, consists of several to tens of thousands of small eyes, and has important influence on the physiology and behavior of the crustacean. The compound eye is also a phenotype which can be observed in the early development stage of shrimp embryos, after the fertilized eggs develop for about 140 hours, the compound eye formation can be observed in the palatinus carinata embryos in the early stage of daphnia larvae, red spots are observed on two sides of the embryos, the spots are gradually increased and converged into an arc line, and the compound eye gradually widens to form a crescent shape along with the development of the embryos. In the later stage of the daphnia larva, compound eyes are developed obviously. It is noteworthy that in insects, many species use the key genes for the formation of compound eyes as marker genes for gene editing operations, such as Drosophila, Tripsammos rubescens, and daphnia. The mutation of the key gene for forming the compound eye can observe obvious phenotype in early embryonic development, does not need complex observation equipment such as a fluorescence microscope and the like, and is suitable as a marker gene for gene editing verification.
The Eyeless gene is a homologous gene of vertebrate Paired box6(Pax6) gene in arthropod, and the coded transcription factor is well conserved in both vertebrates and invertebrates, is a key gene for eye formation and plays an important role in the eye formation and development of animals. The Eyeless gene encodes a protein with two domains that bind to the Homeobox (HOX) and the companion box (PAX) of DNA. The mutation of Pax6/Eyeless gene can cause various symptoms of mammals and arthropods, such as aniridia, ectopia pupillae, dysplasia optic nerve, mouse eye deformity, drosophila eye loss, ectopy, etc. An Eyeless gene (EcEy) gene of palaemon carinicauda is obtained through earlier research, has conserved HOX and PAX structural domains, is proved to cause obvious mutation on compound eye development of palaemon carinicauda through gene editing experiments, can be observed at the early stage of embryonic development, is a marker gene of proper gene editing operation, and can be used as a mark of success or failure of transgenosis. In addition, the sequence and the function of the gene are conserved, the mutation phenotype is obvious, and the gene can also be used for establishing other decapod crustacean gene editing platforms as marker genes for indicating the successful application of the gene editing technology.
Disclosure of Invention
The invention describes a compound eye development regulatory gene EcEy and guide RNA of palaemon carinicauda and application thereof in gene editing research of decapod crustaceans.
The technical scheme of the invention is as follows:
extracting total RNA of the palaemon carinicauda, carrying out reverse transcription to obtain cDNA, and designing a primer EcEy-FL-F based on the nucleotide sequence of the EcEy gene: GAGCCGTTTACTAAAGACCTG and EcEy-FL-R: ACAAAATCCAGACTGAACTACAC are provided. Taking the palaemon carinicauda cDNA as a template to carry out PCR amplification to obtain the full-length sequence of EcEy, wherein the sequence is characterized in that:
SEQ ID No.1：
GAGCCGTTTACTAAAGACCTGGAGGCGAAGACAGGAGCTTAAAAAGGAATCCGTCGAGGTCGCCCACATCTTCACCGTCATCATTAAGGGGCTTCGGTAGCGGCGGCGAGCAAGGCTGGGGGGGTGGGTGCCTGCAGGCTGGCCGCCGCCGTTATGTTGTCCCCAGTAGATGCCGGGCCACATGGACCGCATGGCCCGCATGGTCACCACCCGTGGGGCCCCACCACTTTGGACGACATGTCGCATAAGGACGTGCTCGACTACTCACTGGGACCCTCGGGTAAGGGAGCGATACCCCCCTGGTGGCCAAGGGGGGAAGGGGAGGATGGTCACAGTGGAGTGAACCAACTAGGAGGTGTCTTTGTATCCGGCAGACCATTGCCCGACACCACCAGGCAGAAGATCATCGAGTTGGCTCATTCCGGAGCCAGGCCTTGCGACATCTCCAGAATTCTTCAGGTTTCCAACGGTTGCGTCTCCAAAATACTGGGCAGATACTACGAGACGGGTTCGATCCGTCCCCGAGCAATTGGGGGATCGAAACCTCGCGTAGCCACGGCGGAAGTGGTCGCCAAGATTTCCCAGTTCAAGCGAGAATGTCCTTCAATCTTCGCCTGGGAGATCAGAGACCGACTGCTCTCCGAAGGGGTCTGCTCCTCGGACAACATTCCAAGCGTGTCATCCATCAACCGCGTCCTTCGTAACATCAACTCGAAAGACACCACCGGCAGCGGCTCCGGCGGGCCAGTGGGCGCCGGCGGACCTCCTGGTGGAGGCGCCGGAGGAGGAGCAGGAGGCGGAGGAGGAGGGGGCAGCGCCCTAGCTACTGCAGGAGCTGGTAGCGTCGGAGGAAGCCAGACGATGGGCCTTGGAGGCGGGAACTCCAGCAGCGGCAGTAGTAATTCGAACACGCCCACCAGCACCCCGACGCAGGACCCTATGTACGATAAACTCCGCCTACTGAATGGACAGTCTTCGTGGCCCAGATCACAGTGGTATAGCGGACCAGGCAATGACATCACGGCCCTGCCAGTCGATTCTCCCCAGTCGGCACCTTGTGTTCAGCAAGACATCGTCAAGAAGAGTGACGCGGGCGAGCTTAGCGAGGACAGCAATGCTGGCAGCAGCGAAAACAGTAACAGCGAAGACCAAGCCAGACTTCACTTGAAGAGAAAACTTCAGAGAAACAGAACTTCTTTCACGAATGACCAGATTGATAGTTTGGAAAAAGAATTTGAGAGAACTCATTATCCTGATGTATTTGCTCGCGAGAGACTGGCCGCAAAGATAGGCTTGCCGGAAGCTAGGATACAGGTGTGGTTCAGCAACCGAAGAGCCAAATGGCGGAGAGAAGAAAAGTTAAGGAACCAGCGGAGGGCGGCCGAGGGCATCGCACCTTCCTCCCCCACCAGAATCATCAACAACTTCACGCCTTCTCCCATGTACACCCCATTGCCTCCTCCGCCCATGTCCGTCGCTGACACTTATGGGTCTATGGCAGGCGGAGGCGGATTCGGCATGGGATCGAGTGTGGGCGTAGGGCCAGGAGTGGGCGTGGTGGCCTCCAGCCCCAACTGCCTCCCACAACAGCAGCCCACAACCGTCGTCCACTCTGCGATGACCACGGGCGTGGGACTCGGCCGAGACCATTCTCACAACGCCCACAACCCTTACATGAGTCGGTCTTACGACTCGCTGTACACGCACGCCCGCGCCTCACCCACTTGCCCGCCCATGCTTTATCACCCAGCTTCGCACCACCCACACCAGAACCCACACGCTCCTCACGAGTACAATACGCCCACGCCACCCAATTCACAAGCCGGTCTTCTCTCTCCCGGTGTGTCAGTGCCTGTAGCGGTGCCCGGCCAACACCACGACATGAACACCCAATACTGGACTCGATTGCAGTAGGTGCCAAACGTTTATGGCAAATGTTCAAGGCTCCACCTGGAGGAAAGGAAACAAACGTTTCGAGGTTATAACTCTGTAAACATAACAAAGTGCTTTTGATCTCTCGTCAGAATGCAACTGGTGTTAACTGTGTACAAACGGACGGAGTCTAAAATGTGGATAACGAAACAGAACTGAAAAAAACAAAAGAGGTGCAGAGTGCGTCAGTTACCTTTGTGTGACTCTGGGATCGAAGCGAACGCTAAAGGACCCATCAGTAGATTTCTCATTGGCATCAATATCAAGAAGGATGTTCCATACGATGTTCCGCGAATGAAGGAAACAGAAGGACGTGGTATATACATATTGTGCCTCACACGGAAAAGGTGAAAGGGGTTGGCCTTTTGTTATAAGACAAATTAACACACTTGTGTTTATTTCATTTTACTTATGCTTGTATTATATATAAAACGTGCTTTATATTTCGCAAATTAAATAAAATGAATGAAACTGAACACAGGAATAGAGAATGGATATTAACATAGCTAGCTACAACACCCATGAGTCACTTGATTTTTTTTTTTATCTTAGAAAAATGTGAACAAAAATCTGAAATGGGATTTGTGTAGTTCAGTCTGGATTTTGT
using the online tool CRISPRDIRECT (http://crispr.dbcls.jp/) And (5) selecting the gene editing target site of the EcEy gene. Using guide RNA (gRNA) framework plasmid as a template, and using gRNA-F: TAATACGACTCACTATAGCTCGATGATCTTCTGCCT
GGGTTTTAGAGCTAGAAATAGC and gRNA-R: AAAAAAGCACCGACTCGGTGCCA is used as primer, PCR is carried out to amplify gRNA template. The PCR product of gRNA is transcribed in vitro to synthesize gRNA, and the sequence characteristics are as follows:
SEQ ID No.2：
CUCGAUGAUCUUCUGCCUGGGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUUUU
the application of the EcEy gene of the exopalaemon carinicauda compound eye development regulation gene. The gRNA transcribed in vitro of the palaemon carinicauda is obtained based on the EcEy gene sequence of the palaemon carinicauda, after the commercial Cas9mRNA is mixed, the PAX structural domain of the EcEy gene can be cut by injecting fertilized eggs of the palaemon carinicauda, obvious compound eye development defects are observed in the early embryonic development stage, and the method can be used for marking genes for gene editing to verify whether the gene editing operation is successful or not, and can also be used for establishing other decapod crustacean gene editing platforms.
The invention has the following advantages
1. The invention determines a compound eye development regulation gene EcEy of palaemon carinicauda and the function thereof in the regulation of the compound eye development process of palaemon carinicauda.
2. The invention can obtain the guide RNA for effectively regulating and controlling the compound eye development gene EcEy of the exopalaemon carinicauda, and can be used for verifying whether the gene editing operation is successful or not and developing other decapod animal gene editing platforms.
Drawings
FIG. 1 sequence information of EcEy gene, letters represent the correspondence between the nucleotide sequence and protein sequence of EcEy gene, and shaded parts represent the PAX and HOX domains of EcEy gene.
FIG. 2 alignment of the amino acid sequences of EcEy gene of palaemon carinicauda with other 6 arthropod-homologous genes. (A) And (5) performing multiple sequence alignment. Boxes represent regions of amino acids with sequence similarity higher than 95%. The sequences were from Spodoptera carinica (EcEy, obtained according to the invention), Cypria magna (DpuEy, NCBI accession number EFX75780.1), Triplostegia rubescens (TcEy, ABW23132.1), Drosophila melanogaster (DMey, NP-524628.2), Drosophila pseudocerasus (DsEy, XP-016037330.1), Drosophila melanogaster (DpsEy, XP-001352371.2), Drosophila melanogaster (DvEy, XP-002059744.1), respectively. Asterisks indicate fully conserved amino acid residues. (B) A putative domain of the amino acid sequence of EcEy. The PAX and HOX domains are shown as pentagons, and the boxes represent highly conserved amino acid sequences corresponding to panel (a).
FIG. 3 effect on the process of compound eye formation of palaemon carinicauda embryos after gRNA injection mixed with Cas9 mRNA. There are mainly four phenotypes: (A) only one eye remains; (B) only one eye is left, and the ectopy of compound eyes is accompanied; (C) the two compound eyes are ectopic; (D) the multi-eyepoint phenomenon. CE, compound eye.
FIG. 4 sequencing results of the EcEy gene of the compound-eye mutated Exopalaemon carinicauda. (A) As a result of sequencing, it was found that a sequence deletion of a plurality of bases was generated at the target sequence site of the gRNA. (B) From the results of sequencing peak maps, it can be seen that a significant set of peaks was generated from the sequences after the target sequence site of the gRNA, confirming the edited results. WT, control group; mutations, mutation group; gRNA, guide RNA; PAM, Cas9 protein recognition sequence.
Detailed Description
The present invention will be described in further detail with reference to examples.
The sequence and source sequence information of the compound eye development regulatory gene EcEy of the exopalaemon carinicauda are as follows:
information of SEQ ID No.1
(a) Sequence characterization
Length: 2539 base pairs
Type: nucleotide, its preparation and use
Chain type: double chain
Topology structure: linearity
(b) Molecular type: nucleic acids
Description of the sequence: SEQ ID No.1
GAGCCGTTTACTAAAGACCTGGAGGCGAAGACAGGAGCTTAAAAAGGAATCCGTCGAGGTCGCCCACATCTTCACCGTCATCATTAAGGGGCTTCGGTAGCGGCGGCGAGCAAGGCTGGGGGGGTGGGTGCCTGCAGGCTGGCCGCCGCCGTTATGTTGTCCCCAGTAGATGCCGGGCCACATGGACCGCATGGCCCGCATGGTCACCACCCGTGGGGCCCCACCACTTTGGACGACATGTCGCATAAGGACGTGCTCGACTACTCACTGGGACCCTCGGGTAAGGGAGCGATACCCCCCTGGTGGCCAAGGGGGGAAGGGGAGGATGGTCACAGTGGAGTGAACCAACTAGGAGGTGTCTTTGTATCCGGCAGACCATTGCCCGACACCACCAGGCAGAAGATCATCGAGTTGGCTCATTCCGGAGCCAGGCCTTGCGACATCTCCAGAATTCTTCAGGTTTCCAACGGTTGCGTCTCCAAAATACTGGGCAGATACTACGAGACGGGTTCGATCCGTCCCCGAGCAATTGGGGGATCGAAACCTCGCGTAGCCACGGCGGAAGTGGTCGCCAAGATTTCCCAGTTCAAGCGAGAATGTCCTTCAATCTTCGCCTGGGAGATCAGAGACCGACTGCTCTCCGAAGGGGTCTGCTCCTCGGACAACATTCCAAGCGTGTCATCCATCAACCGCGTCCTTCGTAACATCAACTCGAAAGACACCACCGGCAGCGGCTCCGGCGGGCCAGTGGGCGCCGGCGGACCTCCTGGTGGAGGCGCCGGAGGAGGAGCAGGAGGCGGAGGAGGAGGGGGCAGCGCCCTAGCTACTGCAGGAGCTGGTAGCGTCGGAGGAAGCCAGACGATGGGCCTTGGAGGCGGGAACTCCAGCAGCGGCAGTAGTAATTCGAACACGCCCACCAGCACCCCGACGCAGGACCCTATGTACGATAAACTCCGCCTACTGAATGGACAGTCTTCGTGGCCCAGATCACAGTGGTATAGCGGACCAGGCAATGACATCACGGCCCTGCCAGTCGATTCTCCCCAGTCGGCACCTTGTGTTCAGCAAGACATCGTCAAGAAGAGTGACGCGGGCGAGCTTAGCGAGGACAGCAATGCTGGCAGCAGCGAAAACAGTAACAGCGAAGACCAAGCCAGACTTCACTTGAAGAGAAAACTTCAGAGAAACAGAACTTCTTTCACGAATGACCAGATTGATAGTTTGGAAAAAGAATTTGAGAGAACTCATTATCCTGATGTATTTGCTCGCGAGAGACTGGCCGCAAAGATAGGCTTGCCGGAAGCTAGGATACAGGTGTGGTTCAGCAACCGAAGAGCCAAATGGCGGAGAGAAGAAAAGTTAAGGAACCAGCGGAGGGCGGCCGAGGGCATCGCACCTTCCTCCCCCACCAGAATCATCAACAACTTCACGCCTTCTCCCATGTACACCCCATTGCCTCCTCCGCCCATGTCCGTCGCTGACACTTATGGGTCTATGGCAGGCGGAGGCGGATTCGGCATGGGATCGAGTGTGGGCGTAGGGCCAGGAGTGGGCGTGGTGGCCTCCAGCCCCAACTGCCTCCCACAACAGCAGCCCACAACCGTCGTCCACTCTGCGATGACCACGGGCGTGGGACTCGGCCGAGACCATTCTCACAACGCCCACAACCCTTACATGAGTCGGTCTTACGACTCGCTGTACACGCACGCCCGCGCCTCACCCACTTGCCCGCCCATGCTTTATCACCCAGCTTCGCACCACCCACACCAGAACCCACACGCTCCTCACGAGTACAATACGCCCACGCCACCCAATTCACAAGCCGGTCTTCTCTCTCCCGGTGTGTCAGTGCCTGTAGCGGTGCCCGGCCAACACCACGACATGAACACCCAATACTGGACTCGATTGCAGTAGGTGCCAAACGTTTATGGCAAATGTTCAAGGCTCCACCTGGAGGAAAGGAAACAAACGTTTCGAGGTTATAACTCTGTAAACATAACAAAGTGCTTTTGATCTCTCGTCAGAATGCAACTGGTGTTAACTGTGTACAAACGGACGGAGTCTAAAATGTGGATAACGAAACAGAACTGAAAAAAACAAAAGAGGTGCAGAGTGCGTCAGTTACCTTTGTGTGACTCTGGGATCGAAGCGAACGCTAAAGGACCCATCAGTAGATTTCTCATTGGCATCAATATCAAGAAGGATGTTCCATACGATGTTCCGCGAATGAAGGAAACAGAAGGACGTGGTATATACATATTGTGCCTCACACGGAAAAGGTGAAAGGGGTTGGCCTTTTGTTATAAGACAAATTAACACACTTGTGTTTATTTCATTTTACTTATGCTTGTATTATATATAAAACGTGCTTTATATTTCGCAAATTAAATAAAATGAATGAAACTGAACACAGGAATAGAGAATGGATATTAACATAGCTAGCTACAACACCCATGAGTCACTTGATTTTTTTTTTTATCTTAGAAAAATGTGAACAAAAATCTGAAATGGGATTTGTGTAGTTCAGTCTGGATTTTGT
Information of SEQ ID No.2
(a) Sequence characterization
Length: 102 base
Type: nucleotide, its preparation and use
Chain type: single strand
Topology structure: linearity
(b) Molecular type: ribonucleic acid
Description of the sequence: SEQ ID No.2
CUCGAUGAUCUUCUGCCUGGGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUUUU
Unless otherwise indicated, the techniques used in the examples below, including molecular biology techniques such as RNA extraction, cDNA synthesis, PCR amplification and detection, and gRNA synthesis, are conventional techniques known to those skilled in the art. The instruments, reagents, etc. used are those skilled in the art, and are available from public or commercial sources unless otherwise indicated by the specification.
Example 1 cloning and analysis of the fly-tail white shrimp ommatidium development regulatory gene EcEy
(1) Total RNA extraction of exopalaemon carinicauda
The total RNA extraction method is described in the specification with reference to RNAioso Plus (Takara, cat # 9108). Total RNA from palaemon carinicauda was extracted, RNA concentration was determined using a NanoDrop 2000 spectrophotometer (Thermo), and the quality was checked by agarose gel electrophoresis and stored at-80 ℃.
(2) Reverse transcription of cDNA
First Strand cDNA Synthesis by DNase I digestion and reverse transcription of RNA samples is described in RevertAID First Strand cDNA Synthesis Kit (Thermo, K1622). The size and integrity of the PCR product were checked by gel electrophoresis of the resulting cDNA product.
(3) full-Length cDNA cloning of EcEy Gene
According to sequencing information of a transcriptome of the palaemon carinicauda obtained by earlier sequencing in a laboratory, an obtained EcEy gene nucleic acid sequence is used for designing a primer EcEy-FL-F/R to verify a predicted EcEy gene full-length cDNA sequence, the primer is used for PCR amplification, and the PCR system refers to the use instruction of Ex Taq enzyme (Takara, DRR 001A). PCR reaction procedure: pre-denaturation at 95 ℃ for 5 min; denaturation at 95 ℃ for 30s, annealing at 58 ℃ for 30s, renaturation at 72 ℃ for 2min, and 38 cycles; extension at 72 ℃ for 10 min. After amplification, agarose gel electrophoresis identification is carried out, and then a DNA gel recovery kit (AxyGen, AP-GX-50) is used for purifying and recovering the target fragment.
The primer sequences are as follows:
EcEy-FL-F：GAGCCGTTTACTAAAGACCTG
EcEy-FL-R：ACAAAATCCAGACTGAACTACAC
(4) cloning of the PCR product: connecting the PCR product to a pMD19-T vector (TaKaRa, 6013), transforming Escherichia coli DH5 alpha competent cells (TransGen Biotech, CD201-01), shaking and culturing overnight at 37 ℃, plating, picking a single colony, amplifying the colony by PCR, screening positive clones, and sending to Shanghai engineering biological engineering company for sequencing, wherein the detailed steps are as follows:
a) connection of
The linking system is as follows:
the connection is carried out for more than 4h at 16 ℃.
b) Preparing a flat plate
LB liquid medium formula:
after the medium was sterilized, 0.1% (by volume) of ampicillin was added to the medium in a super clean bench.
c) Transformation of competent cells
The ligation product was added to 100. mu.L of competent cells, mixed well and ice-cooled for 30 min. The heat shock was applied to a water bath at 42 ℃ for 30 seconds and then placed on ice for 2 minutes. 250. mu.L of LB medium was added, centrifuged at 200rpm, and cultured at 37 ℃ for 1 hour.
d) Plates were plated and 40. mu.L of 5-bromo-4-chloro-3-indole-. beta. -D-galactoside (x-gal) and 7. mu.L of Isopropylthiogalactoside (IPTG) were added to each plate, spread evenly and placed in a 37 ℃ incubator until use. 200. mu.L of the bacterial suspension was plated and cultured overnight in a 37 ℃ incubator.
e) Positive clone detection
Placing into a refrigerator at 4 deg.C for 1h, selecting white monoclonal in 10 μ L sterile water in a super clean bench, and vortex mixing. Putting 1 μ L of the mixed solution into a 10 μ L PCR system, and identifying positive clone by using M13 primer, wherein the PCR system is pre-denatured for 5min at 95 ℃ according to a common PCR system; denaturation at 95 ℃ for 30s, annealing at 55 ℃ for 30s, renaturation and extension at 72 ℃ for 1min, and 35 cycles; extension at 72 ℃ for 10 min. And detecting through agarose gel electrophoresis, selecting monoclonals with consistent band sizes, adding the monoclonals into 1.5ml of culture medium containing 0.1% of ampicillin in mass concentration, culturing for 6 hours at 37 ℃, and sending bacterial liquid to Shanghai Bioengineering company for sequencing.
The M13 primer sequences were as follows:
M13F：AGGGTTTTCCCAGTCACG
M13R：GAGCGGATAACAATTTCACAC
(5) sequence analysis: the nucleotide sequence of the sequenced EcEy gene is compared with the nucleotide sequence sequenced by a transcriptome by using NCBI (https:// www.ncbi.nlm.nih.gov /) to verify the correctness, and the result shows that the sequencing results are consistent. The protein sequence of the gene is predicted and analyzed by using ExPASY (http:// web. ExPASy. org/translate), and the result shows that the total length of an open reading frame of the EcEy gene is 1761bp, the 586 amino acid residues are coded (figure 1), the predicted molecular mass is 61.84KD, and the theoretical isoelectric point is 8.86. The present invention further compares the amino acid sequences of EcEy gene of palaemon carinicauda with other arthropod homologous genes, confirming that EcEy has typical PAX and HOX domains and is highly conserved in sequence among arthropods (FIG. 2).
Example 2 gRNA Synthesis and Gene editing experiment of the Gene EcEy regulating the Compound eye development of palaemon carinicauda
(1) gRNA synthesis of exopalaemon carinicauda compound eye development regulatory gene EcEy
a) gRNA framework construction
Firstly, a gRNA framework sequence is designed and synthesized according to the structure of the gRNA:
GTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTTT
the framework sequence was then ligated into a pMD19-T vector (TaKaRa, D102A) to construct the gRNA framework plasmid pMD 19-gRNA.
b) Synthesis of gRNA templates
Using the online tool CRISPRDIRECT (http://crispr.dbcls.jp/) And (4) selecting a gRNA targeting site of the EcEy gene. Corresponding gRNA-F is designed according to the target sequence, and gRNA-R of a reverse primer is designed at the same time.
gRNA-F：
TAATACGACTCACTATAGCTCGATGATCTTCTGCCTGGGTTTTAGAGCTAGAAATAGC
gRNA-R：AAAAAAGCACCGACTCGGTGCCA
The gRNA framework plasmid pMD19-gRNA is used as a template, and gRNA-F and gRNA-R primers are used for carrying out PCR amplification on the gRNA template.
The PCR system was as follows:
PCR cycling program: pre-denaturation at 98 ℃ for 5 min; 10sec at 98 ℃, 5sec at 55 ℃, 30sec at 72 ℃, 35 cycles; extending for 10min at 72 ℃; keeping the temperature at 4 ℃. And detecting the size of the amplified gRNA template by agarose gel electrophoresis of the PCR product, and sequencing the PCR product. The remaining PCR product was used as a template for gRNA in vitro transcription.
c) In vitro transcription and purification of gRNA
The PCR product of the gRNA was transcribed in vitro using a TranscriptAId T7High Yield Transcription Kit (Thermo, K0441) to synthesize the gRNA.
The in vitro transcription system is as follows
The transcription system is reacted for 2h at 37 ℃; then, 1. mu.L of DNase (QIAGEN,79254) was added to the reaction system and digested at 37 ℃ for 15min, the DNA template was removed, and 1. mu.L was taken for electrophoresis. The digested system was extracted with gRNA using phenol/chloroform extraction.
(2) Gene editing experiment of EcEy of exopalaemon carinicauda
a) Feeding female and male shrimps with well developed gonads according to equal number proportion. And after the female shrimps lay eggs, collecting fertilized eggs which are just laid.
b) gRNA and commercial Cas9mRNA (Thermo, A29378) were mixed to a final concentration of 250 ng/. mu.L for EcEy-gRNA and 300 ng/. mu.L for Cas9mRNA, and phenol red was added as an indicator at a final concentration of 0.05% by mass. Performing microinjection on 437 fertilized eggs of exopalaemon carinicauda, wherein the injection amount of each embryo is 0.5 nL; the control group was injected with the same volume of 0.05% phenol red solution, and the control group was injected with 226 fertilized eggs.
c) The injected embryos are placed in a petri dish containing sterile seawater, incubated on a shaker at room temperature, and the sterile seawater is changed every day.
d) And observing the compound eye development condition of the palaemon carinicauda by using a stereomicroscope when the fertilized eggs of the palaemon carinicauda develop to the daphnia-shaped larva stage (140-160 h after injection). 89 surviving subjects (survival rate 20.4%) were observed, with 38 ocular abnormalities and 42.7% deformity rate. The types of mutations are mainly divided into four types: (A) only one eye remains; (B) only one eye remains with a compound eye ectopy; (C) the two compound eyes are ectopic; (D) the multiocular phenomenon (fig. 3). The sequence of the EcEy gene of the malformed individual was detected by Sanger sequencing, and as a result, the malformed individual was confirmed to be an individual in which the EcEy gene was successfully edited (fig. 4). While the control group survived 67 individuals (survival rate 29.6%), no defective phenotype and EcEy gene editing results were observed and detected. The results prove that the EcEy gene regulates the development of compound eyes of palaemon carinicauda, and the mutation of the EcEy gene can cause the deformity of the development of the compound eyes.
The detection primers used were as follows:
EcEy-D-F：AACTAGGAGGTGTCTTTGTA
EcEy-D-R：GTTGGAAACCTGAAGAAT
the results further prove that the CRISPR/Cas9 gene editing technology can be applied to the verification research of the gene function of the palaemon carinicauda, and simultaneously verify that the compound eye development regulatory gene EcEy can be used as a marker gene for indicating whether the gene editing operation of the palaemon carinicauda is successful or not, and is used for indicating the success rate of the gene editing technology.
Example 3 application of the Complex eye development regulatory Gene EcEy of Exopalaemon carinicauda in the establishment of Gene editing platforms for other decapod animals
(1) Homologous sequence acquisition of the EcEy Gene
The EcEy gene of the exopalaemon carinicauda is compared with the published sequencing data of transcriptome of other decapod animals such as Litopenaeus vannamei, red crayfish, Procambrus clarkii and the like, a full-length sequence amplification primer Ey-FL-F/R of the homologous gene is designed according to the homologous conserved sequence (figure 2), and PCR amplification is carried out to obtain the homologous sequence of the EcEy gene of other decapod animals (the detailed steps are the same as the embodiment 1). The example is for giant finger shrimp.
The primer sequences are as follows:
Ey-FL-F：GAGCCGTTTACTAAAGACCTG
Ey-FL-R：ACAAAATCCAGACTGAACTACAC
(2) synthetic gRNA
Based on the homologous sequence of the EcEy gene, using the on-line tool CRISPRDirect (http:// crispr.dbcls.jp/) Selection of a target site for gene editing is performed. And designing corresponding gRNA-F and reverse primer gRNA-R according to the target sequence.
gRNA-F：
TAATACGACTCACTATANNNNNNNNNNNNNNNNNNNNGTTTTAGAGCTAGAAATAGC (20 base sequence designed for target site with single underline)
gRNA-R：AAAAAAGCACCGACTCGGTGCCA
And performing PCR amplification by using the gRNA framework plasmid as a template to obtain a gRNA template. The PCR product of gRNA was transcribed in vitro to synthesize gRNA (detailed procedure same as example 2, step 1).
(3) EcEy homologous Gene editing experiment
a) Obtaining a unicellular fertilized egg of macrobrachium giganteum, mixing gRNA and a commercial Cas9mRNA (Thermo) to ensure that the final concentration of the gRNA is 250 ng/mu L and the final concentration of the Cas9mRNA is 300 ng/mu L, and adding 0.05 percent of phenol red serving as an indicator. Microinjection is carried out on fertilized eggs, and the injection amount of each embryo is 0.5 nL; the control group was injected with the same volume of 0.05% phenol red solution.
b) The injected embryos are placed in a petri dish containing sterile seawater, incubated on a shaker at room temperature, and the sterile seawater is changed every day.
c) And observing the compound eye development condition by using a stereoscopic microscope. If the EcEy homologous gene is successfully edited, the phenotype of the compound eye developmental deformity can be observed. The EcEy homologous gene can be used as a marker gene to verify whether the gene editing operation is successful or not and indicate the success rate of the editing operation; the success of editing was further confirmed by extracting the DNA of the developmental malformed individuals and sequencing the mutant EcEy homologous gene by Sanger sequencing (detailed procedure same as example 2, step 2).
The compound eye development defect phenotype and sequence sequencing result caused by editing the EcEy homologous gene can be used as a characteristic that the CRISPR/Cas9 gene editing technology can be applied to the species and used as a mark established by the gene editing platform of the decapod animals.
The present invention has been described in detail through the specific embodiments and experiments, but some modifications or improvements can be made on the basis of the present invention, for example, by using other gene editing technologies, such as TALEN editing technology, cytidine alkaloid editing technology (CBE), adenine alkaloid editing technology (ABE), CRISPR/Cas 12a editing technology, and using EcEy gene as target gene, the application of the novel gene editing technology in shrimps can be developed. Such modifications are also the subject of the present invention and are readily apparent to those of ordinary skill in the art. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.
Drawings
FIG. 1 sequence information of EcEy gene, letters represent the correspondence between the nucleotide sequence and protein sequence of EcEy gene, and shaded parts represent the PAX and HOX domains of EcEy gene.
FIG. 2 alignment of the amino acid sequences of EcEy gene of palaemon carinicauda with other 6 arthropod-homologous genes. (A) And (5) performing multiple sequence alignment. Boxes represent regions of amino acids with sequence similarity higher than 95%. The sequences were from Spodoptera carinica (EcEy, obtained according to the invention), Cypria magna (DpuEy, NCBI accession number EFX75780.1), Triplostegia rubescens (TcEy, ABW23132.1), Drosophila melanogaster (DMey, NP-524628.2), Drosophila pseudocerasus (DsEy, XP-016037330.1), Drosophila melanogaster (DpsEy, XP-001352371.2), Drosophila melanogaster (DvEy, XP-002059744.1), respectively. Asterisks indicate fully conserved amino acid residues. (B) A putative domain of the amino acid sequence of EcEy. The PAX and HOX domains are shown as pentagons, and the boxes represent highly conserved amino acid sequences corresponding to panel (a).
FIG. 3 effect on the process of forming compound eyes of palaemon carinicauda embryos after gRNA injection mixed with Cas9 mRNA. There are mainly four phenotypes: (A) only one eye remains; (B) only one eye is left, and the ectopy of compound eyes is accompanied; (C) the two compound eyes are ectopic; (D) the multi-eyepoint phenomenon. CE, compound eye.
FIG. 4 sequencing results of the EcEy gene of the compound-eye mutated Exopalaemon carinicauda. (A) As a result of sequencing, it was found that a sequence deletion of a plurality of bases was generated at the target sequence site of the gRNA. (B) From the results of sequencing peak maps, it can be seen that a significant set of peaks was generated from the sequences after the target sequence site of the gRNA, confirming the edited results. WT, control group; mutations, mutation group; gRNA, guide RNA; PAM, Cas9 protein recognition sequence.
Sequence listing
<110> oceanographic institute of Chinese academy of sciences
<120> exopalaemon carinicauda compound eye development regulation gene and guide RNA as well as acquisition and application
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 2539
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 1
gagccgttta ctaaagacct ggaggcgaag acaggagctt aaaaaggaat ccgtcgaggt 60
cgcccacatc ttcaccgtca tcattaaggg gcttcggtag cggcggcgag caaggctggg 120
ggggtgggtg cctgcaggct ggccgccgcc gttatgttgt ccccagtaga tgccgggcca 180
catggaccgc atggcccgca tggtcaccac ccgtggggcc ccaccacttt ggacgacatg 240
tcgcataagg acgtgctcga ctactcactg ggaccctcgg gtaagggagc gatacccccc 300
tggtggccaa ggggggaagg ggaggatggt cacagtggag tgaaccaact aggaggtgtc 360
tttgtatccg gcagaccatt gcccgacacc accaggcaga agatcatcga gttggctcat 420
tccggagcca ggccttgcga catctccaga attcttcagg tttccaacgg ttgcgtctcc 480
aaaatactgg gcagatacta cgagacgggt tcgatccgtc cccgagcaat tgggggatcg 540
aaacctcgcg tagccacggc ggaagtggtc gccaagattt cccagttcaa gcgagaatgt 600
ccttcaatct tcgcctggga gatcagagac cgactgctct ccgaaggggt ctgctcctcg 660
gacaacattc caagcgtgtc atccatcaac cgcgtccttc gtaacatcaa ctcgaaagac 720
accaccggca gcggctccgg cgggccagtg ggcgccggcg gacctcctgg tggaggcgcc 780
ggaggaggag caggaggcgg aggaggaggg ggcagcgccc tagctactgc aggagctggt 840
agcgtcggag gaagccagac gatgggcctt ggaggcggga actccagcag cggcagtagt 900
aattcgaaca cgcccaccag caccccgacg caggacccta tgtacgataa actccgccta 960
ctgaatggac agtcttcgtg gcccagatca cagtggtata gcggaccagg caatgacatc 1020
acggccctgc cagtcgattc tccccagtcg gcaccttgtg ttcagcaaga catcgtcaag 1080
aagagtgacg cgggcgagct tagcgaggac agcaatgctg gcagcagcga aaacagtaac 1140
agcgaagacc aagccagact tcacttgaag agaaaacttc agagaaacag aacttctttc 1200
acgaatgacc agattgatag tttggaaaaa gaatttgaga gaactcatta tcctgatgta 1260
tttgctcgcg agagactggc cgcaaagata ggcttgccgg aagctaggat acaggtgtgg 1320
ttcagcaacc gaagagccaa atggcggaga gaagaaaagt taaggaacca gcggagggcg 1380
gccgagggca tcgcaccttc ctcccccacc agaatcatca acaacttcac gccttctccc 1440
atgtacaccc cattgcctcc tccgcccatg tccgtcgctg acacttatgg gtctatggca 1500
ggcggaggcg gattcggcat gggatcgagt gtgggcgtag ggccaggagt gggcgtggtg 1560
gcctccagcc ccaactgcct cccacaacag cagcccacaa ccgtcgtcca ctctgcgatg 1620
accacgggcg tgggactcgg ccgagaccat tctcacaacg cccacaaccc ttacatgagt 1680
cggtcttacg actcgctgta cacgcacgcc cgcgcctcac ccacttgccc gcccatgctt 1740
tatcacccag cttcgcacca cccacaccag aacccacacg ctcctcacga gtacaatacg 1800
cccacgccac ccaattcaca agccggtctt ctctctcccg gtgtgtcagt gcctgtagcg 1860
gtgcccggcc aacaccacga catgaacacc caatactgga ctcgattgca gtaggtgcca 1920
aacgtttatg gcaaatgttc aaggctccac ctggaggaaa ggaaacaaac gtttcgaggt 1980
tataactctg taaacataac aaagtgcttt tgatctctcg tcagaatgca actggtgtta 2040
actgtgtaca aacggacgga gtctaaaatg tggataacga aacagaactg aaaaaaacaa 2100
aagaggtgca gagtgcgtca gttacctttg tgtgactctg ggatcgaagc gaacgctaaa 2160
ggacccatca gtagatttct cattggcatc aatatcaaga aggatgttcc atacgatgtt 2220
ccgcgaatga aggaaacaga aggacgtggt atatacatat tgtgcctcac acggaaaagg 2280
tgaaaggggt tggccttttg ttataagaca aattaacaca cttgtgttta tttcatttta 2340
cttatgcttg tattatatat aaaacgtgct ttatatttcg caaattaaat aaaatgaatg 2400
aaactgaaca caggaataga gaatggatat taacatagct agctacaaca cccatgagtc 2460
acttgatttt tttttttatc ttagaaaaat gtgaacaaaa atctgaaatg ggatttgtgt 2520
agttcagtct ggattttgt 2539
<210> 2
<211> 102
<212> RNA
<213> Artificial Sequence (Artificial Sequence)
<400> 2
cucgaugauc uucugccugg guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60
cguuaucaac uugaaaaagu ggcaccgagu cggugcuuuu uu 102
Claims (7)
1. The compound eye development regulating gene of palaemon carinicauda is characterized in that: the full-length cDNA sequence of the compound eye development regulatory gene EcEy of palaemon carinicauda is the nucleotide sequence shown as SEQ ID No.1 in the sequence list.
2. A method for obtaining the sequence of the compound eye development regulatory gene EcEy of the exopalaemon carinicauda of claim 1 comprises the following steps:
extracting total RNA of the exopalaemon carinicauda, carrying out reverse transcription to obtain cDNA, and designing a primer EcEy-FL-F based on an EcEy gene sequence: GAGCCGTTTACTAAAGACCTG and EcEy-FL-R: ACAAAATCCAGACTGAACTACAC, respectively; and carrying out PCR amplification by using the palaemon carinicauda cDNA as a template to obtain a full-length cDNA sequence of EcEy.
3. A guide RNA (gRNA) gene of the EcEy gene for controlling the compound eye development of palaemon carinicauda according to claim 1, wherein: is the ribonucleotide sequence shown as SEQ ID No.2 in the sequence list.
4. A method for obtaining gRNA of the compound eye development regulatory gene EcEy of palaemon carinicauda according to claim 3, which comprises the following steps:
a) gRNA framework construction
Firstly, a gRNA framework sequence is designed and synthesized according to the structure of the gRNA:
GTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTTT，
then, the frame sequence is connected into a pMD19-T vector to construct a gRNA plasmid pMD 19-gRNA;
b) synthesis of gRNA templates
Selecting an EcEy gene gRNA targeting site by using an online tool CRISPRDirect; designing corresponding gRNA-F according to the target sequence, and simultaneously designing gRNA-R of a reverse primer;
gRNA-F：
TAATACGACTCACTATAGCTCGATGATCTTCTGCCTGGGTTTTAGAGCTAGAAATAGC
gRNA-R：AAAAAAGCACCGACTCGGTGCCA
carrying out PCR amplification on a gRNA template by taking a gRNA plasmid pMD19-gRNA as a template and gRNA-F and gRNA-R primers;
c) in vitro transcription and purification of gRNA
The PCR product of the gRNA was transcribed in vitro using a TranscriptAId T7High Yield Transcription Kit to synthesize the gRNA.
5. Use of the palaemon carinicauda compound eye development regulatory gene EcEy of claim 1 as a marker gene indicating success or failure of the CRISPR/Cas9 gene editing operation in palaemon carinicauda compound eye development.
6. Use according to claim 5, characterized in that:
the in vitro transcribed guide RNA is designed and obtained based on the EcEy sequence of the exopalaemon carinicauda compound eye development regulatory gene, the commercial Cas9mRNA is mixed, after injecting fertilized eggs of exopalaemon carinicauda, the PAX structural domain of the EcEy gene can be specifically identified and cut, the obvious phenotype of compound eye development defect is observed in the early embryonic development stage, and the method can be used for verifying whether the gene editing operation is successful or not and indicating the success rate of the gene editing technology.
7. Use according to claim 6, characterized in that:
the guide RNA is a guide RNA (gRNA) sequence of the compound eye development regulatory gene EcEy of claim 3.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010633645.4A CN111849997B (en) | 2020-07-02 | 2020-07-02 | Macrobrachium carinicauda compound eye development regulation gene and guide RNA as well as acquisition and application |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010633645.4A CN111849997B (en) | 2020-07-02 | 2020-07-02 | Macrobrachium carinicauda compound eye development regulation gene and guide RNA as well as acquisition and application |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN111849997A CN111849997A (en) | 2020-10-30 |
| CN111849997B true CN111849997B (en) | 2022-09-20 |
Family
ID=73152110
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202010633645.4A Active CN111849997B (en) | 2020-07-02 | 2020-07-02 | Macrobrachium carinicauda compound eye development regulation gene and guide RNA as well as acquisition and application |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN111849997B (en) |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1997021835A2 (en) * | 1995-12-12 | 1997-06-19 | Worcester Polytechnic Institute | Dna markers for shrimp selection |
| CN105505945A (en) * | 2016-01-27 | 2016-04-20 | 中国科学院海洋研究所 | Exopalaemon carinicauda eclosion hormone gene EcEH and application thereof |
| CN111118045A (en) * | 2019-12-31 | 2020-05-08 | 河北大学 | Single-base gene editing system based on exopalaemon carinicauda cytidine deaminase and construction and application thereof |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US9049850B2 (en) * | 2008-02-04 | 2015-06-09 | Ben-Gurion University Of The Negev Research And Development Authority | Insulin-like gene of prawns and uses thereof |
-
2020
- 2020-07-02 CN CN202010633645.4A patent/CN111849997B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1997021835A2 (en) * | 1995-12-12 | 1997-06-19 | Worcester Polytechnic Institute | Dna markers for shrimp selection |
| CN105505945A (en) * | 2016-01-27 | 2016-04-20 | 中国科学院海洋研究所 | Exopalaemon carinicauda eclosion hormone gene EcEH and application thereof |
| CN111118045A (en) * | 2019-12-31 | 2020-05-08 | 河北大学 | Single-base gene editing system based on exopalaemon carinicauda cytidine deaminase and construction and application thereof |
Non-Patent Citations (3)
| Title |
|---|
| CHH family peptides from an "eyeless" deep-sea hydrothermal vent shrimp, Rimicaris kairei: characterization and sequence analysis;Ye-Qing Qian等;《Comp Biochem Physiol B Biochem Mol Biol》;20090505;第154卷(第1期);第37-47页 * |
| Sequence 159 from Patent WO2019158911;FU,X.等;《GenBank》;20190914;第1页 * |
| 缺眼蛋白的结构、功能与疾病;靳梦等;《中国生物化学与分子生物学报》;20171231;第33卷(第12期);第1195-1202页 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN111849997A (en) | 2020-10-30 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Dahm et al. | Learning from small fry: the zebrafish as a genetic model organism for aquaculture fish species | |
| CN105647969B (en) | Method for breeding zebra fish with stat1a gene deletion by gene knockout | |
| Ikmi et al. | TALEN and CRISPR/Cas9-mediated genome editing in the early-branching metazoan Nematostella vectensis | |
| JP5509417B2 (en) | Germ cell marker using fish Vasa gene | |
| Kingsley et al. | New genomic tools for molecular studies of evolutionary change in threespine sticklebacks | |
| CN111394445B (en) | Indel marker for sex identification of channa maculata and application thereof | |
| CN107043782A (en) | A kind of gene knockout method and its sgRNA fragments and application | |
| EP4105334A1 (en) | Intermuscular bone-free crucian strain and cultivation method therefor | |
| CN112725462B (en) | SNP molecular marker combination related to body weight and body size of Longsheng chicken and screened based on whole genome sequencing and application | |
| CA3102740C (en) | Sexual maturation in rainbow trout | |
| Li et al. | Genomic analyses of wild argali, domestic sheep, and their hybrids provide insights into chromosome evolution, phenotypic variation, and germplasm innovation | |
| CN113584079A (en) | Establishment of zebra fish heart specific marker strain applied to calcium ion imaging | |
| Ohde et al. | CRISPR/Cas9-based heritable targeted mutagenesis in Thermobia domestica: a genetic tool in an apterygote development model of wing evolution | |
| Jin et al. | Identification of sex-specific DNA markers: Providing molecular evidence for the ZW sex determination system in the redclaw crayfish (Cherax quadricarinatus) | |
| CN112359104A (en) | Development method and application of sex specific marker of mandarin fish | |
| CN111849997B (en) | Macrobrachium carinicauda compound eye development regulation gene and guide RNA as well as acquisition and application | |
| CN112746070A (en) | Primer pair and kit for early sex identification of chicken and application of primer pair and kit | |
| CN109486844B (en) | Specific labeling method of enterotoxigenic escherichia coli | |
| US20200149063A1 (en) | Methods for gender determination and selection of avian embryos in unhatched eggs | |
| CN113718043B (en) | Specific molecular marker and primer for identifying genetic sex of Chaptera multiflora and method and application thereof | |
| CN113684282B (en) | Method for detecting pig hairless character based on EDAR gene expression quantity and application thereof | |
| CN114480601A (en) | Molecular marker, primer, method and application for identifying genetic sex of hippocampus kelloggi | |
| Liu et al. | Genetic linkage and QTL mapping of ictalurid catfish | |
| CN114591962A (en) | Promoter of lgals3bpb gene and application of promoter in construction of zebra fish model | |
| CN116622717B (en) | Male sex differentiation control gene EcIAG and guide RNA of palaemon carinicauda and application |
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 |