CN112553212A - Schizothorax prenanti clock gene, cloning method and application thereof - Google Patents

Schizothorax prenanti clock gene, cloning method and application thereof Download PDF

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CN112553212A
CN112553212A CN202011479536.8A CN202011479536A CN112553212A CN 112553212 A CN112553212 A CN 112553212A CN 202011479536 A CN202011479536 A CN 202011479536A CN 112553212 A CN112553212 A CN 112553212A
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race
clock gene
schizothorax prenanti
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周建设
王万良
张驰
曾本和
孙帅杰
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Institute of Animal Husbandry and Veterinary Medicine of Tibet Academy of Agriculture and Animal Husbandry Sciences
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Abstract

The invention discloses a schizothorax prenanti clock gene, a cloning method and application thereof. Belongs to the technical field of genetic engineering. Including full-length cDNA of the schizothorax prenanti clock gene, coded protein, cloning method and application. The invention clones the full-length cDNA of the schizothorax prenanti clock gene for the first time, analyzes the tissue expression characteristics, finds that the clock gene has expression in liver, spleen, heart, brain, gonad and muscle tissues no matter in female or male schizothorax prenanti, particularly in female and male individuals, the expression level in the gonad is obviously higher than that in other tissues, shows that the clock gene plays a greater role in the breeding process of the schizothorax prenanti relative to other tissues, lays a foundation for theoretically revealing the important role of the celadon schizothorax prenanti clock gene in the seasonal breeding behavior process, and provides help for the resource conservation of the celadon plateau schizothorax prenanti.

Description

Schizothorax prenanti clock gene, cloning method and application thereof
Technical Field
The invention relates to the technical field of genetic engineering, in particular to a schizothorax prenanti clock gene, a cloning method and application thereof.
Background
Schizothorax prenanti belongs to schizothorax prenanti subfamily, is widely distributed in Yalu Tibetan Bujiang, and is evaluated as 'non-critical species' in Chinese vertebrate red directory, but its kindred species such as schizothorax prenanti, Lassa schizothorax prenanti, etc. are evaluated as 'easily-critical species'. The spawning period of schizothorax prenanti cluster is 3-5 months per year, and obvious seasonal reproduction behavior is shown.
The seasonal reproductive cycle involves the annual regeneration of gonadal tissue, mediated primarily by hormonal regulatory pathways. There is increasing evidence that rhythmicity and reproductive behavior are interrelated, that is, gonad development is periodic in fish like other vertebrates, and that in a given region, there is probably a season of regulation each year, which is the seasonal reproductive behavior of fish.
Research has shown that in various organisms, all physiology and behavior are rhythmic activity controlled by biological clock, and the discovery of transcription factors of biological clock genes in a wide range of tissues besides brain indicates that cell rhythmicity may have a wider biological meaning. Schizothorax biddulphi shows obvious rhythmicity on the reproduction behavior.
However, it is unclear which gene plays an important role in seasonal propagation in such a clear propagation rhythm.
Therefore, the problem that the technicians in the field need to solve is to find out the corresponding regulatory genes and the tissue expression mode thereof to provide help for maintaining schizothorax prenanti resources in Qinghai-Tibet plateau.
Disclosure of Invention
In view of the above, the invention provides a schizothorax prenanti clock gene, a cloning method and an application thereof.
In order to achieve the purpose, the invention adopts the following technical scheme:
a full-length cDNA of a schizothorax prenanti clock gene has a nucleotide sequence shown as SEQ ID NO: 1 is shown.
The invention also provides the protein coded by the full-length cDNA of the schizothorax prenanti clock gene, and the amino acid sequence of the protein is shown as SEQ ID NO. 2.
The invention also provides a cloning method of the full-length cDNA of the schizothorax prenanti clock gene, which comprises 3 'RACE amplification and 5' RACE amplification;
3 'RACE amplification is carried out by taking cDNA of 3' adaptor as a reverse transcription primer as a template, and RC595-F1 and 5.3 'outer as primers for carrying out first round PCR amplification, and by taking the obtained first round PCR amplification product as a template, and by taking RC595-F2 and 5.3' inner as primers for carrying out second round PCR amplification, so as to obtain a second round PCR amplification product;
amplifying the second round PCR amplification product by using specific primers RC595-RT1/RC595-RT2 to obtain cDNA, and treating the cDNA with RNase H and TdT;
performing 5 ' RACE amplification by using cDNA treated by RNase H and TdT as a template and using 5 ' adaptor and RC595-R1 as primers to perform first-round PCR amplification, using the obtained first-round PCR amplification product as a template and using 5.3 ' outer and RC595-R2 as primers to perform second-round PCR amplification;
splicing the 3 'RACE amplification product and the 5' RACE amplification product to obtain full-length cDNA of the clock gene of the schizothorax prenanti;
3' adaptor primer sequence: 5'-GCTGTCAACGATACGCTACGTAACGGCATGACAGTGTTTTTTTTTTTTTTTTTT-3', as shown in SEQ ID NO: 3 is shown in the specification;
RC595-F1 primer sequence: 5'-AGTTCCTACAGGCCCCTCGGCTT-3', as shown in SEQ ID NO: 4 is shown in the specification;
5.3' outer primer sequence: 5'-GCTGTCAACGATACGCTACGTAAC-3', as shown in SEQ ID NO: 5 is shown in the specification;
RC595-F2 primer sequence: 5'-CCAGCTGATCCTTCAGGCAGCGTT-3', as shown in SEQ ID NO: 6 is shown in the specification;
5.3' inner primer sequence: 5'-GCTACGTAACGGCATGACAGTG-3', as shown in SEQ ID NO: 7 is shown in the specification;
RC595-RT1 primer sequence: 5'-CTTAAAGTTTCCGATGAACTTGACATA-3', as shown in SEQ ID NO: 8 is shown in the specification;
RC595-RT2 primer sequence: 5'-GGAGCATGTGGCAACAGAACT-3', as shown in SEQ ID NO: 9 is shown in the figure;
5' adaptor primer sequence: 5 '-GCTGTCAACGATACGCTACGTAACGGCATGACAGTGGGNNGGGNNGGGNNG-3' as set forth in SEQ ID NO: 10 is shown in the figure;
wherein N is hypoxanthine deoxynucleotide;
RC595-R1 primer sequence: 5'-GCAGCATGGTGCCCAGTTCCTTG-3', as shown in SEQ ID NO: 11 is shown in the figure;
RC595-R2 primer sequence: 5'-TCGCCTCTTCTTCTCCGACTTGTTCCTA-3', as shown in SEQ ID NO: shown at 12.
Preferably: the synthesis method of cDNA using 3' adaptor as reverse transcription primer comprises the following steps: and extracting total RNA of each sample, detecting by 1.5% agarose gel electrophoresis, and synthesizing cDNA by taking 3' adaptor as a reverse transcription primer.
Preferably: the first PCR reaction systems of 3 'RACE and 5' RACE were 25. mu.l each, including 2 XGC Buffer I12.5. mu.l, 10. mu.M upstream primer 0.5. mu.l, 10. mu.M downstream primer 0.5. mu.l, 2.5mM dNTP 4. mu.l, ddH2O6.3. mu.l, template 1. mu.l and 5U/. mu.l Taq enzyme 0.2. mu.l; the second PCR reaction systems for 3 'RACE and 5' RACE were both 50. mu.l, including 2 XGC Buffer I25. mu.l, 10. mu.M upstream primer 1. mu.l, 10. mu.M downstream primer 1. mu.l, 2.5mM dNTP 8. mu.l, ddH2O12.5. mu.l, template 2. mu.l and 5U/. mu.l Taq enzyme 0.5. mu.l.
Preferably: the reaction procedure for 3' RACE was: 3min at 95 ℃; 30s at 94 ℃, 30s at 58 ℃ and 60s at 72 ℃ for 33 cycles; 7min at 72 ℃; the reaction procedure for the 5' RACE was: 3min at 95 ℃; 30s at 94 ℃, 30s at 68 ℃ and 60s at 72 ℃ for 33 cycles; 7min at 72 ℃.
The invention also provides application of the schizothorax prenanti clock gene in the seasonal reproductive behavior process.
Preferably: the schizothorax prenanti is the schizothorax prenanti in Qinghai-Tibet plateau.
According to the technical scheme, compared with the prior art, the invention discloses and provides the schizothorax heterodentatus clock gene, the cloning method and the application thereof, and the obtained technical effects are that the schizothorax heterodentatus from Yaluzang Bujiang is taken as a test object, the full-length cDNA sequence of the schizothorax heterodentatus clock gene is firstly cloned by adopting RT-PCR and RACE-PCR technologies, and the expression characteristics of the schizothorax heterodentatus clock gene in different tissues of a schizothorax heterodentatus male and female individual are analyzed by RT-qPCR. The result shows that the total length of the schizothorax prenanti clock gene cDNA is 4291bp, 5 'UTR 539bp, 3' UTR 1046bp, CDS 2706bp, 900 amino acids are coded, and the coded amino acids have a typical Poly-Q structure at the tail end; the tissue expression characteristics are analyzed, the expression of the clock gene in liver, spleen, heart, brain, gonad and muscle tissues is found no matter in female or male schizothorax prenanti, particularly, the expression level in the gonad is obviously higher than that in other tissues in female and male individuals, and the result shows that the clock gene plays a greater role in the reproduction process of the schizothorax prenanti compared with other tissues, thereby laying a foundation for theoretically revealing the important role of the clock gene of the schizothorax prenanti in the seasonal reproduction behavior process and providing help for the resource conservation of the schizothorax prenanti.
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In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, it is obvious that the drawings in the following description are only embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to the provided drawings without creative efforts.
FIG. 1 is a schematic diagram of the result of nested PCR electrophoresis provided by the present invention, wherein a is 5' RACE; b is 3' RACE; and c is Marker.
FIG. 2 is a schematic diagram showing the comparison of the amino acids encoded by the clock gene provided by the present invention, wherein the first row to the fourth row are schizothorax prenanti, caput carpi, sinocyclocheilus grahami and zebra fish in sequence.
FIG. 3 is a schematic diagram showing the expression of clock gene in different tissues according to the present invention.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
The embodiment of the invention discloses a schizothorax prenanti clock gene, a cloning method and application thereof.
In the experimental example, the schizothorax bigardni fish used in the experiment was collected from Yalu Tibetan Bugjiang, the body mass was (1.42 + -0.23) kg/tail, and the body length was (36.34 + -0.34) cm/tail. The fry bag is oxygenated and transported back to the laboratory, and is put into a cement pond after cleaning and disinfection for temporary culture overnight, then the male and female select 3 healthy heterodentate schizothorax fish respectively, and the heart, spleen, gonad, liver, muscle and brain tissues are collected and stored in liquid nitrogen for the research of full-length cloning and tissue distribution of the clock gene.
Example 1
RNA extraction and cDNA Synthesis
Total RNA of each sample was extracted according to Trizol extraction kit (Shanghai bioengineering) instructions and detected by 1.5% agarose gel electrophoresis. cDNA was synthesized using the RevertAID Premium Reverse Transcriptase Reverse transcription kit (Thermo Fisher).
Example 2
Cloning of the Clock Gene of Schizothorax prenanti
Designing a primer:
the CDS sequence of clock gene is called by the sequencing result of the full-length transcriptome of schizothorax prenanti (NCBI transcriptome sequencing result: PRJNA527715) to design specific primers, and the sequences of the primers are shown in Table 1.
Table 1: primer name and sequence
Figure BDA0002836994230000051
3 'RACE and 5' RACE:
nested PCR was performed as shown in Table 2 using cDNA with 3' adaptor as the reverse primer as the template, and the reaction procedure is shown in Table 3. The cDNA was inverted with specific primers RC595-RT1/RC595-RT2, and subjected to RNase H and TdT treatment, followed by nested PCR as shown in Table 2 (see operational procedures in invitrogen life technologies analysis manual: 5' RACE System for Rapid Amplification of cDNA Ends, Version 2.0), and the reaction procedure is shown in Table 3.
Table 2: 3 'RACE and 5' RACE reaction system
Figure BDA0002836994230000061
Table 3: PCR reaction procedure
Figure BDA0002836994230000062
Nested PCR results were visualized by 1% agarose gel electrophoresis (see FIG. 1, where FIG. 1a is 653bp and FIG. 1b is 1317bp, containing part of the CDS). After the target fragment is connected and transformed, the clone body is sent to Shanghai bioengineering for sequencing.
The results show that: the 5 'end and the 3' end of the schizothorax prenanti clock gene are obtained by RACE, the full length of the spliced clock gene cDNA is 4291bp, the 5 'UTR is 539bp, the 3' UTR is 1046bp, the CDS is 2706bp, and the full length cDNA sequence of the schizothorax prenanti clock gene is as follows: CGGGGGAATCCTACACTGCCGTCTCAACCAAAACAAGGCGCAAGAAAATCCCCCTCCTGGACGGCAAATAACCGAGGGTTTCCATCACCATTGGATTTGAGTTTTTGTGTTTGGACTTAAAACGCTTAATGACAATCTACAAGAGCCCTTCTGCAACCTTCCCAACTTAAAGAAGATTTAAGAGCAGATTTACAGTGGTTCAGGAGCATTGCTGCTATTACAGAAATAATGACATGAGAGGTGGCAGGTGCTAACATGTGGTGCCTCCAGTCTCACACCCGAATGAAAGCACATACACCCCACAGGGACCCACAGCAGGCTTTTCTACCTTGACCTCAGAGTCCTCTGATTGACCCTGCACACAAAAACATTCATATTCACTCGCACCTTCTGTCTTGCCCACCGATTGTCCTCATCCTACATCCTGTGGCAAGCTCCTCTCTTCCTTCCTTAGAGCAAGCAGGCCTGGAGGTTACAAATGTCTACCTCACGCTTAAAGGTCCTCTCTCTACAACTGCGACCCTGAACACACTACAAAAATGACCTCTAGCATAGACCGGGATGACAGCAGTATCTTTGATGGGTTGATGGAAGAAGATGAGAAGGACAAAGCAAAAAGAGTTTCTAGGAACAAGTCAGAGAAGAAGAGGCGAGACCAGTTCAATGTTCTCATCAAGGAACTGGGCACCATGCTGCCGGGCAACACCCGCAAGATGGACAAGTCTACCATCCTACAGAAGAGCATCGACTTCCTGCGCAAGCACAAAGAAATTGCTGCACAGTCTGAGTCGAGTGAAATCCGTCAGGACTGGAAGCCACCTTTCCTTAGCAATGAGGAGTTCACACAGCTGATGCTGGAGGCACTAGATGGCTTCTTTTTGGCCATCATGACAGACGGAAACATAATCTATGTCTCAGAAAGTGTCACGTCTTTATTAGAACATCTACCTTCTGATCTTGTGGATCAGAATCTGTTGAATTTCCTTCCACTGGGTGAGCACTCAGAGGTGTATAAAGCTCTATCTACACAGATGCTGGAGGGAGAGACCCTCACCCCAGACTACCTAAAAACAAAGAACCAGTTAGAGTTCTGTTGCCACATGCTCCGGGGCACGATCGACCCGAAAGAGCCGCCCGTTTATGAGTATGTCAAGTTCATCGGAAACTTTAAGTCCCTCAACACTGTGCCTAACTCAACACTTAACGGCTTGGAGGGAGTGATCCAGCGATCGCTGAGGCCCATGTTAGAAGGCCGAGTGTGTTTCATAGCAACTGTGAGGCTAGCCAAGCCTCAGTTTATCAAGGAAATGTGTACTGTGGAGGAGCCCAATGAGGAATTCACCTCCAGACACAGTTTAGAGTGGAAATTCCTCTTCTTGGACCACAGGGCACCCCCCATCATAGGCTACCTACCCTTCGAGGTTCTGGGTACTTCAGGGTACGACTACTATCATGTGGATGATCTGGAGACTCTGGCCAAGTGCCATGAACACTTGATGCAGTATGGTAAGGGGAAGTCGTGCTATTACCGTTTCCTGACTAAAGGTCAGCAGTGGATCTGGCTTCAGACTCACTACTATATCACCTACCACCAGTGGAACTCACGGCCGGAGTTCATCGTCTGCACACACACTGTGGTCAGCTATGCTGAAGTGAGGGCTGAACAGCGCAGGGAACTGGGCATTGAGGAATCACCTCCTGAGATCTCAGCAGACAAGTCTCAGGACTCTGGCTCTGAGTCCCAGTTGAACACCTCCAGCCTGAAGGAGGCGCTAGAGCGATTCGATCACAGCCGCACACCCTCCGCCTCCTCACGGAGCTCCCGCAAATCCTCCTCACACACCGCTGTCTCGGACCCCACGTGTATGTATTACATACCAAAGACAGAAGCCACACAGACTAAGCTACAGACAGACCATAGCACACCCCCCCGCCAAACGGTGACAGCCATTGAGATGACATCACAGCGGAGGTCCTCCATTAGCAGTCAGACGATGAGTTCTCAGAACACAGGACAGACGATGGCAGCATCTCTTGTGTCTCAGCCTCAGCAGCTTCAACCACTTCAGCCCAGTGTGCAGCCGGTGCTGCAGTTTTCCACGCAGATGGATGCGATGCATCATCTTAAGGATCAGCTGGAACAGCGCACACGCATGATTGAGGCCAACATCCAGAGACAACAAGATGAACTGCGGCAAATCCAGGGGGAGCTGCAGAGGGTGCAAGGCCAGGGCTTACAGATGTTCTTGCAGCCCGGAGCTAGTGGTGTCAACCTCGGCTCAGTGCAGCTAACGCAGGGATCATCCGTGCAGCCAGGGGGTGCTCTGTCCATGCAGGGGGCGGTGGTGCCGGCAGGGAGCCTGCAAAGTGGCCTGCAATCCACACACACAGCAACACAACACACTGTCACACAGCATCCTCAGCAGGCACCACCACAACAACAGAACCTTCTCAGAGATCAGAGCTCCACTCTAACTCAGCAGTCTCAGAGGTCGTCTCACACATTGCAGTCTCCTCAGGGAGCATTGCCTGCATCTTTGTATAATACCATGATGATCTCTCAGCCGGCGCAGGCCAACGTTGTCCAGATCTCCACTAGCTTGCCCCAGAACAGCACCCCCAGTGGAGCCGCGGTTGCCACCTTCGCACAAGACCGGCAGATACGGTTCCCTGCTGCCCCACAGCTCCTCACTAAGCTAGTAACCGGCCCAATGGCGTGTGGTGCAGTTATGATGCCTACCACCATGTTCATGGGGCAGGTGGTGACTGCGTTTGCCCCGCAGCAGGGCCAGCCTCAAACAATCAGCATTACCCAGCAGCCGTCTGCGCAGACGCCAGAGCAGCAGGCGCAAACGCAGTCACAGACAGCTACAGGAACAGCCCAACAGCAAGGACAGGCCCAGGCTCAGCTTGCCCAGCAGCAAACTCAGTTCCTACAGGCCCCTCGGCTTTTACATGGCAACCAATCTACCCAGCTGATCCTTCAGGCAGCGTTTCCCCTGCAGCAGCAAGGCACCTTTGCAGCCGCAACACAACAACAACAACAACAGCAGCAGCTGCAACAGCAGCAACAACAACTGCAGCAGCAACAGCAACAGCAGCAACAACAGCTGCAGCAGCAGCATCAACAACAACAACAACAGCTGCAACATCAACATCAACAACAGCAGCAGCAGCTGCAGCAGCAGCTGGCAGCTCATCGCTCAGACAGCATGACGGACCGGTCCAAAGTGCCACCTCAGTAGCTGATCACACGTCAGGGACACAACCGAATCTCCACTGAGCAGTCCTGCAGGCTGGCGGGCTTCTGAGGGGGCTGTTTGGAGCTAAACAATAGCAGTATTTTGTACTGTACCCTCTCAGATACCCTTCCACTGCTTATAGGTGAAGGGCCAGACCTAAACGAGTGTCATTTTTTCACCTCGTGCCCTCTCTGACCTATAGGTAGTTGATCAACGGCTGCTTGCACTGAATTCTGGGAGAGGTCCGACCACGAACGCCGAACTTTCTGCTGGCTGGCGAGGAAGTAGCGGTGTCTGGTTTTCCGCAAACTGTCTCCACCACAATAACCGTGTCCCCTTTGTCTCGTATGCCCAGTGTGTGCCGTCATCCCTCTTTCACAGGTTTAATTTATGCAGAACGAGCCGCAGCGAAGGTTTCAAGACTTGAAGTCGCGCAGGACATACGGCGCCTCTCTTTCTCTTGGCCTCTCTTCCTCTTTCCTCCTCGTTTTACTTGTCGCAAGTAATTATGGGTCAGAAAATATTTTTAAGAATGTCACGTGAATTCAACTTTTATTGTGCAGTTCACCTGTAAAATAATGTGTAAATATATGGACAAAAGGAAAGAGAAACTAATATTTTATATGATGCATGAGACGAAAACCGTAGTCTGTTATTTGTAGCTTTGAATATACTTTTTTTCTAATATTCAGAGGAAAAAAGCTAAACTTAATACACATAATGGCTGATCTCTTCACAGACTTCCTTTTTCCAGTGGGATAGGGGTGAGTGTGTGTATGTGCTTGCACACATGTGTTCGTATGTGTGCGTGTCTGCCGATGCTGGACCCACAGATCGTGTCACAGTTGGTTTAAAACGGCCTTCGTGTTTCAGTAAAACTGCTAAAGGAACAGGCAATCTTTAGATGTTTTGCATTGTGTTATACACCTTAGTAATTGCAAAAAAATATAGATATATTTCAGAGAGATGTTTTACATTTGTAAATAGTTTAAAGAGAAGGCTTCAGATTTAGTATATCATGCGATTAGGCAAAAAAAAAAAAAAAAAAA, SEQ ID NO: 1.
the cDNA sequence of the schizothorax prenanti clock gene contains an initiation codon ATG and a 3' end Poly (A) structure, which indicates that the nucleotide information of the cloned schizothorax prenanti clock gene is complete. The schizothorax prenanti cloc k gene encodes 900 amino acids in total (see FIG. 2 for partial alignment, the specific sequence is MTSSIDRDDSSIFDGLMEEDEKDKAKRVSRNKSEKKRRDQFNVLIKELGTMLPGNTRKMDKSTILQKSIDFLRKHKEIAAQSESSEIRQDWKPPFLSNEEFTQLMLEALDGFFLAIMTDGNIIYVSESVTSLLEHLPSDLVDQNLLNFLPLGEHSEVYKALSTQMLEGETLTPDYLKTKNQLEFCCHMLRGTIDPKEPPVYEYVKFIGNFKSLNTVPNSTLNGLEGVIQRSLRPMLEGRVCFIATVRLAKPQFIKEMCTVEEPNEEFTSRHSLEWKFLFLDHRAPPIIGYLPFEVLGTSGYDYYHVDDLETLAKCHEHLMQYGKGKSCYYRFLTKGQQWIWLQTHYYITYHQWNSRPEFIVCTHTVVSYAEVRAEQRRELGIEESPPEISADKSQDSGSESQLNTSSLKEALERFDHSRTPSASSRSSRKSSSHTAVSDPTCMYYIPKTEATQTKLQTDHSTPPRQSVSAIEMTSQRRSSISSQSMSSQNTGQTMAASLVSQPQQLQPLQPSVQPVLQFSTQMDAMHHLKDQLEQRTRMIEANIQRQQDELRQIQGELQRVQGQGLQMFLQPGASGVNLGSVQLTQGSSVQPGGALSMQGAVVPAGSLQSGLQSTHTATQHTVTQHPQQAPPQQQNLLRDQSSTLTQQSQRSSHTLQSPQGALPASLYNTMMISQPAQANVVQISTSLPQNSTPSGAAVATFAQDRQIRFPAAPQLLTKLVTGPMACGAVMMPTTMFMGQVVTAFAPQQGQPQTISITQQPSAQTPEQQAQTQSQTATGTAQQQGQAQAQLAQQQTQFLQAPRLLHGNQSTQLILQAAFPLQQQGTFAAATQQQQQQQLQQQQQQLQQQQQQQQQQLQQQHQQQQQQLQHQHQQQQQQLQQQLAAHRSDSMTDRSKVPPQ, SEQ ID No.2), and the encoded amino acids have a typical Poly-Q structure at the end.
Example 3
cDNA was synthesized from heart, spleen, gonad, liver, muscle and brain tissues collected from each of 3 schizothorax prenanti of 3 sexes by 1.2, specific primers (clock-qPCR-f: 5'-GCCACCTTCGCACAAGACC-3', SEQ ID NO: 13; clock-qPCR-r: 5'-ATAACTGCACCACACGCCAT-3', SEQ ID NO: 14) were designed using Oligo software, and internal reference primers were designed using the schizothorax prenanti β -actin sequence to perform RT-qPCR.
The expression distribution of the clock gene in heart, spleen, gonad, liver, muscle and brain of the schizothorax prenanti is detected by RT-qPCR, and the result is shown in FIG. 3. In female fish, the relative expression level difference of liver and whole brain, spleen and heart clock genes is not significant (p >0.05), the expression level difference between other tissues is significant (p <0.01), and the relative expression level in gonad is the highest and is significantly higher than that of other tissues; secondly, muscle tissue is adopted, and the relative expression quantity of the muscle tissue is obviously lower than that of the gonad and is obviously higher than that of other tissues; the relative expression in the whole brain is significantly lower than that in the gonads and muscles and significantly higher than that in the spleen and heart. In male fish, the relative expression quantity difference of clock genes in liver and heart, liver and whole brain, heart and whole brain, gonad and muscle is not significant (p is greater than 0.05), the expression quantity difference between other tissues is significant (p is less than 0.01), and the relative expression quantity in gonad is the highest and is significantly higher than that of other tissues except muscle; the relative expression of spleen tissues is the lowest, and is significantly lower than that of other tissues.
The embodiments in the present description are described in a progressive manner, each embodiment focuses on differences from other embodiments, and the same and similar parts among the embodiments are referred to each other.
The previous description of the disclosed embodiments is provided to enable any person skilled in the art to make or use the present invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other embodiments without departing from the spirit or scope of the invention. Thus, the present invention is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.
Sequence listing
<110> institute of aquatic science of academy of agriculture and animal husbandry in autonomous region of Tibet
<120> schizothorax heterodentatus clock gene, cloning method and application thereof
<160> 14
<170> SIPOSequenceListing 1.0
<210> 1
<211> 4291
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 1
cgggggaatc ctacactgcc gtctcaacca aaacaaggcg caagaaaatc cccctcctgg 60
acggcaaata accgagggtt tccatcacca ttggatttga gtttttgtgt ttggacttaa 120
aacgcttaat gacaatctac aagagccctt ctgcaacctt cccaacttaa agaagattta 180
agagcagatt tacagtggtt caggagcatt gctgctatta cagaaataat gacatgagag 240
gtggcaggtg ctaacatgtg gtgcctccag tctcacaccc gaatgaaagc acatacaccc 300
cacagggacc cacagcaggc ttttctacct tgacctcaga gtcctctgat tgaccctgca 360
cacaaaaaca ttcatattca ctcgcacctt ctgtcttgcc caccgattgt cctcatccta 420
catcctgtgg caagctcctc tcttccttcc ttagagcaag caggcctgga ggttacaaat 480
gtctacctca cgcttaaagg tcctctctct acaactgcga ccctgaacac actacaaaaa 540
tgacctctag catagaccgg gatgacagca gtatctttga tgggttgatg gaagaagatg 600
agaaggacaa agcaaaaaga gtttctagga acaagtcaga gaagaagagg cgagaccagt 660
tcaatgttct catcaaggaa ctgggcacca tgctgccggg caacacccgc aagatggaca 720
agtctaccat cctacagaag agcatcgact tcctgcgcaa gcacaaagaa attgctgcac 780
agtctgagtc gagtgaaatc cgtcaggact ggaagccacc tttccttagc aatgaggagt 840
tcacacagct gatgctggag gcactagatg gcttcttttt ggccatcatg acagacggaa 900
acataatcta tgtctcagaa agtgtcacgt ctttattaga acatctacct tctgatcttg 960
tggatcagaa tctgttgaat ttccttccac tgggtgagca ctcagaggtg tataaagctc 1020
tatctacaca gatgctggag ggagagaccc tcaccccaga ctacctaaaa acaaagaacc 1080
agttagagtt ctgttgccac atgctccggg gcacgatcga cccgaaagag ccgcccgttt 1140
atgagtatgt caagttcatc ggaaacttta agtccctcaa cactgtgcct aactcaacac 1200
ttaacggctt ggagggagtg atccagcgat cgctgaggcc catgttagaa ggccgagtgt 1260
gtttcatagc aactgtgagg ctagccaagc ctcagtttat caaggaaatg tgtactgtgg 1320
aggagcccaa tgaggaattc acctccagac acagtttaga gtggaaattc ctcttcttgg 1380
accacagggc accccccatc ataggctacc tacccttcga ggttctgggt acttcagggt 1440
acgactacta tcatgtggat gatctggaga ctctggccaa gtgccatgaa cacttgatgc 1500
agtatggtaa ggggaagtcg tgctattacc gtttcctgac taaaggtcag cagtggatct 1560
ggcttcagac tcactactat atcacctacc accagtggaa ctcacggccg gagttcatcg 1620
tctgcacaca cactgtggtc agctatgctg aagtgagggc tgaacagcgc agggaactgg 1680
gcattgagga atcacctcct gagatctcag cagacaagtc tcaggactct ggctctgagt 1740
cccagttgaa cacctccagc ctgaaggagg cgctagagcg attcgatcac agccgcacac 1800
cctccgcctc ctcacggagc tcccgcaaat cctcctcaca caccgctgtc tcggacccca 1860
cgtgtatgta ttacatacca aagacagaag ccacacagac taagctacag acagaccata 1920
gcacaccccc ccgccaaacg gtgacagcca ttgagatgac atcacagcgg aggtcctcca 1980
ttagcagtca gacgatgagt tctcagaaca caggacagac gatggcagca tctcttgtgt 2040
ctcagcctca gcagcttcaa ccacttcagc ccagtgtgca gccggtgctg cagttttcca 2100
cgcagatgga tgcgatgcat catcttaagg atcagctgga acagcgcaca cgcatgattg 2160
aggccaacat ccagagacaa caagatgaac tgcggcaaat ccagggggag ctgcagaggg 2220
tgcaaggcca gggcttacag atgttcttgc agcccggagc tagtggtgtc aacctcggct 2280
cagtgcagct aacgcaggga tcatccgtgc agccaggggg tgctctgtcc atgcaggggg 2340
cggtggtgcc ggcagggagc ctgcaaagtg gcctgcaatc cacacacaca gcaacacaac 2400
acactgtcac acagcatcct cagcaggcac caccacaaca acagaacctt ctcagagatc 2460
agagctccac tctaactcag cagtctcaga ggtcgtctca cacattgcag tctcctcagg 2520
gagcattgcc tgcatctttg tataatacca tgatgatctc tcagccggcg caggccaacg 2580
ttgtccagat ctccactagc ttgccccaga acagcacccc cagtggagcc gcggttgcca 2640
ccttcgcaca agaccggcag atacggttcc ctgctgcccc acagctcctc actaagctag 2700
taaccggccc aatggcgtgt ggtgcagtta tgatgcctac caccatgttc atggggcagg 2760
tggtgactgc gtttgccccg cagcagggcc agcctcaaac aatcagcatt acccagcagc 2820
cgtctgcgca gacgccagag cagcaggcgc aaacgcagtc acagacagct acaggaacag 2880
cccaacagca aggacaggcc caggctcagc ttgcccagca gcaaactcag ttcctacagg 2940
cccctcggct tttacatggc aaccaatcta cccagctgat ccttcaggca gcgtttcccc 3000
tgcagcagca aggcaccttt gcagccgcaa cacaacaaca acaacaacag cagcagctgc 3060
aacagcagca acaacaactg cagcagcaac agcaacagca gcaacaacag ctgcagcagc 3120
agcatcaaca acaacaacaa cagctgcaac atcaacatca acaacagcag cagcagctgc 3180
agcagcagct ggcagctcat cgctcagaca gcatgacgga ccggtccaaa gtgccacctc 3240
agtagctgat cacacgtcag ggacacaacc gaatctccac tgagcagtcc tgcaggctgg 3300
cgggcttctg agggggctgt ttggagctaa acaatagcag tattttgtac tgtaccctct 3360
cagataccct tccactgctt ataggtgaag ggccagacct aaacgagtgt cattttttca 3420
cctcgtgccc tctctgacct ataggtagtt gatcaacggc tgcttgcact gaattctggg 3480
agaggtccga ccacgaacgc cgaactttct gctggctggc gaggaagtag cggtgtctgg 3540
ttttccgcaa actgtctcca ccacaataac cgtgtcccct ttgtctcgta tgcccagtgt 3600
gtgccgtcat ccctctttca caggtttaat ttatgcagaa cgagccgcag cgaaggtttc 3660
aagacttgaa gtcgcgcagg acatacggcg cctctctttc tcttggcctc tcttcctctt 3720
tcctcctcgt tttacttgtc gcaagtaatt atgggtcaga aaatattttt aagaatgtca 3780
cgtgaattca acttttattg tgcagttcac ctgtaaaata atgtgtaaat atatggacaa 3840
aaggaaagag aaactaatat tttatatgat gcatgagacg aaaaccgtag tctgttattt 3900
gtagctttga atatactttt tttctaatat tcagaggaaa aaagctaaac ttaatacaca 3960
taatggctga tctcttcaca gacttccttt ttccagtggg ataggggtga gtgtgtgtat 4020
gtgcttgcac acatgtgttc gtatgtgtgc gtgtctgccg atgctggacc cacagatcgt 4080
gtcacagttg gtttaaaacg gccttcgtgt ttcagtaaaa ctgctaaagg aacaggcaat 4140
ctttagatgt tttgcattgt gttatacacc ttagtaattg caaaaaaata tagatatatt 4200
tcagagagat gttttacatt tgtaaatagt ttaaagagaa ggcttcagat ttagtatatc 4260
atgcgattag gcaaaaaaaa aaaaaaaaaa a 4291
<210> 2
<211> 900
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 2
Met Thr Ser Ser Ile Asp Arg Asp Asp Ser Ser Ile Phe Asp Gly Leu
1 5 10 15
Met Glu Glu Asp Glu Lys Asp Lys Ala Lys Arg Val Ser Arg Asn Lys
20 25 30
Ser Glu Lys Lys Arg Arg Asp Gln Phe Asn Val Leu Ile Lys Glu Leu
35 40 45
Gly Thr Met Leu Pro Gly Asn Thr Arg Lys Met Asp Lys Ser Thr Ile
50 55 60
Leu Gln Lys Ser Ile Asp Phe Leu Arg Lys His Lys Glu Ile Ala Ala
65 70 75 80
Gln Ser Glu Ser Ser Glu Ile Arg Gln Asp Trp Lys Pro Pro Phe Leu
85 90 95
Ser Asn Glu Glu Phe Thr Gln Leu Met Leu Glu Ala Leu Asp Gly Phe
100 105 110
Phe Leu Ala Ile Met Thr Asp Gly Asn Ile Ile Tyr Val Ser Glu Ser
115 120 125
Val Thr Ser Leu Leu Glu His Leu Pro Ser Asp Leu Val Asp Gln Asn
130 135 140
Leu Leu Asn Phe Leu Pro Leu Gly Glu His Ser Glu Val Tyr Lys Ala
145 150 155 160
Leu Ser Thr Gln Met Leu Glu Gly Glu Thr Leu Thr Pro Asp Tyr Leu
165 170 175
Lys Thr Lys Asn Gln Leu Glu Phe Cys Cys His Met Leu Arg Gly Thr
180 185 190
Ile Asp Pro Lys Glu Pro Pro Val Tyr Glu Tyr Val Lys Phe Ile Gly
195 200 205
Asn Phe Lys Ser Leu Asn Thr Val Pro Asn Ser Thr Leu Asn Gly Leu
210 215 220
Glu Gly Val Ile Gln Arg Ser Leu Arg Pro Met Leu Glu Gly Arg Val
225 230 235 240
Cys Phe Ile Ala Thr Val Arg Leu Ala Lys Pro Gln Phe Ile Lys Glu
245 250 255
Met Cys Thr Val Glu Glu Pro Asn Glu Glu Phe Thr Ser Arg His Ser
260 265 270
Leu Glu Trp Lys Phe Leu Phe Leu Asp His Arg Ala Pro Pro Ile Ile
275 280 285
Gly Tyr Leu Pro Phe Glu Val Leu Gly Thr Ser Gly Tyr Asp Tyr Tyr
290 295 300
His Val Asp Asp Leu Glu Thr Leu Ala Lys Cys His Glu His Leu Met
305 310 315 320
Gln Tyr Gly Lys Gly Lys Ser Cys Tyr Tyr Arg Phe Leu Thr Lys Gly
325 330 335
Gln Gln Trp Ile Trp Leu Gln Thr His Tyr Tyr Ile Thr Tyr His Gln
340 345 350
Trp Asn Ser Arg Pro Glu Phe Ile Val Cys Thr His Thr Val Val Ser
355 360 365
Tyr Ala Glu Val Arg Ala Glu Gln Arg Arg Glu Leu Gly Ile Glu Glu
370 375 380
Ser Pro Pro Glu Ile Ser Ala Asp Lys Ser Gln Asp Ser Gly Ser Glu
385 390 395 400
Ser Gln Leu Asn Thr Ser Ser Leu Lys Glu Ala Leu Glu Arg Phe Asp
405 410 415
His Ser Arg Thr Pro Ser Ala Ser Ser Arg Ser Ser Arg Lys Ser Ser
420 425 430
Ser His Thr Ala Val Ser Asp Pro Thr Cys Met Tyr Tyr Ile Pro Lys
435 440 445
Thr Glu Ala Thr Gln Thr Lys Leu Gln Thr Asp His Ser Thr Pro Pro
450 455 460
Arg Gln Ser Val Ser Ala Ile Glu Met Thr Ser Gln Arg Arg Ser Ser
465 470 475 480
Ile Ser Ser Gln Ser Met Ser Ser Gln Asn Thr Gly Gln Thr Met Ala
485 490 495
Ala Ser Leu Val Ser Gln Pro Gln Gln Leu Gln Pro Leu Gln Pro Ser
500 505 510
Val Gln Pro Val Leu Gln Phe Ser Thr Gln Met Asp Ala Met His His
515 520 525
Leu Lys Asp Gln Leu Glu Gln Arg Thr Arg Met Ile Glu Ala Asn Ile
530 535 540
Gln Arg Gln Gln Asp Glu Leu Arg Gln Ile Gln Gly Glu Leu Gln Arg
545 550 555 560
Val Gln Gly Gln Gly Leu Gln Met Phe Leu Gln Pro Gly Ala Ser Gly
565 570 575
Val Asn Leu Gly Ser Val Gln Leu Thr Gln Gly Ser Ser Val Gln Pro
580 585 590
Gly Gly Ala Leu Ser Met Gln Gly Ala Val Val Pro Ala Gly Ser Leu
595 600 605
Gln Ser Gly Leu Gln Ser Thr His Thr Ala Thr Gln His Thr Val Thr
610 615 620
Gln His Pro Gln Gln Ala Pro Pro Gln Gln Gln Asn Leu Leu Arg Asp
625 630 635 640
Gln Ser Ser Thr Leu Thr Gln Gln Ser Gln Arg Ser Ser His Thr Leu
645 650 655
Gln Ser Pro Gln Gly Ala Leu Pro Ala Ser Leu Tyr Asn Thr Met Met
660 665 670
Ile Ser Gln Pro Ala Gln Ala Asn Val Val Gln Ile Ser Thr Ser Leu
675 680 685
Pro Gln Asn Ser Thr Pro Ser Gly Ala Ala Val Ala Thr Phe Ala Gln
690 695 700
Asp Arg Gln Ile Arg Phe Pro Ala Ala Pro Gln Leu Leu Thr Lys Leu
705 710 715 720
Val Thr Gly Pro Met Ala Cys Gly Ala Val Met Met Pro Thr Thr Met
725 730 735
Phe Met Gly Gln Val Val Thr Ala Phe Ala Pro Gln Gln Gly Gln Pro
740 745 750
Gln Thr Ile Ser Ile Thr Gln Gln Pro Ser Ala Gln Thr Pro Glu Gln
755 760 765
Gln Ala Gln Thr Gln Ser Gln Thr Ala Thr Gly Thr Ala Gln Gln Gln
770 775 780
Gly Gln Ala Gln Ala Gln Leu Ala Gln Gln Gln Thr Gln Phe Leu Gln
785 790 795 800
Ala Pro Arg Leu Leu His Gly Asn Gln Ser Thr Gln Leu Ile Leu Gln
805 810 815
Ala Ala Phe Pro Leu Gln Gln Gln Gly Thr Phe Ala Ala Ala Thr Gln
820 825 830
Gln Gln Gln Gln Gln Gln Leu Gln Gln Gln Gln Gln Gln Leu Gln Gln
835 840 845
Gln Gln Gln Gln Gln Gln Gln Gln Leu Gln Gln Gln His Gln Gln Gln
850 855 860
Gln Gln Gln Leu Gln His Gln His Gln Gln Gln Gln Gln Gln Leu Gln
865 870 875 880
Gln Gln Leu Ala Ala His Arg Ser Asp Ser Met Thr Asp Arg Ser Lys
885 890 895
Val Pro Pro Gln
900
<210> 3
<211> 54
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 3
gctgtcaacg atacgctacg taacggcatg acagtgtttt tttttttttt tttt 54
<210> 4
<211> 23
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 4
agttcctaca ggcccctcgg ctt 23
<210> 5
<211> 24
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 5
gctgtcaacg atacgctacg taac 24
<210> 6
<211> 24
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 6
ccagctgatc cttcaggcag cgtt 24
<210> 7
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 7
gctacgtaac ggcatgacag tg 22
<210> 8
<211> 27
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 8
cttaaagttt ccgatgaact tgacata 27
<210> 9
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 9
ggagcatgtg gcaacagaac t 21
<210> 10
<211> 51
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221> misc_feature
<222> (39)..(40)
<223> n is hypoxanthine deoxynucleotide
<220>
<221> misc_feature
<222> (44)..(45)
<223> n is hypoxanthine deoxynucleotide
<220>
<221> misc_feature
<222> (49)..(50)
<223> n is hypoxanthine deoxynucleotide
<400> 10
gctgtcaacg atacgctacg taacggcatg acagtgggnn gggnngggnn g 51
<210> 11
<211> 23
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 11
gcagcatggt gcccagttcc ttg 23
<210> 12
<211> 28
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 12
tcgcctcttc ttctccgact tgttccta 28
<210> 13
<211> 19
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 13
gccaccttcg cacaagacc 19
<210> 14
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 14
ataactgcac cacacgccat 20

Claims (8)

1. A full-length cDNA of a schizothorax prenanti clock gene is characterized in that the nucleotide sequence is shown as SEQ ID NO: 1 is shown.
2. The protein encoded by the full-length cDNA of the schizothorax prenanti clock gene of claim 1, wherein the amino acid sequence thereof is represented by SEQ ID NO. 2.
3. The method for cloning the full-length cDNA of the clock gene of Schizothorax prenanti of claim 1, comprising 3 'RACE amplification and 5' RACE amplification;
the 3 'RACE amplification uses cDNA of 3' adaptor as a reverse transcription primer as a template, uses RC595-F1 and 5.3 'outer as primers to carry out first round PCR amplification, uses the obtained first round PCR amplification product as a template, and uses RC595-F2 and 5.3' inner as primers to carry out second round PCR amplification, so as to obtain a second round PCR amplification product;
amplifying the second round PCR amplification product by using specific primers RC595-RT1/RC595-RT2 to obtain cDNA, and treating the cDNA with RNase H and TdT;
the 5 ' RACE amplification uses cDNA treated by RNase H and TdT as a template, 5 ' adaptor and RC595-R1 as primers to carry out first round PCR amplification, uses the obtained first round PCR amplification product as a template, and uses 5.3 ' outer and RC595-R2 as primers to carry out second round PCR amplification;
splicing the 3 'RACE amplification product and the 5' RACE amplification product to obtain full-length cDNA of the clock gene of the schizothorax prenanti;
the 3' adaptor primer sequence: 5'-GCTGTCAACGATACGCTACGTAACGGCATGACAGTGTTTTTTTTTTTTTTTTTT-3', as shown in SEQ ID NO: 3 is shown in the specification;
the RC595-F1 primer sequence: 5'-AGTTCCTACAGGCCCCTCGGCTT-3', as shown in SEQ ID NO: 4 is shown in the specification;
the 5.3' outer primer sequence: 5'-GCTGTCAACGATACGCTACGTAAC-3', as shown in SEQ ID NO: 5 is shown in the specification;
the RC595-F2 primer sequence: 5'-CCAGCTGATCCTTCAGGCAGCGTT-3', as shown in SEQ ID NO: 6 is shown in the specification;
the 5.3' inner primer sequence: 5'-GCTACGTAACGGCATGACAGTG-3', as shown in SEQ ID NO: 7 is shown in the specification;
the RC595-RT1 primer sequence: 5'-CTTAAAGTTTCCGATGAACTTGACATA-3', as shown in SEQ ID NO: 8 is shown in the specification;
the RC595-RT2 primer sequence: 5'-GGAGCATGTGGCAACAGAACT-3', as shown in SEQ ID NO: 9 is shown in the figure;
the 5' adaptor primer sequence: 5 '-GCTGTCAACGATACGCTACGTAACGGCATGACAGTGGGNNGGGNNGGGNNG-3' as set forth in SEQ ID NO: 10 is shown in the figure;
wherein N is hypoxanthine deoxynucleotide;
the RC595-R1 primer sequence: 5'-GCAGCATGGTGCCCAGTTCCTTG-3', as shown in SEQ ID NO: 11 is shown in the figure;
the RC595-R2 primer sequence: 5'-TCGCCTCTTCTTCTCCGACTTGTTCCTA-3', as shown in SEQ ID NO: shown at 12.
4. The cloning method according to claim 3, wherein the cDNA using 3' adaptor as reverse transcription primer is synthesized by the following steps: and extracting total RNA of each sample, detecting by 1.5% agarose gel electrophoresis, and synthesizing cDNA by taking 3' adaptor as a reverse transcription primer.
5. The cloning method of claim 3, wherein the 3 'RACE and 5' RACE first PCR reaction systems are each 25 μ l, and comprise 2 XGC Buffer I12.5 μ l, 10 μ M upstream primer 0.5 μ l, 10 μ M downstream primer 0.5 μ l, 2.5mM dNTP 4 μ l, ddH2O6.3. mu.l, template 1. mu.l and 5U/. mu.l Taq enzyme 0.2. mu.l; the second round PCR reaction systems of the 3 'RACE and the 5' RACE are both 50 ul, and comprise 2X GC Buffer I25 ul, 10 uM upstream primer 1 ul, 10 uM downstream primer 1 ul, 2.5mM dNTP 8 ul, ddH2O12.5. mu.l, template 2. mu.l and 5U/. mu.l Taq enzyme 0.5. mu.l.
6. The cloning method of claim 3, wherein said 3' RACE is performed by the reaction program of: 3min at 95 ℃; 30s at 94 ℃, 30s at 58 ℃ and 60s at 72 ℃ for 33 cycles; 7min at 72 ℃; the reaction procedure for the 5' RACE was: 3min at 95 ℃; 30s at 94 ℃, 30s at 68 ℃ and 60s at 72 ℃ for 33 cycles; 7min at 72 ℃.
7. Use of the schizothorax prenanti clock gene according to claim 1 during seasonal reproductive performance.
8. The use of claim 7, wherein: the schizothorax prenanti is the schizothorax prenanti in Qinghai-Tibet plateau.
CN202011479536.8A 2020-12-15 2020-12-15 Schizothorax prenanti clock gene, cloning method and application thereof Pending CN112553212A (en)

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Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110511938A (en) * 2019-09-05 2019-11-29 水利部中国科学院水工程生态研究所 A kind of short hairs schizothoracin heat stress proteins HSP70 gene, detection method and its application

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110511938A (en) * 2019-09-05 2019-11-29 水利部中国科学院水工程生态研究所 A kind of short hairs schizothoracin heat stress proteins HSP70 gene, detection method and its application

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
NCBI: "XM_016294269.1", 《NCBI》 *
周建设等: "异齿裂腹鱼(Schizothorax oconnori)CLOCK蛋白结构和功能的生物信息学分析", 《西藏农业科技》 *
商振达等: "异齿裂腹鱼CCK基因的cDNA克隆及其摄食功能", 《中国水产科学》 *

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