Goldfish hypoxia inducible factor HIF-1 α gene, cloning method and application
阅读说明：本技术 一种金鱼缺氧诱导因子HIF-1α基因、克隆方法及应用 (Goldfish hypoxia inducible factor HIF-1 α gene, cloning method and application ) 是由 李春艳 姜巨峰 刘克明 孟一耕 蔡超 刘肖莲 白晓慧 于 2019-10-30 设计创作，主要内容包括：本发明涉及一种金鱼缺氧诱导因子HIF-1α基因,所述金鱼缺氧诱导因子HIF-1α基因的核苷酸序列为SEQ ID NO.1。本发明首次公开了金鱼缺氧诱导因子HIF-1α基因的核苷酸序列和氨基酸序列,并通过qPCR技术验证该基因在金鱼低氧胁迫中所发挥的作用,利用实时荧光定量qPCR对金鱼缺氧诱导因子HIF-1α在肝脏、脑、心脏和鳃组织中的表达模式进行分析,结果表明金鱼在低氧胁迫条件下,四种组织中的HIF-1α基因表达模式不同,可以为鱼类低氧适应机制、低氧信号传导途径及培育耐低氧鱼类新品种提供基础数据和参考。(The invention relates to a goldfish hypoxia inducible factor HIF-1 α gene, wherein the nucleotide sequence of the goldfish hypoxia inducible factor HIF-1 α gene is SEQ ID NO. 1. the invention discloses the nucleotide sequence and amino acid sequence of the goldfish hypoxia inducible factor HIF-1 α gene for the first time, verifies the function of the gene in the low oxygen stress of goldfish by qPCR technology, analyzes the expression modes of the goldfish hypoxia inducible factor HIF-1 α in liver, brain, heart and gill tissues by using real-time fluorescent quantitative qPCR, and shows that the HIF-1 α gene expression modes in the four tissues are different under the low oxygen stress condition of the goldfish, and can provide basic data and reference for a fish low oxygen adaptation mechanism, a low oxygen signal conduction path and the cultivation of a new species of low oxygen resistant fish.)
1. A goldfish hypoxia inducible factor HIF-1 α gene is characterized in that the nucleotide sequence of the goldfish hypoxia inducible factor HIF-1 α gene is SEQ ID NO. 1.
2. The amino acid encoded by the goldfish hypoxia inducible factor HIF-1 α gene of claim 1, wherein the sequence of said amino acid is SEQ ID No. 2.
3. The method for cloning the gene of Goldfish hypoxia inducible factor HIF-1 α according to claim 1, comprising the steps of:
step 1: extracting total RNA of liver, gill, brain and heart tissues of the goldfish, and performing reverse transcription to obtain cDNA;
step 2, downloading mRNA sequence and protein sequence of carp hypoxia inducible factor HIF-1 α gene from NCBI database, comparing with goldfish transcriptome sequence, wherein the goldfish transcriptome sequence is SEQ ID NO.3, calling goldfish HIF-1 α gene sequence, designing 3 to verify the called transcriptome sequence by using primer, carrying out PCR amplification by using goldfish cDNA as template, recovering glue, purifying and sequencing PCR product, and splicing sequence to obtain goldfish HIF-1 α gene intermediate segment;
step 3, obtaining the 5' end sequence of the HIF-1 α gene;
step 4, obtaining the 3' end sequence of the HIF-1 α gene;
splicing the sequence fragments obtained in the steps 2-4 to obtain the full-length sequence of the HIF-1 α gene;
and 6, predicting the ORF of the open reading frame of the HIF-1 α gene by using FGENESH and ORF Finder software according to the full-length sequence of the gene.
4. The method for cloning the HIF-1 α gene of Goldfish hypoxia inducible factor in the claim 3, wherein the 3 pairs of primer sequences in the step 2 are SEQ ID NO.4, SEQ ID NO.5, SEQ ID NO.6, SEQ ID NO.7, SEQ ID NO.8 and SEQ ID NO. 9.
5. The method for cloning the HIF-1 α gene of Goldfish hypoxia inducible factor according to claim 3, wherein the primers of SEQ ID NO.10, SEQ ID NO.11 and SEQ ID NO.12 are used in step 3.
6. The method for cloning the HIF-1 α gene of the goldfish hypoxia inducible factor as claimed in claim 3, wherein the primers of SEQ ID No.13 and SEQ ID No.14 are used in step 4.
7. The method for cloning the gene of Goldfish hypoxia inducible factor HIF-1 α according to any of claims 3 to 6, wherein the open reading frame in step 6 has a length of 2343bp and encodes 780 amino acids.
8. The use of the goldfish hypoxia inducible factor HIF-1 α gene according to claim 1 for the regulation in a hypoxic environment.
9. The use of the amino acid encoded by the goldfish hypoxia inducible factor HIF-1 α gene according to claim 1 for regulating hypoxia.
Technical Field
The invention belongs to the technical field of molecular biology, and particularly relates to a goldfish hypoxia inducible factor HIF-1 α gene, a cloning method and application.
Background
Oxygen is essential to most organisms. Generally, human beings and other mammals have poor oxygen tolerance, and long-term hypoxia can cause body tissue failure and even death. However, marine invertebrates and fish have a strong resistance to hypoxia, and can survive a period of hypoxia or even anaerobic conditions. In order to cope with an anoxic environment, these organisms can regulate physiological processes such as glucose transport, glycolysis, and red blood cell production by controlling the expression of related genes, thereby enhancing the body's tolerance to hypoxia.
HIF-1 α is the active subunit of HIF-1, under the condition of hypoxia, HIF-1 α regulates the organism by activating erythropoietin, glucose transport protein and glycolytic enzyme, thus increasing the exchange of oxygen or promoting metabolism to adapt to the hypoxic environment.
The goldfish (Carassius auratus) belongs to one of crucian carps in the phylum of chordata, class of teleostomia and family of Cyprinidae, has extremely strong hypoxia tolerance and is an ideal model organism for hypoxia tolerance research.
Through searching, no patent publication related to the present patent application has been found.
Disclosure of Invention
The invention aims to overcome the defects in the prior art, provides a goldfish hypoxia inducible factor HIF-1 α gene, a cloning method and application thereof, and can provide basic data and reference for a fish hypoxia adaptation mechanism, a hypoxia signal transduction path and the cultivation of a new hypoxia-resistant fish variety.
The technical scheme adopted by the invention for solving the technical problems is as follows:
a goldfish hypoxia inducible factor HIF-1 α gene, the nucleotide sequence of the goldfish hypoxia inducible factor HIF-1 α gene is SEQ ID NO. 1.
The amino acid sequence of the amino acid coded by the goldfish hypoxia inducible factor HIF-1 α gene is SEQ ID NO. 2.
The method for cloning the goldfish hypoxia inducible factor HIF-1 α gene comprises the following steps:
step 1: extracting total RNA of liver, gill, brain and heart tissues of the goldfish, and performing reverse transcription to obtain cDNA;
step 4, obtaining the 3' end sequence of the HIF-1 α gene;
splicing the sequence fragments obtained in the steps 2-4 to obtain the full-length sequence of the HIF-1 α gene;
and 6, predicting the ORF of the open reading frame of the HIF-1 α gene by using FGENESH and ORF Finder software according to the full-length sequence of the gene.
And the 3 pairs of primer sequences in the
In addition, primers are required in the
In addition, primers are required in the step 4, and the sequences of the primers are shown in SEQ ID NO.13 and SEQ ID NO. 14.
Furthermore, the open reading frame in step 6 is 2343bp long and encodes 780 amino acids.
The goldfish hypoxia inducible factor HIF-1 α gene is applied to the regulation under the low oxygen environment.
The amino acid coded by the goldfish hypoxia inducible factor HIF-1 α gene is applied to the regulation and control under the low oxygen environment.
The invention has the advantages and positive effects that:
1. the invention discloses the nucleotide sequence and amino acid sequence of goldfish hypoxia inducible factor HIF-1 α gene for the first time, and verifies the function of the gene in goldfish hypoxia stress by qPCR technology, and analyzes the expression modes of the goldfish hypoxia inducible factor HIF-1 α in liver, brain, heart and gill tissues by using real-time fluorescence quantitative qPCR, and the results show that the HIF-1 α gene expression modes in the four tissues are different under the condition of hypoxia stress of goldfish, and can provide basic data and reference for a fish hypoxia adaptation mechanism, a hypoxia signal conduction path and the cultivation of a new fish tolerant variety.
2. The method comprises the steps of extracting total RNA from liver, gill, brain and heart tissues of goldfish, carrying out reverse transcription to obtain cDNA, using a sequence comparison method to call partial sequence of goldfish HIF-1 α gene from the existing transcriptome data, and cloning by using an RACE method to obtain the full-length gene, wherein the open reading frame of the gene is 2343bp long and encodes 780 amino acids.
Drawings
FIG. 1 is a diagram of an intermediate amplified fragment of the HIF-1 α gene of Goldfish according to the present invention;
FIG. 2 is a diagram of 5' RACE amplification in the present invention;
FIG. 3 is a 3' RACE amplification plot of the present invention;
FIG. 4 is an expression diagram of a goldfish hypoxia stress test gene HIF-1 α (also called a differential expression diagram of goldfish HIF-1 α gene in different tissues under different dissolved oxygen conditions) in the invention, wherein capital letters in the diagram are test groups, C is a control group, A is a hypoxia stress group, R is a reoxygenation group, and lowercase letters represent the difference significance of the same tissue under different test conditions.
Detailed Description
The following detailed description of the embodiments of the present invention is provided for the purpose of illustration and not limitation, and should not be construed as limiting the scope of the invention.
The raw materials used in the invention are conventional commercial products unless otherwise specified; the methods used in the present invention are conventional in the art unless otherwise specified.
A goldfish hypoxia inducible factor HIF-1 α gene, the nucleotide sequence of the goldfish hypoxia inducible factor HIF-1 α gene is SEQ ID NO. 1.
The amino acid sequence of the amino acid coded by the goldfish hypoxia inducible factor HIF-1 α gene is SEQ ID NO. 2.
The method for cloning the goldfish hypoxia inducible factor HIF-1 α gene comprises the following steps:
step 1: extracting total RNA of liver, gill, brain and heart tissues of the goldfish, and performing reverse transcription to obtain cDNA;
step 4, obtaining the 3' end sequence of the HIF-1 α gene;
splicing the sequence fragments obtained in the steps 2-4 to obtain the full-length sequence of the HIF-1 α gene;
and 6, predicting the ORF of the open reading frame of the HIF-1 α gene by using FGENESH and ORF Finder software according to the full-length sequence of the gene.
Preferably, the 3 pairs of primers in
Preferably, primers are used in
Preferably, the primer sequence shown in SEQ ID NO.13 and SEQ ID NO.14 is used in the step 4.
Preferably, the open reading frame in the step 6 is 2343bp in length and encodes 780 amino acids.
The goldfish hypoxia inducible factor HIF-1 α gene can be applied to the regulation under the low oxygen environment.
The amino acid coded by the goldfish hypoxia inducible factor HIF-1 α gene can be applied to the regulation under the low oxygen environment.
The preparation and detection are as follows:
first, obtaining gene sequence of goldfish hypoxia inducible factor HIF-1 α gene coding region
Step 1: synthesis of Goldfish cDNA
Dissecting and separating liver, gill, brain and heart tissues of goldfish, and freezing and storing in a refrigerator at-80 deg.C. After grinding the tissue with liquid nitrogen, total RNA was extracted using TRIZOLReagent (Introvigen) kit, and the specific procedures were performed according to the actual and instructions. The concentration is detected by using an ultramicro ultraviolet spectrophotometer.
The First Strand of cDNA was synthesized using total RNA as template and reverse transcription kit, reverse Aid First Strand cDNA Synthesis kit (Fermentas corporation), and stored at-20 ℃ for further use, with the specific procedures being referred to the kit instructions.
Obtaining mRNA sequence (GenBank: EU144225.1) and protein sequence (GenBank: ABV59209.1) of carp HIF-1 α gene from NCBI, comparing the mRNA sequence and protein sequence of carp HIF-1 α gene with goldfish transcription group data (SEQID NO.3), adjusting mRNA sequence of goldfish HIF-1 α gene, firstly carrying out sequence verification on the adjusted goldfish mRNA sequence, carrying out verification in three sections, verifying primers (B3151, B3152, B3153) to be shown in Table 1, designing the primers by using Primer Premier5.0 software, synthesizing by bio-engineering (Shanghai) GmbH, detecting PCR product by using 1.0% agarose gel electrophoresis, and obtaining PCR amplification result of the three sections as shown in figure 1. the purified product is sent to bio-engineering (Shanghai) GmbH for sequencing, and splicing and sequencing the obtained three sections to obtain intermediate section sequence of HIF- α gene.
Using the intermediate fragment sequence results, 5' RACE primers (GSP1, GSP2, GSP3) were designed using Primer Premier5.0 software, see Table 1. The detection was carried out using a 5' RACE System for Rapid Amplification of cDNA Ends, Version 2.0 kit from Invitrogen. The amplification result shows that the length of the product sequence of the non-coding region at the 5' end is 261bp, and the amplification result is shown in figure 2.
Step 4 acquisition of the 3' -terminal sequence of the HIF-1 α Gene
Using the results of the intermediate fragment sequences, 3' RACE primers (C238-1, C238-2) were designed using Primer Premier5.0 software, see Table 1. Using SMARTerTMRACE cDNA Amplification Kit. The amplification result shows that the length of the product sequence of the 3' end non-coding region is 1263bp, and the amplification result is shown in figure 3.
Table 1 verification of primer names and sequences
And 5: full-length splicing of gene sequences
The middle section sequence, 5 'RACE sequence and 3' RACE sequence of the obtained HIF-1 α gene are analyzed and spliced by software, and the obtained HIF-1 α gene coding region sequence has the full length of 2343bp, as shown in the following (SEQ ID NO. 1):
ATGGATACTGGAGTTGTCACTGAAAAGAAAAGGGTGAGCTCGGAGCGCAGGAAGGAGAAGTCCAGGGATGCAGCGCGATCTCGCAGGGGAAAGGAGTCTGAGGTGTTCTACGAGTTAGCACACCAGCTCCCCCTGCCACACAATGTCACCTCCCACCTGGACAAAGCCTCCATCATGAGGCTGACCATCAGCTATCTGCGCATGAGGAAGCTGCTCAGCTCTGATGAATCTGAGAAGGAGAATGAGGAGGAGAGTCAGCTGAACAGCTTTTATCTGAAGGCTCTGGAGGGTTTCCTCATGGTCCTGTCTGAAGATGGAGACATGGTTTATCTCTCTGAGAATGTCAGCAAGAGCATGGGCCTCTCACAGTTTGATCTGACTGGTCACAGCATCTTTGAATTTTCACACCCATGTGACCATGAGGAGCTGAGAGAGATGCTGGTCTACAGGACAGGATCCAAAAAGACCAAGGAACAAAACACAGAGCGCAGCTTCTTCCTGCGCATGAAGTGCACACTCACTAGCAGAGGGCGCACTGTCAATATCAAGTCTGCCACGTGGAAGGTTCTTCACTGCACTGGTCATGTGCGTGTGCAGGAGTGCGACGAGGGTTCGGGAGACTCTGGCTTTAAAGAGCCTCCTCTGACCTACCTTGTGCTCATCTGTGAGCCCATTCCTCATCCCTCGAACATCGAGGTGCCTCTGGACAGCAAGACCTTCCTTAGCCGCCACACTCTGGACATGAAGTTTTCCTACTGTGATGAAAGAATCACAGAGCTGATGGGATATGAGCCAGATGATCTGCTGAACAAGTCTGTGTATGAATACTATCATGCCCTGGATTCAGATCACCTTACCAAGACACATCACAACTTGTTCGCAAAGGTTCAACTTTGTTCGCAGGGCCAGGCCACCACAGGCCAGTACCGCATGCTGGCTAAGAAAGGTGGTTTTGTGTGGGTGGAGACTCAGGCCACTGTTATCTACAATCCCAAGAATTCTCAGCCACAGTGCATTGTGTGCGTCAACTACGTTCTCAGTGGCATTGTGGAGGGGGATGTTGTCCTGTCCTTGCAGCAGACCATGACCGAACCCGAGGCTGAGGAGAAAGAGAACCAGGAGATGGAAGAGGAGGCCTCTGAGGTGGACATACTCAAACTCTTCAAGCCAGAAAGCCTCAAGTGTCCCGTGAAGAGCTCTGAACTTTATGAGAAGCTGAAAGAGGAGCCTGAGGCTCTCAATGTCTTGGCACCTTCTGCAGGAGACACCATCATCTCTCTGGACTTCAACAATTCAGACTCTGATATGCAGCTGCTGAAGGAGGTGCCCCTCTACAATGACGTCATGCTGCCCTCCAGCAGCGAGAAGCTGCCCATCAGTCTGTCTCCTCTGACGCCCATTGACTCCACCCCTGTTCTGACCAAACTAGACACCGGAGCGGAGGACTTCCCTTTCTGCTCTGCCTCTGATCGTGGTCCAGACTCTGCAAACTCATCTTCCACATCTGGACTGGGCTCCCCGGGGCCCAACAGCCCCATGGATTACTGTTTCCAAGTAGACTCCGACATCAATTCTGAATTCAAACTGGACCTGGTTGAGAAACTGTTTGCTATTGATACTGAGGCAAAGACCCCTTTTAGCTCCCAGGCCATGGAGGATCTCGACCTAGAGATGCTGGCTCCATACATCCCAATGGATGATGACTTCCAGCTGCGTATTCCCTCTCCGCTGGATCCGCTCCCCTCTGGCCCTCACTCTGTGTCCACCATGAGCTCCCTGTTCCAACCCTTGCCCTCCCCTGTGTCTCCAGCCTCTTCCTCCAGCAGCACAGTGAAACAGGAGCCGTCATCTCAGGCCCCTTCACCCCTGCACCTGCTGCAGGAGGTGTGCAATGCTCCTGTCTCACCCTTCAGTGGCAGTCGGGACGTTTCCCCTGCTCGGTCTCCCACCCCACAGAACAGCAATCAGCTCAACAACAGAGAGCTGTCTCCAAAGATGATAGCTGTCCAGAACGCTCAGCGTAAGAGGAAGCTGGAGGAAGTGACATCACTTTCTGAGGCTGTTGGATTGAGTGCTTTGCTTCAGAGTGTGGACAGTGCTATAGAGCCTGGAAAGAGGGCAAAGGTTTTAGAGCTTAAAGGATCAAGTGTGCTTGGAGGAAACAAAACCATTCTCATACTGCCCTCTGATGTGGCCAGTCGTCTGTTGTGCAGTTCTTTAGAGAGCAGCCATGGGCTGCCTCAGCTAACACGTTACGACTGCGAAGTCAACGCTCCTGTGCAGGACCGCCATCATCTGCTGCAGGGAGAGGAGCTCCTGCGTGCTCTGGACCAAGTCAACTGA
step 6: coding region prediction
Using FGENESH software
(http:// linux1. software. softberry. com/berry. phtml:
MDTGVVTEKKRVSSERRKEKSRDAARSRRGKESEVFYELAHQLPLPHNVTSHLDKASIMRLTISYLRMRKLLSSDESEKENEEESQLNSFYLKALEGFLMVLSEDGDMVYLSENVSKSMGLSQFDLTGHSIFEFSHPCDHEELREMLVYRTGSKKTKEQNTERSFFLRMKCTLTSRGRTVNIKSATWKVLHCTGHVRVQECDEGSGDSGFKEPPLTYLVLICEPIPHPSNIEVPLDSKTFLSRHTLDMKFSYCDERITELMGYEPDDLLNKSVYEYYHALDSDHLTKTHHNLFAKVQLCSQGQATTGQYRMLAKKGGFVWVETQATVIYNPKNSQPQCIVCVNYVLSGIVEGDVVLSLQQTMTEPEAEEKENQEMEEEASEVDILKLFKPESLKCPVKSSELYEKLKEEPEALNVLAPSAGDTIISLDFNNSDSDMQLLKEVPLYNDVMLPSSSEKLPISLSPLTPIDSTPVLTKLDTGAEDFPFCSASDRGPDSANSSSTSGLGSPGPNSPMDYCFQVDSDINSEFKLDLVEKLFAIDTEAKTPFSSQAMEDLDLEMLAPYIPMDDDFQLRIPSPLDPLPSGPHSVSTMSSLFQPLPSPVSPASSSSSTVKQEPSSQAPSPLHLLQEVCNAPVSPFSGSRDVSPARSPTPQNSNQLNNRELSPKMIAVQNAQRKRKLEEVTSLSEAVGLSALLQSVDSAIEPGKRAKVLELKGSSVLGGNKTILILPSDVASRLLCSSLESSHGLPQLTRYDCEVNAPVQDRHHLLQGEELLRALDQVN*
secondly, the expression of the goldfish hypoxia inducible factor HIF-1 α in different tissues under the condition of hypoxia stress
Step 1: goldfish hypoxia stress test
The test fish is obtained from the flower, bird, fish and insect market, has a body mass of 53 + -2 g and a full length of 14 + -2 cm. After being transported back, the test fish is placed in a circulating water aquaculture system in a laboratory for temporary culture. The water temperature in the circulating system is 18 +/-0.5 ℃, and the dissolved oxygen is 8.01 mg/L. Feed feeding is started after 1 day of adaptation, 9 a.m. every day: 00 and 16 pm: 00 feeding granular baits respectively, and domesticating for 7 days under the condition. And selecting strong and vigorous individuals from the temporarily cultured fishes for testing.
The test was divided into a control group (C), a hypoxic group (H) and a recovery group (R), each group was set up with 3 parallels, each placed with 10 crucian carps in parallel. The control group was oxygenated normally with dissolved oxygen maintained at about 8.01mg/L, and the hypoxic group was oxygenated with nitrogen to reduce the dissolved oxygen level to about 1.04 mg/L. Sampling was performed 12h after hypoxia stress. And opening the sealing device after the recovery group is stressed by hypoxia for 12 hours, normally inflating to enable dissolved oxygen to recover to about 7.85mg/L, and sampling after the recovery group is inflated for 12 hours. Taking 5 of each group, taking liver, branchia, brain and heart tissue, preserving in RNAlater, standing overnight at 4 deg.C, and transferring to-80 deg.C for cryopreservation.
Step 2: the steps of RNA extraction and cDNA synthesis are the same as the first step.
Quantitative primers are designed and synthesized by using Primer 5.0 software, and the expression of the HIF-1 α gene in liver, gill, heart and brain tissues is detected by taking the Carp β -Actin gene as an internal reference, wherein the internal reference Primer (Carp-Actin) and the fluorescence quantitative Primer of the target gene (HIF-1 α) used in the part are shown in a table 2.
The reaction system used was 15. mu.L, with 0.2. mu. mol/L of each of the upstream and downstream primers, and 50ng of template, 1 XSSYBRPremix ExTaq (TaKaRa). The amplification condition is pre-denaturation at 94 ℃ for 5 min; denaturation at 94 ℃ for 15s, annealing at 60 ℃ for 15s, extension at 72 ℃ for 20s, 40 cycles; extension at 72 ℃ for 5 min. The instrument used was BIO-RAD CFX ConnectTMFluorescent quantitative PCR detection system.
And 4, step 4: data analysis
All experiments were set up for 3 replicates and results were utilized
TABLE 2 primers used in qPCR experiments
A sequence table:
SEQUENCE LISTING
SEQ ID NO 1：
ATGGATACTGGAGTTGTCACTGAAAAGAAAAGGGTGAGCTCGGAGCGCAGGAAGGAGAAGTCCAGGGATGCAGCGCGATCTCGCAGGGGAAAGGAGTCTGAGGTGTTCTACGAGTTAGCACACCAGCTCCCCCTGCCACACAATGTCACCTCCCACCTGGACAAAGCCTCCATCATGAGGCTGACCATCAGCTATCTGCGCATGAGGAAGCTGCTCAGCTCTGATGAATCTGAGAAGGAGAATGAGGAGGAGAGTCAGCTGAACAGCTTTTATCTGAAGGCTCTGGAGGGTTTCCTCATGGTCCTGTCTGAAGATGGAGACATGGTTTATCTCTCTGAGAATGTCAGCAAGAGCATGGGCCTCTCACAGTTTGATCTGACTGGTCACAGCATCTTTGAATTTTCACACCCATGTGACCATGAGGAGCTGAGAGAGATGCTGGTCTACAGGACAGGATCCAAAAAGACCAAGGAACAAAACACAGAGCGCAGCTTCTTCCTGCGCATGAAGTGCACACTCACTAGCAGAGGGCGCACTGTCAATATCAAGTCTGCCACGTGGAAGGTTCTTCACTGCACTGGTCATGTGCGTGTGCAGGAGTGCGACGAGGGTTCGGGAGACTCTGGCTTTAAAGAGCCTCCTCTGACCTACCTTGTGCTCATCTGTGAGCCCATTCCTCATCCCTCGAACATCGAGGTGCCTCTGGACAGCAAGACCTTCCTTAGCCGCCACACTCTGGACATGAAGTTTTCCTACTGTGATGAAAGAATCACAGAGCTGATGGGATATGAGCCAGATGATCTGCTGAACAAGTCTGTGTATGAATACTATCATGCCCTGGATTCAGATCACCTTACCAAGACACATCACAACTTGTTCGCAAAGGTTCAACTTTGTTCGCAGGGCCAGGCCACCACAGGCCAGTACCGCATGCTGGCTAAGAAAGGTGGTTTTGTGTGGGTGGAGACTCAGGCCACTGTTATCTACAATCCCAAGAATTCTCAGCCACAGTGCATTGTGTGCGTCAACTACGTTCTCAGTGGCATTGTGGAGGGGGATGTTGTCCTGTCCTTGCAGCAGACCATGACCGAACCCGAGGCTGAGGAGAAAGAGAACCAGGAGATGGAAGAGGAGGCCTCTGAGGTGGACATACTCAAACTCTTCAAGCCAGAAAGCCTCAAGTGTCCCGTGAAGAGCTCTGAACTTTATGAGAAGCTGAAAGAGGAGCCTGAGGCTCTCAATGTCTTGGCACCTTCTGCAGGAGACACCATCATCTCTCTGGACTTCAACAATTCAGACTCTGATATGCAGCTGCTGAAGGAGGTGCCCCTCTACAATGACGTCATGCTGCCCTCCAGCAGCGAGAAGCTGCCCATCAGTCTGTCTCCTCTGACGCCCATTGACTCCACCCCTGTTCTGACCAAACTAGACACCGGAGCGGAGGACTTCCCTTTCTGCTCTGCCTCTGATCGTGGTCCAGACTCTGCAAACTCATCTTCCACATCTGGACTGGGCTCCCCGGGGCCCAACAGCCCCATGGATTACTGTTTCCAAGTAGACTCCGACATCAATTCTGAATTCAAACTGGACCTGGTTGAGAAACTGTTTGCTATTGATACTGAGGCAAAGACCCCTTTTAGCTCCCAGGCCATGGAGGATCTCGACCTAGAGATGCTGGCTCCATACATCCCAATGGATGATGACTTCCAGCTGCGTATTCCCTCTCCGCTGGATCCGCTCCCCTCTGGCCCTCACTCTGTGTCCACCATGAGCTCCCTGTTCCAACCCTTGCCCTCCCCTGTGTCTCCAGCCTCTTCCTCCAGCAGCACAGTGAAACAGGAGCCGTCATCTCAGGCCCCTTCACCCCTGCACCTGCTGCAGGAGGTGTGCAATGCTCCTGTCTCACCCTTCAGTGGCAGTCGGGACGTTTCCCCTGCTCGGTCTCCCACCCCACAGAACAGCAATCAGCTCAACAACAGAGAGCTGTCTCCAAAGATGATAGCTGTCCAGAACGCTCAGCGTAAGAGGAAGCTGGAGGAAGTGACATCACTTTCTGAGGCTGTTGGATTGAGTGCTTTGCTTCAGAGTGTGGACAGTGCTATAGAGCCTGGAAAGAGGGCAAAGGTTTTAGAGCTTAAAGGATCAAGTGTGCTTGGAGGAAACAAAACCATTCTCATACTGCCCTCTGATGTGGCCAGTCGTCTGTTGTGCAGTTCTTTAGAGAGCAGCCATGGGCTGCCTCAGCTAACACGTTACGACTGCGAAGTCAACGCTCCTGTGCAGGACCGCCATCATCTGCTGCAGGGAGAGGAGCTCCTGCGTGCTCTGGACCAAGTCAACTGA
SEQ ID NO 2：
MDTGVVTEKKRVSSERRKEKSRDAARSRRGKESEVFYELAHQLPLPHNVTSHLDKASIMRLTISYLRMRKLLSSDESEKENEEESQLNSFYLKALEGFLMVLSEDGDMVYLSENVSKSMGLSQFDLTGHSIFEFSHPCDHEELREMLVYRTGSKKTKEQNTERSFFLRMKCTLTSRGRTVNIKSATWKVLHCTGHVRVQECDEGSGDSGFKEPPLTYLVLICEPIPHPSNIEVPLDSKTFLSRHTLDMKFSYCDERITELMGYEPDDLLNKSVYEYYHALDSDHLTKTHHNLFAKVQLCSQGQATTGQYRMLAKKGGFVWVETQATVIYNPKNSQPQCIVCVNYVLSGIVEGDVVLSLQQTMTEPEAEEKENQEMEEEASEVDILKLFKPESLKCPVKSSELYEKLKEEPEALNVLAPSAGDTIISLDFNNSDSDMQLLKEVPLYNDVMLPSSSEKLPISLSPLTPIDSTPVLTKLDTGAEDFPFCSASDRGPDSANSSSTSGLGSPGPNSPMDYCFQVDSDINSEFKLDLVEKLFAIDTEAKTPFSSQAMEDLDLEMLAPYIPMDDDFQLRIPSPLDPLPSGPHSVSTMSSLFQPLPSPVSPASSSSSTVKQEPSSQAPSPLHLLQEVCNAPVSPFSGSRDVSPARSPTPQNSNQLNNRELSPKMIAVQNAQRKRKLEEVTSLSEAVGLSALLQSVDSAIEPGKRAKVLELKGSSVLGGNKTILILPSDVASRLLCSSLESSHGLPQLTRYDCEVNAPVQDRHHLLQGEELLRALDQVN*
SEQ ID NO 3：
ATGGATACTGGAGTTGTCACTGAAAAGAAAAGGGTGAGCTCGGAGCGCAGGAAGGAGAAGTCCAGGGATGCAGCGCGATCTCGCAGGGGAAAGGAGTCTGAGGTGTTCTACGAGTTAGCACACCAGCTCCCCCTGCCACACAATGTCAACTCCCACCTGGACAAAGCCTCCATCATGAGGCTGACCATCAGCTATCTGCGCATGAGGAAGCTGCTCAGCTCTGATACTCCTGTAGTCATATATGAATCTGAGAAGGAGAATGAGGAGGAGAGTCAGCTGAACAGCTTTTATCTGAAGGCCCTGGAGGGTTTCCTCATGGTCCTGTCTGAAGATGGAGACATGGTTTATCTCTCTGAGAATGTCAGCAAGAGCATGGGCCTCTCACAGTTTGATCTGACTGGTCACAGCATCTTTGAATTTTCACACCCATGTGACCATGAGGAGCTGAGAGAGATGCTGGTCTACAGGACAGGATCCAAAAAGACCAAGGAACAAAACACAGACGCGCAGCTCTTCCTGCGCATGAAGTGCACACTCACTAGCAGAGGTCGCACTGTCAATATAAAGTCTGCTACGTGGAAGGTTCTTCACTGCACTGGTCATGTGCGTGTGCAGGAGTGCGACGAGGGTTCGGGAGACTCTGGCTTTAAAGAGCCTCCTCTGACCTACCTTGTGCTCATCTGTGAGCCCATTCCTCATCCCTCGAACATCGAGGTGCCTCTGGACAGCAAGACCTTCCTTAGCCGCCACACTCTGGACATGACCCCCAGGATAAGTTCAGTTGAAAGGATTTGTTGTGGAAATGATTGTAAATGGATCACAGAGCTGATGGGATATGAGCCAGATGATCTGCTGAACAGGTCTGTGTACGAATACTATCATGCCCTGGATTCAGATCACCTTACCAAGACACATCACAACTTGTTTGCAAAGGGCCAGGCCACCACAGGCCAGTACCGCATGCTGGCTAAGAAAGGTGGTTTTGTGTGGGTGGAGACTCAGGCCACTGTTATCTACAACCCCAAGAATTCTCAGCCACAGTGCATTGTGTGCGTCAACTACGTTCTCAGGGCTGCAACTAACGATTATTTTGATAATCGATTAATCGTTTTGCCCATTTTTTCCCCAACAACGAATCGATTACTAAATTATGGCATTGTGGAGGGGGATGTTGTCCTGTCCTTGCAGCAGACCATGACTGAGCCCAAGGCTGAGGAGAAAGAGAACCAGAAGATGGAAGATGAGGCCTCTGAGGTGGACATACTCAAACTCTTCAAGCCAGAAAGCCTCAAGTGTCCCATGAAGAGCCCTGACCTGTATGAGAAGCTGAAAGAGGAGCCCGAGGCTCTCAATGTGTTGGCACCTTCTGCAGGAGACACCATCATCTCTCTGGACTTCAACAACTCAGATTCTGACATGCAGCTGCTGAAGGAGGTGCCCCTCTACAATGATGTCATGCTGCCCTCCAGCAGCGAGAAGCTGCCCATCAGTCTGTCTCCTCTGACGCCCACCGACTCCACCCCTGTTCTGACGGGGTCTGATCGTGGTCCAGACTCCACAAACACACCTTCCACATCTGGACTGGGCTCCTCGGGGCCTAACAGTCCCATGGATTACTGTTTCCAAGTAGACTCAGACATCAATTCTGAATTCAAACTGGACCTGGTTGAGAAACTGTTTGCTATCGATACCGAGGCAAAGACCCCTTTTAGCTCCCAGGACATGGAGGATCTTGACCTAGAGATGCTGGCTCCATACATCCCAATGGATGACGACTTCCAGCTGCGCATCCCCTCTCCGCTGGATCCGCTCCCCTCTGGCCCTCACTCTGTCCTCTTCCTCCAGCAGCACAGTGAACAGGAGCCGTCATCTCAGGGCCCTTCGCCCTTGCACCTGCTGCAGGAGGTGTGCAATGCCCCTGTCTCACCCTTCAGTGGCAGTCGGGACGTTTCCCCTGCTCGGTCCCCCACCCCACAGAGCAGCAATCAGCTCAACAAGAGAGAGCTGTCTCCAAAGATGTTGGCTATCCAGAACGCCCAACGCAAGAGGAAGCTGGAGGAAGTGACATCACTTTCTGAGGCTGTTGGACTGAGTGTGGACAGTGCTATAGAGCCTGGAAAGAGGGCAAAGGTTTTAGAGCTTAAAGGATCAAGTGTGCTTGGAGGAAACAAAACCATTCTCATACTGCCCTCTGATGTGGCCAGTCGTCTGTTGTGCAGTTCTTTAGAGAGCTGCAGTGGGCTGCCTCAGCTAACACGTTACGACTGCGAAGTCAACGCTCCCGTGCAGGACCGCCATCATCTGCTGCAGGGAGAGGAGCTCCTGCGTGCTCTGGACCAAGTCAAC
SEQ ID NO 4(B3151-F)：ATGGATACTGGAGTTGTC
SEQ ID NO 5(B3151-R)：ACAATGCACTGTGGCTGA
SEQ ID NO 6(B3152-F)：TGGATTCAGATCACCTTACC
SEQ ID NO 7(B3152-R)：GGTCTTTGCCTCGGTATC
SEQ ID NO 8(B3153-F)：CCTCCAGCAGCGAGAAGC
SEQ ID NO 9(B3153-R)：GTTGACTTGGTCCAGAGC
SEQ ID NO 10(GSP1)：CTGGTGTGCTAACTCG
SEQ ID NO 11(GSP2)：ACCTCAGACTCCTTTCCC
SEQ ID NO 12(GSP3)：GCTGCATCCCTGGACTTC
SEQ ID NO 13(C238-1)：GCCCTCTGATGTGGCCAGTCGTCT
SEQ ID NO 14(C238-2)：AGCTAACACGTTACGACTGCGAAG
SEQ ID NO 15(HIF-1αF)：CTCACTAGCAGAGGTCGCAC
SEQ ID NO 16(HIF-1αR)：GGGATGAGGAATGGGCTCAC
SEQ ID NO 17(Carp-Actin F)：TGCAAAGCCGGATTCGCTGG
SEQ ID NO 18(Carp-Actin R)：AGTTGGTGACAATACCGTGC
although the embodiments of the present invention have been disclosed for illustrative purposes, those skilled in the art will appreciate that: various substitutions, changes and modifications are possible without departing from the spirit and scope of the invention and the appended claims, and therefore the scope of the invention is not limited to the disclosure of the embodiments and the accompanying drawings.
Sequence listing
<110> Tianjin City Water production research institute
<120> goldfish hypoxia inducible factor HIF-1 α gene, cloning method and application
<160>18
<170>SIPOSequenceListing 1.0
<210>1
<211>2343
<212>DNA/RNA
<213> nucleotide sequence of Goldfish hypoxia inducible factor HIF-1 α Gene (Unknown)
<400>1
atggatactg gagttgtcac tgaaaagaaa agggtgagct cggagcgcag gaaggagaag 60
tccagggatg cagcgcgatc tcgcagggga aaggagtctg aggtgttcta cgagttagca 120
caccagctcc ccctgccaca caatgtcacc tcccacctgg acaaagcctc catcatgagg 180
ctgaccatca gctatctgcg catgaggaag ctgctcagct ctgatgaatc tgagaaggag 240
aatgaggagg agagtcagct gaacagcttt tatctgaagg ctctggaggg tttcctcatg 300
gtcctgtctg aagatggaga catggtttat ctctctgaga atgtcagcaa gagcatgggc 360
ctctcacagt ttgatctgac tggtcacagc atctttgaat tttcacaccc atgtgaccat 420
gaggagctga gagagatgct ggtctacagg acaggatcca aaaagaccaa ggaacaaaac 480
acagagcgca gcttcttcct gcgcatgaag tgcacactca ctagcagagg gcgcactgtc 540
aatatcaagt ctgccacgtg gaaggttctt cactgcactg gtcatgtgcg tgtgcaggag 600
tgcgacgagg gttcgggaga ctctggcttt aaagagcctc ctctgaccta ccttgtgctc 660
atctgtgagc ccattcctca tccctcgaac atcgaggtgc ctctggacag caagaccttc 720
cttagccgcc acactctgga catgaagttt tcctactgtg atgaaagaat cacagagctg 780
atgggatatg agccagatga tctgctgaac aagtctgtgt atgaatacta tcatgccctg 840
gattcagatc accttaccaa gacacatcac aacttgttcg caaaggttca actttgttcg 900
cagggccagg ccaccacagg ccagtaccgc atgctggcta agaaaggtgg ttttgtgtgg 960
gtggagactc aggccactgt tatctacaat cccaagaatt ctcagccaca gtgcattgtg 1020
tgcgtcaact acgttctcag tggcattgtg gagggggatg ttgtcctgtc cttgcagcag 1080
accatgaccg aacccgaggc tgaggagaaa gagaaccagg agatggaaga ggaggcctct 1140
gaggtggaca tactcaaact cttcaagcca gaaagcctca agtgtcccgt gaagagctct 1200
gaactttatg agaagctgaa agaggagcct gaggctctca atgtcttggc accttctgca 1260
ggagacacca tcatctctct ggacttcaac aattcagact ctgatatgca gctgctgaag 1320
gaggtgcccc tctacaatga cgtcatgctg ccctccagca gcgagaagct gcccatcagt 1380
ctgtctcctc tgacgcccat tgactccacc cctgttctga ccaaactaga caccggagcg 1440
gaggacttcc ctttctgctc tgcctctgat cgtggtccag actctgcaaa ctcatcttcc 1500
acatctggac tgggctcccc ggggcccaac agccccatgg attactgttt ccaagtagac 1560
tccgacatca attctgaatt caaactggac ctggttgaga aactgtttgc tattgatact 1620
gaggcaaaga ccccttttag ctcccaggcc atggaggatc tcgacctaga gatgctggct 1680
ccatacatcc caatggatga tgacttccag ctgcgtattc cctctccgct ggatccgctc 1740
ccctctggcc ctcactctgt gtccaccatg agctccctgt tccaaccctt gccctcccct 1800
gtgtctccag cctcttcctc cagcagcaca gtgaaacagg agccgtcatc tcaggcccct 1860
tcacccctgc acctgctgca ggaggtgtgc aatgctcctg tctcaccctt cagtggcagt 1920
cgggacgttt cccctgctcg gtctcccacc ccacagaaca gcaatcagct caacaacaga 1980
gagctgtctc caaagatgat agctgtccag aacgctcagc gtaagaggaa gctggaggaa 2040
gtgacatcac tttctgaggc tgttggattg agtgctttgc ttcagagtgt ggacagtgct 2100
atagagcctg gaaagagggc aaaggtttta gagcttaaag gatcaagtgt gcttggagga 2160
aacaaaacca ttctcatact gccctctgat gtggccagtc gtctgttgtg cagttcttta 2220
gagagcagcc atgggctgcc tcagctaaca cgttacgact gcgaagtcaa cgctcctgtg 2280
caggaccgcc atcatctgct gcagggagag gagctcctgc gtgctctgga ccaagtcaac 2340
tga 2343
<210>2
<211>780
<212>PRT
<213> amino acid sequence (Unknown)
<400>2
Met Asp Thr Gly Val Val Thr Glu Lys Lys Arg Val Ser Ser Glu Arg
1 5 10 15
Arg Lys Glu Lys Ser Arg Asp Ala Ala Arg Ser Arg Arg Gly Lys Glu
20 25 30
Ser Glu Val Phe Tyr Glu Leu Ala His Gln Leu Pro Leu Pro His Asn
35 40 45
Val Thr Ser His Leu Asp Lys Ala Ser Ile Met Arg Leu Thr Ile Ser
50 55 60
Tyr Leu Arg Met Arg Lys Leu Leu Ser Ser Asp Glu Ser Glu Lys Glu
65 70 75 80
Asn Glu Glu Glu Ser Gln Leu Asn Ser Phe Tyr Leu Lys Ala Leu Glu
85 90 95
Gly Phe Leu Met Val Leu Ser Glu Asp Gly Asp Met Val Tyr Leu Ser
100 105 110
Glu Asn Val Ser Lys Ser Met Gly Leu Ser Gln Phe Asp Leu Thr Gly
115 120 125
His Ser Ile Phe Glu Phe Ser His Pro Cys Asp His Glu Glu Leu Arg
130 135 140
Glu Met Leu Val Tyr Arg Thr Gly Ser Lys Lys Thr Lys Glu Gln Asn
145 150 155 160
Thr Glu Arg Ser Phe Phe Leu Arg Met Lys Cys Thr Leu Thr Ser Arg
165 170 175
Gly Arg Thr Val Asn Ile Lys Ser Ala Thr Trp Lys Val Leu His Cys
180 185 190
Thr Gly His Val Arg Val Gln Glu Cys Asp Glu Gly Ser Gly Asp Ser
195 200 205
Gly Phe Lys Glu Pro Pro Leu Thr Tyr Leu Val Leu Ile Cys Glu Pro
210 215 220
Ile Pro His Pro Ser Asn Ile Glu Val Pro Leu Asp Ser Lys Thr Phe
225 230 235 240
Leu Ser Arg His Thr Leu Asp Met Lys Phe Ser Tyr Cys Asp Glu Arg
245 250 255
Ile Thr Glu Leu Met Gly Tyr Glu Pro Asp Asp Leu Leu Asn Lys Ser
260 265 270
Val Tyr Glu Tyr Tyr His Ala Leu Asp Ser Asp His Leu Thr Lys Thr
275 280 285
His His Asn Leu Phe Ala Lys Val Gln Leu Cys Ser Gln Gly Gln Ala
290 295 300
Thr Thr Gly Gln Tyr Arg Met Leu Ala Lys Lys Gly Gly Phe Val Trp
305 310 315 320
Val Glu Thr Gln Ala Thr Val Ile Tyr Asn Pro Lys Asn Ser Gln Pro
325 330 335
Gln Cys Ile Val Cys Val Asn Tyr Val Leu Ser Gly Ile Val Glu Gly
340 345 350
Asp Val Val Leu Ser Leu Gln Gln Thr Met Thr Glu Pro Glu Ala Glu
355 360 365
Glu Lys Glu Asn Gln Glu Met Glu Glu Glu Ala Ser Glu Val Asp Ile
370 375 380
Leu Lys Leu Phe Lys Pro Glu Ser Leu Lys Cys Pro Val Lys Ser Ser
385 390 395 400
Glu Leu Tyr Glu Lys Leu Lys Glu Glu Pro Glu Ala Leu Asn Val Leu
405 410 415
Ala Pro Ser Ala Gly Asp Thr Ile Ile Ser Leu Asp Phe Asn Asn Ser
420 425 430
Asp Ser Asp Met Gln Leu Leu Lys Glu Val Pro Leu Tyr Asn Asp Val
435 440 445
Met Leu Pro Ser Ser Ser Glu Lys Leu Pro Ile Ser Leu Ser Pro Leu
450 455 460
Thr Pro Ile Asp Ser Thr Pro Val Leu Thr Lys Leu Asp Thr Gly Ala
465 470 475 480
Glu Asp Phe Pro Phe Cys Ser Ala Ser Asp Arg Gly Pro Asp Ser Ala
485 490 495
Asn Ser Ser Ser Thr Ser Gly Leu Gly Ser Pro Gly Pro Asn Ser Pro
500 505 510
Met Asp Tyr Cys Phe Gln Val Asp Ser Asp Ile Asn Ser Glu Phe Lys
515 520 525
Leu Asp Leu Val Glu Lys Leu Phe Ala Ile Asp Thr Glu Ala Lys Thr
530 535 540
Pro Phe Ser Ser Gln Ala Met Glu Asp Leu Asp Leu Glu Met Leu Ala
545 550 555 560
Pro Tyr Ile Pro Met Asp Asp Asp Phe Gln Leu Arg Ile Pro Ser Pro
565 570 575
Leu Asp Pro Leu Pro Ser Gly Pro His Ser Val Ser Thr Met Ser Ser
580 585 590
Leu Phe Gln Pro Leu Pro Ser Pro Val Ser Pro Ala Ser Ser Ser Ser
595 600 605
Ser Thr Val Lys Gln Glu Pro Ser Ser Gln Ala Pro Ser Pro Leu His
610 615 620
Leu Leu Gln Glu Val Cys Asn Ala Pro Val Ser Pro Phe Ser Gly Ser
625 630 635 640
Arg Asp Val Ser Pro Ala Arg Ser Pro Thr Pro Gln Asn Ser Asn Gln
645 650 655
Leu Asn Asn Arg Glu Leu Ser Pro Lys Met Ile Ala Val Gln Asn Ala
660 665 670
Gln Arg Lys Arg Lys Leu Glu Glu Val Thr Ser Leu Ser Glu Ala Val
675 680 685
Gly Leu Ser Ala Leu Leu Gln Ser Val Asp Ser Ala Ile Glu Pro Gly
690 695 700
Lys Arg Ala Lys Val Leu Glu Leu Lys Gly Ser Ser Val Leu Gly Gly
705 710 715 720
Asn Lys Thr Ile Leu Ile Leu Pro Ser Asp Val Ala Ser Arg Leu Leu
725 730 735
Cys Ser Ser Leu Glu Ser Ser His Gly Leu Pro Gln Leu Thr Arg Tyr
740 745 750
Asp Cys Glu Val Asn Ala Pro Val Gln Asp Arg His His Leu Leu Gln
755 760 765
Gly Glu Glu Leu Leu Arg Ala Leu Asp Gln Val Asn
770 775 780
<210>3
<211>2346
<212>DNA/RNA
<213> sequence of Goldfish transcriptome sequence (Unknown)
<400>3
atggatactg gagttgtcac tgaaaagaaa agggtgagct cggagcgcag gaaggagaag 60
tccagggatg cagcgcgatc tcgcagggga aaggagtctg aggtgttcta cgagttagca 120
caccagctcc ccctgccaca caatgtcaac tcccacctgg acaaagcctc catcatgagg 180
ctgaccatca gctatctgcg catgaggaag ctgctcagct ctgatactcc tgtagtcata 240
tatgaatctg agaaggagaa tgaggaggag agtcagctga acagctttta tctgaaggcc 300
ctggagggtt tcctcatggt cctgtctgaa gatggagaca tggtttatct ctctgagaat 360
gtcagcaaga gcatgggcct ctcacagttt gatctgactg gtcacagcat ctttgaattt 420
tcacacccat gtgaccatga ggagctgaga gagatgctgg tctacaggac aggatccaaa 480
aagaccaagg aacaaaacac agacgcgcag ctcttcctgc gcatgaagtg cacactcact 540
agcagaggtc gcactgtcaa tataaagtct gctacgtgga aggttcttca ctgcactggt 600
catgtgcgtg tgcaggagtg cgacgagggt tcgggagact ctggctttaa agagcctcct 660
ctgacctacc ttgtgctcat ctgtgagccc attcctcatc cctcgaacat cgaggtgcct 720
ctggacagca agaccttcct tagccgccac actctggaca tgacccccag gataagttca 780
gttgaaagga tttgttgtgg aaatgattgt aaatggatca cagagctgat gggatatgag 840
ccagatgatc tgctgaacag gtctgtgtac gaatactatc atgccctgga ttcagatcac 900
cttaccaaga cacatcacaa cttgtttgca aagggccagg ccaccacagg ccagtaccgc 960
atgctggcta agaaaggtgg ttttgtgtgg gtggagactc aggccactgt tatctacaac 1020
cccaagaatt ctcagccaca gtgcattgtg tgcgtcaact acgttctcag ggctgcaact 1080
aacgattatt ttgataatcg attaatcgtt ttgcccattt tttccccaac aacgaatcga 1140
ttactaaatt atggcattgt ggagggggat gttgtcctgt ccttgcagca gaccatgact 1200
gagcccaagg ctgaggagaa agagaaccag aagatggaag atgaggcctc tgaggtggac 1260
atactcaaac tcttcaagcc agaaagcctc aagtgtccca tgaagagccc tgacctgtat 1320
gagaagctga aagaggagcc cgaggctctc aatgtgttgg caccttctgc aggagacacc 1380
atcatctctc tggacttcaa caactcagat tctgacatgc agctgctgaa ggaggtgccc 1440
ctctacaatg atgtcatgct gccctccagc agcgagaagc tgcccatcag tctgtctcct 1500
ctgacgccca ccgactccac ccctgttctg acggggtctg atcgtggtcc agactccaca 1560
aacacacctt ccacatctgg actgggctcc tcggggccta acagtcccat ggattactgt 1620
ttccaagtag actcagacat caattctgaa ttcaaactgg acctggttga gaaactgttt 1680
gctatcgata ccgaggcaaa gacccctttt agctcccagg acatggagga tcttgaccta 1740
gagatgctgg ctccatacat cccaatggat gacgacttcc agctgcgcat cccctctccg 1800
ctggatccgc tcccctctgg ccctcactct gtcctcttcc tccagcagca cagtgaacag 1860
gagccgtcat ctcagggccc ttcgcccttg cacctgctgc aggaggtgtg caatgcccct 1920
gtctcaccct tcagtggcag tcgggacgtt tcccctgctc ggtcccccac cccacagagc 1980
agcaatcagc tcaacaagag agagctgtct ccaaagatgt tggctatcca gaacgcccaa 2040
cgcaagagga agctggagga agtgacatca ctttctgagg ctgttggact gagtgtggac 2100
agtgctatag agcctggaaa gagggcaaag gttttagagc ttaaaggatc aagtgtgctt 2160
ggaggaaaca aaaccattct catactgccc tctgatgtgg ccagtcgtct gttgtgcagt 2220
tctttagaga gctgcagtgg gctgcctcag ctaacacgtt acgactgcga agtcaacgct 2280
cccgtgcagg accgccatca tctgctgcag ggagaggagc tcctgcgtgc tctggaccaa 2340
gtcaac 2346
<210>4
<211>18
<212>DNA/RNA
<213>B3151- F(Unknown)
<400>4
atggatactg gagttgtc 18
<210>5
<211>18
<212>DNA/RNA
<213>B3151- R(Unknown)
<400>5
acaatgcact gtggctga 18
<210>6
<211>20
<212>DNA/RNA
<213>B3152- F(Unknown)
<400>6
tggattcaga tcaccttacc 20
<210>7
<211>18
<212>DNA/RNA
<213>B3152- R(Unknown)
<400>7
ggtctttgcc tcggtatc 18
<210>8
<211>18
<212>DNA/RNA
<213>B3153 -F(Unknown)
<400>8
cctccagcag cgagaagc 18
<210>9
<211>18
<212>DNA/RNA
<213>B3153 -R(Unknown)
<400>9
gttgacttgg tccagagc 18
<210>10
<211>16
<212>DNA/RNA
<213>GSP1(Unknown)
<400>10
ctggtgtgct aactcg 16
<210>11
<211>18
<212>DNA/RNA
<213>GSP2(Unknown)
<400>11
acctcagact cctttccc 18
<210>12
<211>18
<212>DNA/RNA
<213>GSP3(Unknown)
<400>12
gctgcatccc tggacttc 18
<210>13
<211>24
<212>DNA/RNA
<213>C238-1 (Unknown)
<400>13
gccctctgat gtggccagtc gtct 24
<210>14
<211>24
<212>DNA/RNA
<213>C238-2(Unknown)
<400>14
agctaacacg ttacgactgc gaag 24
<210>15
<211>20
<212>DNA/RNA
<213>HIF-1αF(Unknown)
<400>15
ctcactagca gaggtcgcac 20
<210>16
<211>20
<212>DNA/RNA
<213>HIF-1αR(Unknown)
<400>16
gggatgagga atgggctcac 20
<210>17
<211>20
<212>DNA/RNA
<213>Carp-Actin F(Unknown)
<400>17
tgcaaagccg gattcgctgg 20
<210>18
<211>20
<212>DNA/RNA
<213>Carp-Actin R(Unknown)
<400>18
agttggtgac aataccgtgc 20
- 上一篇：一种医用注射器针头装配设备
- 下一篇：一种海地瓜生物活性肽的制备方法