CN1928086A - Gene sequence of sparus latus houttuyn actin - Google Patents
Gene sequence of sparus latus houttuyn actin Download PDFInfo
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- CN1928086A CN1928086A CN 200610037560 CN200610037560A CN1928086A CN 1928086 A CN1928086 A CN 1928086A CN 200610037560 CN200610037560 CN 200610037560 CN 200610037560 A CN200610037560 A CN 200610037560A CN 1928086 A CN1928086 A CN 1928086A
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Abstract
The present invention designs degenerating primer in the conservation region of CDS sequence in kindred species actin gene to extract total RNA from spleen of yellow-fin Sparus latus, and clones the expression sequence of cyclin B gene of yellow-fin Sparus latus through PCR process amplification, . Obtaining the expression sequence makes it possible to obtain recombinant protein with immunological activity through extracorporeal recombination and to lay the theoretic foundation for further function study.
Description
Technical field
The present invention relates to gene clone field in the molecular biology, relate in particular to yellowfin houttuyn actin gene order.
Background technology
Actin muscle is a kind of special albumen, is defined by a kind of molecular moter, moves with cell, cell fission and wound healing be relevant; The while Actin muscle also participates in DNA and transcribes, and can convene rna plymerase ii, serves as a molecular moter when the rna plymerase ii transcriptional start.If Actin muscle is suppressed, just transcribes and to begin.It is all extremely important that DNA transcribes all activities of this process pair cell.These functions will make us effectively intervene when genetic transcription makes mistakes; Actin muscle can be used as cofactor, has the function of the viral enzyme of degradation of cell skeleton.
Along with the development of mariculture industry, the disease problem is on the rise at present, therefore self start with from the fish body, and the functional gene that the clone is relevant, the active vitro recombination albumen of tool of acquisition purifying, improving fish body resistance against diseases is the trend of current disease control.So far also less to the gene studies of yellowfin porgy cell aspect, especially the research to actin gene also belongs to blank.
Summary of the invention
The objective of the invention is by degenerate primer, and with the amplification of PCR method, be cloned into the full expressed sequence of yellowfin houttuyn actin gene in conjunction with the RACE technology with the conserved regions design of the CDS sequence of nearly source species (rainbow trout, lefteye flounder, carp) actin gene.The gene order of gained is:
Nucleotide sequence (1125 bases):
atggatgatgaaatcgccgcactggttgttgacaacggatccggtatgtgcaaagccggt
ttcgccggagacgacgcccctcgtgctgtcttcccctccatcgtcggtcgccccaggcat
cagggtgtgatggtgggtatgggccagaaggacagctacgttggtgatgaggcccagagc
aagagaggtatcctgaccctgaagtaccccatcgagcacggtattgtgaccaactgggat
gacatggagaagatctggcatcacaccttctacaacgagctgagagttgcccctgaggag
caccctgtcctgctcacagaggcccccctgaaccccaaagccaacagggagaagatgacc
cagatcatgttcgagaccttcaacacccctgccatgtacgttgccatccaggctgtgctg
tccctgtatgcctctggtcgtaccactggtatcgtcatggactccggtgatggtgtgacc
cacacagtgcccatctatgagggctatgccctgccccacgccatcctgcgtctggacttg
gccggccgcgacctcacagactacctcatgaagatcctgacagagcgtggctactccttc
accaccacagccgagagggaaatcgtgcgtgacatcaaggagaagctgtgctatgtcgcc
ctggacttcgagcaggagatgggcactgctgcctcctcctcctccctggagaagagctat
gagctgcctgacggacaggtcatcaccatcggcaatgagaggttccgttgcccagaggcc
ctcttccagccatccttcctcggtatggagtcctgcggaatccatgagaccacctacaac
agcatcatgaagtgtgatgtcgacatccgtaaggacctgtatgccaacacagtgctgtct
ggaggtaccaccatgtaccctggcatcgctgacaggatgcagaaggagatcacagccctg
gccccatccaccatgaagattaagatcattgccccacctgagcgtaaatactctgtctgg
atcggaggctccatcctggcctccctgtccaccttccagcagatgtggatcagcaagcag
gagtacgatgagtccggcccctccatcgtccaccgcaaatgcttc
Aminoacid sequence (375 amino acid):
Met?Asp?Asp?Glu?Ile?Ala?Ala?Leu?Val?Val?Asp?Asn?Gly?Ser?Gly?Met
Cys?Lys?Ala?Gly?Phe?Ala?Gly?Asp?Asp?Ala?Pro?Arg?Ala?Val?Phe?Pro
Ser?Ile?Val?Gly?Arg?Pro?Arg?His?Gln?Gly?Val?Met?Val?Gly?Met?Gly
Gln?Lys?Asp?Ser?Tyr?Val?Gly?Asp?Glu?Ala?Gln?Ser?Lys?Arg?Gly?Ile
Leu?Thr?Leu?Lys?Tyr?Pro?Ile?Glu?His?Gly?Ile?Val?Thr?Asn?Trp?Asp
Asp?Met?Glu?Lys?Ile?Trp?His?His?Thr?Phe?Tyr?Asn?Glu?Leu?Arg?Val
Ala?Pro?Lys?Lys?His?Pro?Val?Leu?Leu?Thr?Glu?Ala?Pro?Leu?Asn?Pro
Lys?Ala?Asn?Arg?Glu?Lys?Met?Thr?Gln?Ile?Met?Phe?Glu?Thr?Phe?Asn
Thr?Pro?Ala?Met?Tyr?Val?Ala?Ile?Gln?Ala?Val?Leu?Ser?Leu?Tyr?Ala
Ser?Gly?Arg?Thr?Thr?Gly?Ile?Val?Met?Asp?Ser?Gly?Asp?Gly?Val?Thr
His?Thr?Val?Pro?Ile?Tyr?Glu?Gly?Tyr?Ala?Leu?Pro?His?Ala?Ile?Leu
Arg?Leu?Asp?Leu?Ala?Gly?Arg?Asp?Leu?Thr?Asp?Tyr?Leu?Met?Lys?Ile
Leu?Thr?Glu?Arg?Gly?Tyr?Ser?Phe?Thr?Thr?Thr?Ala?Glu?Arg?Glu?Ile
Val?Arg?Asp?Ile?Lys?Glu?Lys?Leu?Cys?Tyr?Val?Ala?Leu?Asp?Phe?Glu
Gln?Glu?Met?Gly?Thr?Ala?Ala?Ser?Ser?Ser?Ser?Lys?Glu?Lys?Ser?Tyr
Glu?Leu?Pro?Asp?Gly?Gln?Val?Ile?Thr?Ile?Gly?Asn?Glu?Arg?Phe?Arg
Cys?Pro?Glu?Ala?Leu?Phe?Gln?Pro?Ser?Phe?Leu?Gly?Met?Glu?Ser?Cys
Gly?Ile?His?Glu?Thr?Thr?Tyr?Asn?Ser?Ile?Met?Lys?Cys?Asp?Val?Asp
Ile?Arg?Lys?Asp?Leu?Tyr?Ala?Asn?Thr?Val?Leu?Ser?Gly?Gly?Thr?Thr
Met?Tyr?Pro?Gly?Ile?Ala?Asp?Arg?Met?Gln?Lys?Glu?Ile?Thr?Ala?Leu
Ala?Pro?Ser?Thr?Met?Lys?Ile?Lys?Ile?Ile?Ala?Pro?Pro?Glu?Arg?Lys
Tyr?Ser?Val?Trp?Ile?Gly?Gly?Ser?Ile?Leu?Ala?Ser?Leu?Ser?Thr?Phe
Gln?Gln?Met?Trp?Ile?Ser?Lys?Gln?Glu?Tyr?Asp?Glu?Ser?Gly?Pro?Ser
Ile?Val?His?Arg?Lys?Cys?Phe
Above-mentioned purpose is achieved through the following technical solutions:
Dissect the yellowfin porgy and take out spleen, extract total RNA, make it to mix, carry out reverse transcription, the synthetic first chain cDNA with reverse transcription primer (oligo-dT joint primer).
From GenBank, download nearly source species (as rainbow trout, lefteye flounder, carp etc.) Actin muscle homologous gene CDS sequence, utilize Clustal W software to carry out the multisequencing comparison, determine conserved regions, according to conserved regions sequences Design degenerate primer, the amplified fragments size is about 1125bp.Adopt β-second eyeball phosphoramidite chemical method to carry out that DNA is synthetic to obtain following primer sequence after the design of primers:
F：5’ATg?gA(A/T)g(A/g)(T/C)gAA?ATC?gCC?gC?3’
R：5’TTA?gAA?gCA?TTT(g/A)Cg?gTg?gA 3’。
As template, carry out pcr amplification with degenerate primer with the synthetic first chain cDNA, institute's amplification PCR products detects with 1.2% agarose gel electrophoresis, and purifying reclaims the purpose product from gel.Then with the PCR product cloning of purifying in the pMD-18T carrier, transformed into escherichia coli JM-109 competent cell, the picking positive colony extracts plasmid DNA.After degenerated primer PCR detects, will have the segmental plasmid DNA of insertion and carry out two-way order-checking with the M13 universal primer.Order-checking institute calling sequence utilizes Clustal W software to splice can obtain yellowfin houttuyn actin gene.
Resulting PCR product is after carrying out separation detection on 1.2% the agarose gel electrophoresis, behind the PCR product purification, be cloned in the pMD-18T carrier transformed into escherichia coli JM-109 competent cell, the picking positive colony checks order to plasmid DNA with 3730 sequenators.Sequencing result BLAST software (
Http:// www.ncbi.nim.nih.gov/) carry out homology mensuration, be defined as yellowfin houttuyn actin dna homolog sequence.Comparison result:
Score E
Sequences?producing?significant?alignments： (Bits) Value
gi|56554009|gb|AAV97945.1|beta?actin?2[Rivulus?marmoratus]...
755 0.0
gi|52547951|gb|AAR97600.2|beta?actin[Epinepheius?coioides]...
753 0.0
gi|40362701|gb|AAR84618.1|beta?actin[Acanthopagrus?schlegelii]
753 0.0
gi|57977261|dbj|BAD88412.1|beta?cytoplasmic?actin[Pagrus?major
752 0.0
gi|19309743|emb|CAD27237.1|beta-actin[oncorhynchus?mykiss]...
752 0.0
gi|45709360|gb|AAH67566.1|Bactin2[Danio?rerio]＞gi|2822456|...
752 0.0
According to the report of Iron1995,, prove that promptly sequence is with a kind of gene order as long as then have absolute homology between two sequences with E-Value value after the comparison of BLAST software less than 0.005 between two sequences.After comparing result: target gene sequences BLAST comparison, with the E value of the Actin muscle of other species definitely less than 0.005, so prove that the goal gene of being cloned is the homologous sequence of Actin muscle.
The acquisition of this gene order not only makes and obtains to have immunocompetent recombinant protein by extracorporeal recombination and become possibility, and provides theoretical basis for further inquiring into its function.Actin muscle can be used as the internal reference of the expression in vivo rule of other functional genes of research, come the intravital again levels of replication of regulatory gene, therefore obtain yellowfin houttuyn actin gene order and make the expression in vivo regulation rule of further research functional gene become possibility.
Embodiment
The present invention is further elaborated below by following embodiment, but content of the present invention is not limited to this fully.
1. the extraction of total RNA
Get fresh and alive healthy yellowfin porgy (the about 400g of body weight) and in the laboratory, behind the temporarily foster 2d (about 24 ℃ of water temperature, air-pump inflating), inject lipopolysaccharides (LPS, 10 μ g/mL) 200 μ L from the thoracic cavity.After stimulating 6h, dissect the fish body and take out the about 100mg of spleen, (Gibco carries out homogenate in Japan), extracts total RNA according to the test kit operation instruction to put into 1mL Trizol respectively.
2.cDNA first chain is synthetic
Getting the total RNA 5 μ g of yellowfin porgy mixes with reverse transcription primer (oligo-dT joint primer) 1 μ L (10pmol/L), behind 70 ℃ of heating 5min, place on ice immediately, add 5 * buffer then, 2.5mmol/L dNTP mixed solution, Ribonuclease Inhibitor, M-MLV ThermoScript II, reaction system are 25 μ L.Reaction process is 42 ℃ of 60min, 70 ℃ of 15min, and it is standby to put into-80 ℃ of preservations at last.
3. degenerate primer design considerations, primer synthetic method
From GenBank, download nearly source species (as rainbow trout, lefteye flounder, carp etc.) Actin muscle homologous gene CDS sequence, utilize Clustal W software to carry out the multisequencing comparison, determine conserved regions, according to conserved regions sequences Design degenerate primer, the amplified fragments size is about 1125bp.Adopting β-second eyeball phosphoramidite chemical method to carry out DNA after the design of primers synthesizes.
Primer sequence is:
F：5’ATg?gA(A/T)g(A/g)(T/C)gAA?ATC?gCC?gC?3’
R：5’TTA?gAA?gCA?TTT(g/A)Cg?gTg?gA?3’。
4. the clone of yellowfin houttuyn actin gene cDNA complete sequence
With the degenerate primer of the conserved regions design of nearly source species actin gene CDS sequence, the amplified fragments size is about 1125bp.
As template, carry out pcr amplification with degenerate primer with the above-mentioned synthetic first chain cDNA, reaction system is: 10x PCR reaction buffer 5 μ L, 25mmol/L MgCl
23 μ L, 2.5mmol/L dNTP 2 μ L, each 2 μ L of 10nmol/L primer dTF and dTR, Taq enzyme 1.25U is supplemented to 50 μ L with PCR water with reaction system.Reaction conditions is: 1 circulation, 94 ℃ of sex change 5min; 35 circulations: 94 ℃ of sex change 45s, 54 ℃ of annealing 45s, 72 ℃ are extended 45s; 1 circulation, 72 ℃ are extended 10min; 4 ℃ of insulations.Institute's amplification PCR products detects with 1.2% agarose gel electrophoresis, and purifying reclaims the purpose product from gel.Then with the PCR product cloning of purifying in the pMD-18T carrier, transformed into escherichia coli JM-109 competent cell, the picking positive colony extracts plasmid DNA.After degenerated primer PCR detects, will have the segmental plasmid DNA of insertion and carry out two-way order-checking with the M13 universal primer.Order-checking institute calling sequence utilizes Clustal W software to splice.
5. to the mensuration of yellowfin houttuyn actin gene
Resulting PCR product is after carrying out separation detection on 1.2% the agarose gel electrophoresis, behind the PCR product purification, be cloned in the pMD-18T carrier transformed into escherichia coli JM-109 competent cell, the picking positive colony checks order to plasmid DNA with 3730 sequenators.Sequencing result BLAST software (
Http:// www.ncbi.nim.nih.gov/) carry out homology mensuration, be defined as yellowfin houttuyn actin dna homolog sequence.Comparison result:
Score E
Sequences?producing?significant?alignments： (Bits) Value
gi|56554009|gb|AAV97945.1|beta?actin?2[Rivulus?marmoratus]...
755 0.0
gi|52547951|gb|AAR97600.2|beta?actin[Epinephelus?coioides]...
753 0.0
gi|40362701|gb|AAR84618.1|beta?actin[Acanthopagrus?schlegelii]
753 0.0
gi|57977261|dbj|BAD88412.1|beta?cytoplasmic?actin[Pagrus?major
752 0.0
gi|19309743|emb|CAD27237.1|beta-actin[Oncorhynchus?mykiss]...
752 0.0
gi|45709360|gb|AAH67566.1|Bactin2[Danio?rerio]＞gi|2822456|....
752 0.0
According to the report of Iron1995,, prove that promptly sequence is with a kind of gene order as long as then have absolute homology between two sequences with E-Value value after the comparison of BLAST software less than 0.005 between two sequences.After seeing target gene sequences BLAST comparison from comparing result, with the E value of the Actin muscle of other species definitely less than 0.005, so prove that the goal gene of being cloned is the homologous sequence of Actin muscle.
Sequence table
＜110〉Nanhai Aquatic Inst., Chinese Aquatic Scientific Research Inst
＜120〉gene order of yellowfin houttuyn actin
<160>2
<210>1
<211>1125
<212>RNA
＜213〉yellowfin porgy (Sparus latus)
<220>
<221>CDS
<222>(1)...(1125)
<400>1
atg?gat?gat?gaa?atc?gcc?gca?ctg?gtt?gtt?gac?aac?gga?tcc?ggt?atg 48
Met?Asp?Asp?Glu?Ile?Ala?Ala?Leu?Val?Val?Asp?Asn?Gly?Ser?Gly?Met 16
1 7 13
tgc?aaa?gcc?ggt?ttc?gcc?gga?gac?gac?gcc?cct?cgt?gct?gtc?ttc?ccc 96
Cys?Lys?Ala?Gly?Phe?Ala?Gly?Asp?Asp?Ala?Pro?Arg?Ala?Val?Phe?Pro 32
17 23 29
tcc?atc?gtc?ggt?cgc?ccc?agg?cat?cag?ggt?gtg?atg?gtg?ggt?atg?ggc 144
Ser?Ile?Val?Gly?Arg?Pro?Arg?His?Gln?Gly?Val?Met?Val?Gly?Met?Gly 48
33 39 45
cag?aag?gac?agc?tac?gtt?ggt?gat?gag?gcc?cag?agc?aag?aga?ggt?atc 192
Gln?Lys?Asp?Ser?Tyr?Val?Gly?Asp?Glu?Ala?Gln?Ser?Lys?Arg?Gly?Ile 64
49 55 61
ctg?acc?ctg?aag?tac?ccc?atc?gag?cac?ggt?att?gtg?acc?aac?tgg?gat 240
Leu?Thr?Leu?Lys?Tyr?Pro?Ile?Glu?His?Gly?Ile?Val?Thr?Asn?Trp?Asp 80
65 71 77
gac?atg?gag?aag?atc?tgg?cat?cac?acc?ttc?tac?aac?gag?ctg?aga?gtt 288
Asp?Met?Glu?Lys?Ile?Trp?His?His?Thr?Phe?Tyr?Asn?Glu?Leu?Arg?Val 96
81 87 93
gcc?cct?gag?gag?cac?cct?gtc?ctg?ctc?aca?gag?gcc?ccc?ctg?aac?ccc 336
Ala?Pro?Lys?Lys?His?Pro?Val?Leu?Leu?Thr?Glu?Ala?Pro?Leu?Asn?Pro 112
97 103 109
aaa?gcc?aac?agg?gag?aag?atg?acc?cag?atc?atg?ttc?gag?acc?ttc?aac 384
Lys?Ala?Asn?Arg?Glu?Lys?Met?Thr?Gln?Ile?Met?Phe?Glu?Thr?Phe?Asn 128
113 119 125
acc?cct?gcc?atg?tac?gtt?gcc?atc?cag?gct?gtg?ctg?tcc?ctg?tat?gcc 432
Thr?Pro?Ala?Met?Tyr?Val?Ala?Ile?Gln?Ala?Val?Leu?Ser?Leu?Tyr?Ala 144
129 135 141
tct?ggt?cgt?acc?act?ggt?atc?gtc?atg?gac?tcc?ggt?gat?ggt?gtg?acc 480
Ser?Gly?Arg?Thr?Thr?Gly?Ile?Val?Met?Asp?Ser?Gly?Asp?Gly?Val?Thr 160
145 151 157
cac?aca?gtg?ccc?atc?tat?gag?ggc?tat?gcc?ctg?ccc?cac?gcc?atc?ctg 528
His?Thr?Val?Pro?Ile?Tyr?Glu?Gly?Tyr?Ala?Leu?Pro?His?Ala?Ile?Leu 176
161 167 173
cgt?ctg?gac?ttg?gcc?ggc?cgc?gac?ctc?aca?gac?tac?ctc?atg?aag?atc 576
Arg?Leu?Asp?Leu?Ala?Gly?Arg?Asp?Leu?Thr?Asp?Tyr?Leu?Met?Lys?Ile 192
177 183 189
ctg?aca?gag?cgt?ggc?tac?tcc?ttc?acc?acc?aca?gcc?gag?agg?gaa?atc 624
Leu?Thr?Glu?Arg?Gly?Tyr?Ser?Phe?Thr?Thr?Thr?Ala?Glu?Arg?Glu?Ile 208
193 199 205
gtg?cgt?gac?atc?aag?gag?aag?ctg?tgc?tat?gtc?gcc?ctg?gac?ttc?gag 672
Val?Arg?Asp?Ile?Lys?Glu?Lys?Leu?Cys?Tyr?Val?Ala?Leu?Asp?Phe?Glu 224
209 215 221
cag?gag?atg?ggc?act?gct?gcc?tcc?tcc?tcc?tcc?ctg?gag?aag?agc?tat 720
Gln?Glu?Met?Gly?Thr?Ala?Ala?Ser?Ser?Ser?Ser?Lys?Glu?Lys?Ser?Tyr 240
225 231 237
gag?ctg?cct?gac?gga?cag?gtc?atc?acc?atc?ggc?aat?gag?agg?ttc?cgt 768
Glu?Leu?Pro?Asp?Gly?Gln?Val?Ile?Thr?Ile?Gly?Asn?Glu?Arg?Phe?Arg 256
241 247 253
tgc?cca?gag?gcc?ctc?ttc?cag?cca?tcc?ttc?ctc?ggt?atg?gag?tcc?tgc 816
Cys?Pro?Glu?Ala?Leu?Phe?Gln?Pro?Ser?Phe?Leu?Gly?Met?Glu?Ser?Cys 272
257 263 269
gga?atc?cat?gag?acc?acc?tac?aac?agc?atc?atg?aag?tgt?gat?gtc?gac 864
Gly?Ile?His?Glu?Thr?Thr?Tyr?Asn?Ser?Ile?Met?Lys?Cys?Asp?Val?Asp 288
273 279 285
atc?cgt?aag?gac?ctg?tat?gcc?aac?aca?gtg?ctg?tct?gga?ggt?acc?acc 912
Ile?Arg?Lys?Asp?Leu?Tyr?Ala?Asn?Thr?Val?Leu?Ser?Gly?Gly?Thr?Thr 304
289 295 301
atg?tac?cct?ggc?atc?gct?gac?agg?atg?cag?aag?gag?atc?aca?gcc?ctg 960
Met?Tyr?Pro?Gly?Ile?Ala?Asp?Arg?Met?Gln?Lys?Glu?Ile?Thr?Ala?Leu 320
305 311 317
gcc?ccg?tcc?acc?atg?aag?att?aag?atc?att?gcc?cca?cct?gag?cgt?aaa 1008
Ala?Pro?Ser?Thr?Met?Lys?Ile?Lys?Ile?Ile?Ala?Pro?Pro?Glu?Arg?Lys 336
321 327 333
tac?tct?gtc?tgg?atc?gga?ggc?tcc?atc?ctg?gcc?tcc?ctg?tcc?acc?ttc 1056
Tyr?Ser?Val?Trp?Ile?Gly?Gly?Ser?Ile?Leu?Ala?Ser?Leu?Ser?Thr?Phe 352
337 343 349
cag?cag?atg?tgg?atc?agc?aag?cag?gag?tac?gat?gag?tcc?ggc?ccc?tcc 1104
Gln?Gln?Met?Trp?Ile?Ser?Lys?Gln?Glu?Tyr?Asp?Glu?Ser?Gly?Pro?Ser 368
353 359 365
atc?gtc?cac?cgc?aaa?tgc?ttc 1125
Ile?Val?His?Arg?Lys?Cys?Phe 375
369 375
<210>2
<2ll>375
<212>PRT
＜213〉yellowfin porgy (Sparus latus)
<400>2
Met?Asp?Asp?Glu?Ile?Ala?Ala?Leu?Val?Val?Asp?Asn?Gly?Ser?Gly?Met 16
1 7 13
Cys?Lys?Ala?Gly?Phe?Ala?Gly?Asp?Asp?Ala?Pro?Arg?Ala?Val?Phe?Pro 32
17 23 29
Ser?Ile?Val?Gly?Arg?Pro?Arg?hIs?Gln?Gly?Val?Met?Val?Gly?Met?Gly 48
33 39 45
Gln?Lys?Asp?Ser?Tyr?Val?Gly?Asp?Glu?Ala?Gln?Ser?Lys?Arg?Gly?Ile 64
49 55 61
Leu?Thr?Leu?Lys?Tyr?Pro?Ile?Glu?His?Gly?Ile?Val?Thr?Asn?Trp?Asp 80
65 71 77
Asp?Met?Glu?Lys?Ile?Trp?His?His?Thr?Phe?Tyr?Asn?Glu?Leu?Arg?Val 96
81 87 93
Ala?Pro?Lys?Lys?His?Pro?Val?Leu?Leu?Thr?Glu?Ala?Pro?Leu?Asn?Pro 112
97 103 109
Lys?Ala?Asn?Arg?Glu?Lys?Met?Thr?Gln?Ile?Met?Phe?Glu?Thr?Phe?Asn 128
113 119 125
Thr?Pro?Ala?Met?Tyr?Val?Ala?Ile?Gln?Ala?Val?Leu?Ser?Leu?Tyr?Ala 144
129 135 141
Ser?Gly?Arg?Thr?Thr?Gly?Ile?Val?Met?Asp?Ser?Gly?Asp?Gly?Val?Thr 160
145 151 157
His?Thr?Val?Pro?Ile?Tyr?Glu?Gly?Tyr?Ala?Leu?Pro?His?Ala?Ile?Leu 176
161 167 173
Arg?Leu?Asp?Leu?Ala?Gly?Arg?Asp?Leu?Thr?Asp?Tyr?Leu?Met?Lys?Ile 192
177 183 189
Leu?Thr?Glu?Arg?Gly?Tyr?Ser?Phe?Thr?Thr?Thr?Ala?Glu?Arg?Glu?Ile 208
193 199 205
Val?Arg?Asp?Ile?Lys?Glu?Lys?Leu?Cys?Tyr?Val?Ala?Leu?Asp?Phe?Glu 224
209 215 221
Gln?Glu?Met?Gly?Thr?Ala?Ala?Ser?Ser?Ser?Ser?Lys?Glu?Lys?Ser?Tyr 240
225 231 237
Glu?Leu?Pro?Asp?Gly?Gln?Val?Ile?Thr?Ile?Gly?Asn?Glu?Arg?Phe?Arg 256
241 247 253
Cys?Pro?Glu?Ala?Leu?Phe?Gln?Pro?Ser?Phe?Leu?Gly?Met?Glu?Ser?Cys 272
257 263 269
Gly?Ile?His?Glu?Thr?Thr?Tyr?Asn?Ser?Ile?Met?Lys?Cys?Asp?Val?Asp 288
273 279 285
Ile?Arg?Lys?Asp?Leu?Tyr?Ala?Asn?Thr?Val?Leu?Ser?Gly?Gly?Thr?Thr 304
289 295 301
Met?Tyr?Pro?Gly?Ile?Ala?Asp?Arg?Met?Gln?Lys?Glu?Ile?Thr?Ala?Leu 320
305 311 317
Ala?Pro?Ser?Thr?Met?Lys?Ile?Lys?Ile?Ile?Ala?Pro?Pro?Glu?Arg?Lys 336
321 327 333
Tyr?Ser?Val?Trp?Ile?Gly?Gly?Ser?Ile?Leu?Ala?Ser?Leu?Ser?Thr?Phe 352
337 343 349
Gln?Gln?Met?Trp?Ile?Ser?Lys?Gln?Glu?Tyr?Asp?Glu?Ser?Gly?Pro?Ser 368
353 359 365
Ile?Val?His?Arg?Lys?Cys?Phe 375
369 375
Claims (1)
1, a kind of yellowfin houttuyn actin gene is characterized in that having following RNA Nucleotide and amino acid sequence corresponding:
atg?gat?gat?gaa?atc?gcc?gca?ctg?gtt?gtt?gac?aac?gga?tcc?ggt?atg 48
Met?Asp?Asp?Glu?Ile?Ala?Ala?Leu?Val?Val?Asp?Asn?Gly?Ser?Gly?Met 16
1 7 13
tgc?aaa?gcc?ggt?ttc?gcc?gga?gac?gac?gcc?cct?cgt?gct?gtc?ttc?ccc 96
Cys?Lys?Ala?Gly?Phe?Ala?Gly?Asp?Asp?Ala?Pro?Arg?Ala?Val?Phe?Pro 32
17 23 29
tcc?atc?gtc?ggt?cgc?ccc?agg?cat?cag?ggt?gtg?atg?gtg?ggt?atg?ggc 144
Ser?Ile?Val?Gly?Arg?Pro?Arg?His?Gln?Gly?Val?Met?Val?Gly?Met?Gly 48
33 39 45
cag?aag?gac?agc?tac?gtt?ggt?gat?gag?gcc?cag?agc?aag?aga?ggt?atc 192
Gln?Lys?Asp?Ser?Tyr?Val?Gly?Asp?Glu?Ala?Gln?Ser?Lys?Arg?Gly?Ile 64
49 55 61
ctg?acc?ctg?aag?tac?ccc?atc?gag?cac?ggt?att?gtg?acc?aac?tgg?gat 240
Leu?Thr?Leu?Lys?Tyr?Pro?Ile?Glu?His?Gly?Ile?Val?Thr?Asn?Trp?Asp 80
65 71 77
gac?atg?gag?aag?atc?tgg?cat?cac?acc?ttc?tac?aac?gag?ctg?aga?gtt 288
Asp?Met?Glu?Lys?Ile?Trp?His?His?Thr?Phe?Tyr?Asn?Glu?Leu?Arg?Val 96
81 87 93
gcc?cct?gag?gag?cac?cct?gtc?ctg?ctc?aca?gag?gcc?ccc?ctg?aac?ccc 336
Ala?Pro?Lys?Lys?His?Pro?Val?Leu?Leu?Thr?Glu?Ala?Pro?Leu?Asn?Pro 112
97 103 109
aaa?gcc?aac?agg?gag?aag?atg?acc?cag?atc?atg?ttc?gag?acc?ttc?aac 384
Lys?Ala?Asn?Arg?Glu?Lys?Met?Thr?Gln?Ile?Met?Phe?Glu?Thr?Phe?Asn 128
113 119 125
acc?cct?gcc?atg?tac?gtt?gcc?atc?cag?gct?gtg?ctg?tcc?ctg?tat?gcc 432
Thr?Pro?Ala?Met?Tyr?Val?Ala?Ile?Gln?Ala?Val?Leu?Ser?Leu?Tyr?Ala 144
129 135 141
tct?ggt?cgt?acc?act?ggt?atc?gtc?atg?gac?tcc?ggt?gat?ggt?gtg?acc 480
Ser?Gly?Arg?Thr?Thr?Gly?Ile?Val?Met?Asp?Ser?Gly?Asp?Gly?Val?Thr 160
145 151 157
cac?aca?gtg?ccc?atc?tat?gag?ggc?tat?gcc?ctg?ccc?cac?gcc?atc?ctg 528
His?Thr?Val?Pro?Ile?Tyr?Glu?Gly?Tyr?Ala?Leu?Pro?His?Ala?Ile?Leu 176
161 167 173
cgt?ctg?gac?ttg?gcc?ggc?cgc?gac?ctc?aca?gac?tac?ctc?atg?aag?atc 576
Arg?Leu?Asp?Leu?Ala?Gly?Arg?Asp?Leu?Thr?Asp?Tyr?Leu?Met?Lys?Ile 192
177 183 189
ctg?aca?gag?cgt?ggc?tac?tcc?ttc?acc?acc?aca?gcc?gag?agg?gaa?atc 624
Leu?Thr?Glu?Arg?Gly?Tyr?Ser?Phe?Thr?Thr?Thr?Ala?Glu?Arg?Glu?Ile 208
193 199 205
gtg?cgt?gac?atc?aag?gag?aag?ctg?tgc?tat?gtc?gcc?ctg?gac?ttc?gag 672
Val?Arg?Asp?Ile?Lys?Glu?Lys?Leu?Cys?Tyr?Val?Ala?Leu?Asp?Phe?Glu 224
209 215 221
cag?gag?atg?ggc?act?gct?gcc?tcc?tcc?tcc?tcc?ctg?gag?aag?agc?tat 720
Gln?Glu?Met?Gly?Thr?Ala?Ala?Ser?Ser?Ser?Ser?Lys?Glu?Lys?Ser?Tyr 240
225 231 237
gag?ctg?cct?gac?gga?cag?gtc?atc?acc?atc?ggc?aat?gag?agg?ttc?cgt 768
Glu?Leu?Pro?Asp?Gly?Gln?Val?Ile?Thr?Ile?Gly?Asn?Glu?Arg?Phe?Arg 256
241 247 253
tgc?cca?gag?gcc?ctc?ttc?cag?cca?tcc?ttc?ctc?ggt?atg?gag?tcc?tgc 816
Cys?Pro?Glu?Ala?Leu?Phe?Gln?Pro?Ser?Phe?Leu?Gly?Met?Glu?Ser?Cys 272
257 263 269
gga?atc?cat?gag?acc?acc?tac?aac?agc?atc?atg?aag?tgt?gat?gtc?gac 864
Gly?Ile?His?Glu?Thr?Thr?Tyr?Asn?Ser?Ile?Met?Lys?Cys?Asp?Val?Asp 288
273 279 285
atc?cgt?aag?gac?ctg?tat?gcc?aac?aca?gtg?ctg?tct?gga?ggt?acc?acc 912
Ile?Arg?Lys?Asp?Leu?Tyr?Ala?Asn?Thr?Val?Leu?Ser?Gly?Gly?Thr?Thr 304
289 295 301
atg?tac?cct?ggc?atc?gct?gac?agg?atg?cag?aag?gag?atc?aca?gcc?ctg 960
Met?Tyr?Pro?Gly?Ile?Ala?Asp?Arg?Met?Gln?Lys?Glu?Ile?Thr?Ala?Leu 320
305 311 317
gcc?cca?tcc?acc?atg?aag?att?aag?atc?att?gcc?cca?cct?gag?cgt?aaa 1008
Ala?Pro?Ser?Thr?Met?Lys?Ile?Lys?Ile?Ile?Ala?Pro?Pro?Glu?Arg?Lys 336
321 327 333
tac?tct?gtc?tgg?atc?gga?ggc?tcc?atc?ctg?gcc?tcc?ctg?tcc?acc?ttc 1056
Tyr?Ser?Val?Trp?Ile?Gly?Gly?Ser?Ile?Leu?Ala?Ser?Leu?Ser?Thr?Phe 352
337 343 349
cag?cag?atg?tgg?atc?agc?aag?cag?gag?tac?gat?gag?tcc?ggc?ccc?tcc 1104
Gln?Gln?Met?Trp?Ile?Ser?Lys?Gln?Glu?Tyr?Asp?Glu?Ser?Gly?Pro?Ser 368
353 359 365
atc?gtc?cac?cgc?aaa?tgc?ttc 1125
Ile?Val?His?Arg?Lys?Cys?Phe 375
369 375
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|---|---|---|---|
| CN 200610037560 CN1928086A (en) | 2006-09-07 | 2006-09-07 | Gene sequence of sparus latus houttuyn actin |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
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