CN1482253A

CN1482253A - Growth hormone gene of southern siluroid

Info

Publication number: CN1482253A
Application number: CNA03128373XA
Authority: CN
Inventors: 平宋; 宋平
Original assignee: Wuhan University WHU
Current assignee: Wuhan University WHU
Priority date: 2003-07-24
Filing date: 2003-07-24
Publication date: 2004-03-17

Abstract

The growth hormone gene of Southern catfish is growth hormone cDNA full length sequence of Southern catfish obtained with hypophysis mRNA as template and through RT-PCR, 5'RACE and 3'RACE method. The sequence may be used as destination gene segment for transgenic fish breeding, and may be also inserted into expression vector, such as pET-30a(t), pPIC9K, etc to constitute engineering bacterium, such as BL21(DE3), BL21, etc. to produce growth hormone. The method is simple and low in cost, can speed fish's growth and may be used in aquatic cultivation.

Description

The Silurus meridionalis growth hormone gene

Technical field

The invention belongs to biological technical field, particularly relate to the Silurus meridionalis growth hormone gene.

Background technology

Silurus meridionalis Chen (Silurus meridionalis) belongs to SILURIFORMES, catfish section, and catfish belongs to, and is commonly called as the river catfish, the great river catfish.Main product is in the korneforos, great river of the Yangtze valley, be a kind of be the large-scale economic fish of staple food with the fish, about 2～5 kilograms of common individual weight, maximum individuality can reach more than 30 kilograms.It with the very wide the sort of little individual catfish (being commonly called as native catfish, catfish cripple) that distributes be belong to together not of the same race.Silurus meridionalis Chen contains meat rate height, and protein and vitamin contents are abundant, fine and tender taste, and delicious flavour is one of the place of production masses senior fish of very praising highly.Sichuan Province aquatic products institute promptly carried out moving the investigative test that domesticating and pond are propagated artificially to it since 1985, the result shows, Silurus meridionalis Chen has growth and (supports under the condition in the pond soon, the fry of April breeding then to the end of the year average cabrage can reach 0.4～0.5 kilogram, can grow in 1 year to 1.5 kilograms, reach 2.7 kilograms on the 3rd year), culture-cycle short (year fry is raised can become marketable fish in 5～6 months), adapt to strong (China south of low temperature ability, can both survive the winter in the open air naturally in the north), feeding habits can change (be converted into by special living fish shrimp and eat compound feed), consumption market wide (all being in great demand both at home and abroad), economic benefit higher (the mu profit is to culture 3～6 times of conventional variety) also possesses the advantages such as competition potential of foreign exchange earning.

(growth hormone is that it is a kind of somatomedin with extensive physiological function by a kind of single peptide chain protein of leaf eosinophil excretory hormone between hypophysis GH) to tethelin.Can influence several all types of organizations and cells of exhaling, it mainly acts on is growth and the differentiation that stimulates bone, chondrocyte; Conditioning protein, sugar and fatty metabolism.Tethelin performance function is mainly by two approach: the one, and inducing hepatocyte, myocyte produce somatomdein, work indirectly through somatomdein again; The 2nd, directly act on target cell and produce physiological effect.Any mode all needs at first to be excited a series of biochemical events and finally produced biological effect by receptor-mediated again with the combination of cell surface specific receptors.(Sun?et?al.Foreign?Medical?Sectoin?ofPathophysiology?and?Clinical?Medicine?1999，19(1))

Fish growth hormone (fish growth hormone, fGH) be polypeptide hormone by a kind of single subunit of fish pituitary gland mesenchymal cell excretory, its basic function be promote the fish bulk-growth grow and participate in multiple short anabolic action (Pan steps on etc. the Taiwan Straits, 2001, Vol.20 supplementary issue 85-89), the fish growth promotor that is considered to tool potentiality.Because fish growth hormone can be accelerated fish growth greatly, in aquaculture field great application value is arranged, and causes people's attention day by day.(Pan steps on etc. the Taiwan Straits, 2001, Vol.20 supplementary issue 85-89) present, fish growth hormone research has the separated and clone of recombinant human growth hormone cDNA of Oncorhynchi, rainbow trout, coho, common eel, tuna, tilapia, carp, lefteye flounder, porgy, grass carp, Atlantic Ocean mandarin fish etc. gradually deeply to molecular level; Wherein common eel, tilapia, tuna, rainbow trout efficiently express in engineering bacteria or zooblast.(Xu Bin etc. Oceanologia et Limnologia Sinica, 1997 Vol28, No5 553-556) the fish growth hormone research of using in aquaculture is carried out widely, because content own is seldom in the fish body for fish growth hormone, will be by in the fish body, directly extracting, cost is bigger, and it is loaded down with trivial details that technology also compares, and the applied research of all present stages mainly concentrates on two aspects: the breeding of the long hormone gene fish of reincarnation and the research of albonubes somatotropic hormone gene engineering.(Pan steps on etc. the Taiwan Straits, 2001, Vol.20 supplementary issue 85-89) and the prerequisite of using above biotechnology is the global cDNA sequence that at first obtains fish growth hormone, this is the basis of all work.

The fish growth hormone gene is the goal gene that is used for the genetically engineered fish breeding the earliest.The recombinant DNA molecules that the utilization transgenic technology will contain albonubes somatotropic hormone gene imports in the zygote of fish, make it in the fish body with karyomit(e) generation recombination.Like this growth hormone gene of external source just can be in the fish body stable existence and expressed and produce corresponding biological function (Pan steps on etc. the Taiwan Straits, 2001, Vol.20 supplementary issue 85-89).

1985, Zhu Zuoyan etc. at first adopted the external source hgh gene microinjection to import in the goldfish zygote, make its integrate and expressing human tethelin (Xu Bin etc. Oceanologia et Limnologia Sinica, 1997 Vol.28, No5553-556).The research of the long hormone gene fish of reincarnation has obtained sizable progress since this.The importing of growth hormone genes such as loach, crucian, black carp, rainbow trout, ditch Nian, salmon, triangular bream is all achieved success in succession, also obtained genetically engineered fish can with foreign gene to the evidence of filial generation genetic stability (Pan steps on etc. the Taiwan Straits, 2001, Vol.20 supplementary issue 85-89).Zhang etc. (1990) import rainbow trout to carp, and fast and this specific character can heredity than common fish for the speed of growth.(Zhang P.Mol Reprod Devel, 1990,25 (1): 3-13) Du etc. (1992) imports Atlantic salmon with full albonubes somatotropic hormone gene, successfully obtain genetically engineered fish (Du doubly than the big 4-6 of control group fish, S.et al., 1992 Biotechnology, 10:176-181).Devlin etc. (1994) import coho with the full albonubes somatotropic hormone gene of sockeye, have obtained than control group sashimi (raw fish) long fast 11 times genetically engineered fish (Devlin, R H.et al., 1994, Nature, 371 (6 494): 209-210).. Liang Liqun etc. (1994) import crucian with external source Oncorhynchi growth hormone gene (dna fragmentation of the about 3.5kb in coding region comprises intron), and confirm its to crucian have growth promoting function (Liang Liqun etc. biotechnology, 4 (5): 24-26,1994).Tsai etc. (1995), Xu etc. (1995) adopt sperm to carry or electricimpulse is sent out full albonubes somatotropic hormone gene is imported respectively in loach and the lefteye flounder, have also obtained than the long fast 2.5 times genetically engineered fish of control group sashimi (raw fish).(Tsai，H.J?etal.，1995，Can.J.Aquat.Sci.，52(4)：776-787.Xu，B.et?al.，1995?Proceeding?of?FourthAsian?Fisheries?Forum，Edited?and?Published?by?Editorial?Committee?of?Journal?ofFisheries?of?China(Shanghai)，p.80.)

By the exogenous growth hormone gene being imported the acceptor fish and making it to express, can reach and make the cultured fishes purpose of growth fast, the particularly use of full fish gene element (promotor and the albonubes somatotropic hormone gene that are efficiently expressed by fish self constitute), both can improve expression level, and can eliminate mammals tethelin again and introduce the edible doubt that the fish body produces the human consumer.The final breakthrough of genetically engineered fish research on theoretical and method, to shortening the culture-cycle of main economic fish, improve output and economic benefit, and even to whole aquaculture will produce deep change (Xu Bin etc. Oceanologia et Limnologia Sinica, 1997 Vol.28, No5 553-556).

Fish growth hormone is a single polypeptide, there is not subunit, do not need glycosylation modifiedly, and disulfide linkage is less, easily renaturation, be specially adapted to carry out gene engineering expression (Pan steps on etc. the Taiwan Straits, 2001, the Vol.20 supplementary issue), and recombinant human growth hormone has identical biological activity with the spontaneous growth hormone, introduce the fish body and can not produce accumulation, do not influence the moiety of fish body.(Xu Bin etc. Oceanologia et Limnologia Sinica, 1997 Vol.28, No5 553-556) therefore, change albonubes somatotropic hormone gene over to the expressive host bacterium through vitro recombination, make up the engineering bacteria of stably express, foreign gene is great expression in host bacterial, can obtain corresponding tethelin behind the purifying.Experimental results show that the reorganization fish growth hormone of producing with gene engineering method has biologic activity, can by oral through enteron aisle by the fish bulk absorption, have very strong short fish bulk-growth effect (Pan steps on etc. the Taiwan Straits, 2001, Vol.20 supplementary issue).By improving technology, reducing cost is expected to be applied to culture fishery as fodder additives, produces considerable economic.

The gene engineering expression of the fish growth hormone that carries out mainly is to express by intestinal bacteria and yeast at present.Transform salmon growth hormone gene cDNA with round pcr clear wait (1999) of letter, again colibacillus expression plasmid pBV220 is arrived in improved gene clone, make up reorganization salmon growth hormone gene expression plasmid pBVGH18, and in intestinal bacteria, obtain to express, expression amount accounts for 10% of cell solubility total protein.Expression product after preliminary purification and renaturation as the fodder additives tilapia of throwing something and feeding, after raising through feeding of five weeks, the long rate of increase of body weight of experiment fish and body is compared respectively according to fast 119.5% and 64.5% (white person of outstanding talent etc., Journal of Agricultural Biotechnology, 1998,6 (4). letter is clear, white person of outstanding talent's fresh water fishery, 1999,29 (3): 3-5.).Chen Dan etc. (1998) have successfully expressed perch tethelin in methanol yeast.By technology clone the perch growth hormone gene, carry out vitro recombination with the carrier pHIL-D of the alcohol oxidase AOX gene promoter that contains methanol yeast, make up pHIL-GH yeast integrated plasmid, albonubes somatotropic hormone gene is under the control of AOX gene promoter like this, by with electrotransformation albonubes somatotropic hormone gene being incorporated on the zymic karyomit(e).Under the inducing of methyl alcohol, the AOX gene promoter has started the expression of albonubes somatotropic hormone gene, and the expression amount in shake-flask culture has reached 0.1 (m/m), can also improve output greatly through large-scale fermentation culture.In feeding the experiment of raising tilapia, obtained good promotes growth effect.(Chen Dan, Wang Wei. biological chemistry and biophysics progress, 1998,25 (1): 140-148.) two treasured, Chen Rongzhong, Yang Feng etc. (2001) yeast after with fermentation is made dry powder and is fed as fodder additives and raise perch, large yellow croaker, and the weighted average of experimental group increases more than 20% than control group.(two treasured, Chen Rongzhong, Yang Feng etc. perch tethelin is at the expression pilot experiment .2001 in yeast). Ma Jin etc. (1999) transform the rainbow trout growth hormone cDNA with round pcr, with improved gene clone to the intestinal bacteria yeast shuttle plasmid pMA91 that contains yeast PRK promotor, transformed saccharomyces cerevisiae Y33, expression amount accounts for 3% of cell total soluble protein, and expression product has tangible growth promoting function as the fodder additives tilapia of having thrown something and fed.(horse advances, white person of outstanding talent. the biotechnology journal, 1999,15 (4): 434-438.) Wu Aizhen etc. (1997) is with PRPL, and the Trp promotor is at expression in escherichia coli carp growth hormone recombinant DNA, through high density fermentation, the extraction of inclusion body, recover natural structure, every liter of fermentation can get fGH 2g, adds fGH and obtained good effect of increasing production in artificial diet.(Wu Aizhen, Sun Yukun. the biotechnology journal, 1997,13 (2): 200-205) white person of outstanding talent waits (1999) also to carry out the prokaryotic expression of carp growth hormone, has obtained good effect in the short growth experiment to tilapia.(white person of outstanding talent, horse advances. Chinese biological chemistry and molecular biology journal, 1999,15 (3): 409-412)

Summary of the invention

The object of the present invention is to provide the growth hormone gene of a kind of Silurus meridionalis Chen (Silurus meridionalis), adopt the method for reverse transcription, article one chain of synthetic cDNA, method with 5 ' RACE and 3 ' RACE obtains Silurus meridionalis growth hormone cDNA full length sequence again, method is easy, with low cost, accelerated fish growth, have using value in aquaculture field.

The present invention extracts total RNA from the pituitary gland of Silurus meridionalis Chen, adopt the method for reverse transcription, article one chain of synthetic cDNA, it is characterized in that it is that template is through RT-PCR with mRNA pituitary, the method of 5 ' RACE and 3 ' RACE obtains Silurus meridionalis growth hormone cDNA full length sequence, see sequence table one, its nucleotide sequence and deduced amino acid are seen sequence table two.In order to clone Silurus meridionalis growth hormone cDNA, codeword triplet (dna sequence dna) the design forward degenerated primer GHFC (5 ' CTG TTR YCT GAD GAM CGC 3 ') of the tethelin aminoacid sequence conserved regions LLPEER of known other the 23 kinds of fish of foundation; Design reverse degenerated primer GHRA (5 ' TCT ACA GGG TRC AGT TKG 3 ') according to the tethelin aminoacid sequence conserved regions SNCTL of known other 20 kinds of fish and the codeword triplet (dna sequence dna) of terminator codon.Article one chain with hypophysis cDNA is a template, obtains a treaty 0.4kbDNA master tape through 30 thermal cycling amplifications; Through the agarose gel electrophoresis analysis, this clip size is consistent with the clip size that known other animal is compared between two conserved regions.To cloning vector pGEM-T easy, positive colony (clone SsmB2) confirms that through digestion with restriction enzyme and dna sequence analysis this cloned sequence size is 444bp with this fragment cloning; Confirm that through inquiry this cloned sequence is a growth hormone cDNA portion homologous molecule.

In 30 thermal cycling amplification procedures, the renaturation process temperature that adopts of promptly annealing is most important.If the renaturation temperature is too high, primer can not with template renaturation well, amplification efficiency can be very low; If the renaturation temperature is too low, primer will produce non-special renaturation, thereby cause the amplification of nonspecific dna fragmentation, can't obtain the target DNA amplified fragments.We determine the clear bright annealing temperature of DNA cloning band and are optimum annealing temperature for 55 ℃ on the comparative analysis test base to various annealing temperatures (50 ℃, 52 ℃, 55 ℃, 58 ℃, 60 ℃).

In order further to clone this growth hormone cDNA 3 ' end, design gene specific primer GHFS1 according to above-mentioned cDNA fragment.Adopt special primer GHFS1 and universal primer AUAP, with cDNA is that template amplification goes out about 0.8kbDNA fragment, through clone (clone SsmB1-2) and dna sequence analysis, confirm that this cloned sequence is that complete Silurus meridionalis growth hormone cDNA3 ' holds its size to be 854bp.

Design gene specific primer GSP1 and GSP2 according to Silurus meridionalis growth hormone cDNA 444bp fragment sequence at last, to have Poly (C) hypophysis cDNA is template, adopt the RACE method to amplify the 603bp fragment, through clone (clone SsmB9) and dna sequence analysis, confirm that this cloned sequence is complete Silurus meridionalis growth hormone cDNA5 ' end.

By three amplifications and clone, successfully obtain the Silurus meridionalis growth hormone global cDNA.Its total length 1070nt (nucleotide) (not comprising poly (A)), the long 52nt of 5 ' end non-coding region reads the long 603nt of frame (ORF), and 3 ' holds the long 415nt of non-coding region.. at 21nt place, Poly (A) upstream, an eukaryotic cell conservative property sequence A ATAAA is arranged, this shears and adds the signal of Poly (A) for carrying out mRNA precursor 3 ' end.Form wherein 27 of acidic amino acids (13.5%) from the Silurus meridionalis growth hormone precursor that the cDNA sequence is derived by 200 amino acid, 23 of basic aminoacidss (11.5%), polare Aminosaeren 54 (27%) and 71 of hydrophobic amino acids (35.5%), its iso-electric point is 5.94.

(http://www.ncbi.nlm.nih.gov/) retrieves nucleotide sequence database (Genbank) and amino acid sequence database respectively in the NCBI website, being found to the nucleotide sequence that had not both had Silurus meridionalis growth hormone does not so far have the submission of its aminoacid sequence yet, confirms that we are the nucleotide sequence of cloning Silurus meridionalis growth hormone first really.When we search for BlastN and BlastP respectively in the NCBI website, find the particularly similarity all very high (seeing sequence table three) of SILURIFORMES fish growth hormone gene coding region and aminoacid sequence of the aminoacid sequence of clone's the coding region of Silurus meridionalis growth hormone nucleotide sequence and supposition in view of the above and other fish, thereby what can determine substantially that we clone is the tethelin nucleotide sequence of Silurus meridionalis Chen really.The target gene fragment that this sequence can be used as the genetically engineered fish breeding also can be inserted into expression vector (pET-30a (t), pPIC9K etc.) construction expression engineering bacteria (BL21 (DE3), BL21 etc.), produce tethelin, therefore in aquaculture, containing huge using value.

The present invention compared with prior art, have the following advantages and effect: raise normal diet and compare with feeding with traditional fish breeding, utilization transgenic breeding and fish growth hormone genetically engineered in aquaculture, method is easy, with low cost, accelerated the economic fish growth, increased output of aquatic products greatly, significantly improve the economic benefit of aquaculture, in aquaculture, had huge using value.Along with deepening continuously of research, the solution of variety of issue in the actual production, the growth hormone gene of the various economic classes that the clone comes out will produce huge economic benefit in aquaculture.

Description of drawings

Fig. 1, RT-PCR result (A) and RT-PCR cloned sequence (B) the M.LambdaDNA/HindIII/Eco.RI molecular criteria of cutting through enzyme: 1,5 end RT-PCR; 2, adopt the RT-PCR of primer GHFC and GHRA; 3,3 end RT-PCR; 4,5 and 6 be respectively clone SsmB, 9SsmB2 and SsmB1-2.

Embodiment

1, the extraction of Silurus meridionalis Chen pituitary gland RNA and cDNA first chain thereof is synthetic

Pituitary gland adopts guanidinium isothiocyanate-phenol-one step of chloroform extraction process to extract total RNA, be dissolved in an amount of distilled water through DNase I (TAKARA BIO) processing, phenol and chloroform extracting, dehydrated alcohol post precipitation after glass homogenizer homogenate.The total RNA that handled through DNase I with 1.5 μ g is a template, adopts primer 3AP (5 ' GGC CAC GCG TCG ACT AGT ACT (T) ₁₆3 '), with reversed transcriptive enzyme M-MLV (GIBCO BRL) synthetic first chain cDNA in 30 μ l reaction volumes.Chomczynski，P.，Sacchi，N.(1987)Single-step?method?of?RNA?isolationby?acid?guanidinium?thiocyanate-phenol-chloroform?extraction，Anal.Biochem.，162：156-159。

2, the segmental amplification of Silurus meridionalis Chen GH cDNA part

Conservative region according to the tethelin sequence of having delivered has designed pair of degenerate primers: forward primer GHFC (5 ' CTG TTR YCT GAD GAM CGC 3 ') and reverse primer GHRA (5 ' TCT ACA GGG TRCAGT TKG 3 ').Total cDNA 0.1 μ l is that template is carried out pcr amplification with the reverse transcription synthetic, contains 10mM TrisHCl (pH=8.3), 1.5mM MgCl in the 50 μ l reaction volumes ₂, 0.1% gelatin, the Taq archaeal dna polymerase (Promega) of 2 units, four kinds of each 200 μ M of dNTP, every kind of each 100pM of primer.The PCR condition is: 94 ℃ of pre-sex change 5min, and 94 ℃ of sex change 1min then, 55 ℃ of annealing 45s, 72 ℃ are extended 45s, and after totally 30 circulations, 72 ℃ are extended 7min.

3, the amplification of Silurus meridionalis growth hormone cDNA3 ' end (downstream area) and 5 ' end (upstream region)

Adopt the 3 ' end (downstream area) and 5 ' of 3 ' end and 5 ' end RACE (Rapid amplification of the cDNA ends) the Silurus meridionalis growth hormone cDNA that increases successively to hold (upstream region) respectively to obtain complete cDNA sequence.The special primer that adopts among the RACE is to design respectively according to the fragment that RT-PCR increases.

Growth hormone cDNA 3 ' end amplification: the first chain cDNA is a template with the 3AP synthetic, adopt gene specific primer GHFS1 (5 ' AGC TGA GCA AGA TCT TCC 3 ') and grappling universal primer AUAP (5 ' GGCCAC GCG TCG ACT AGT AC 3 ') to increase, its PCR reaction conditions and reaction content are all with " 2 ".

Hypophysis cDNA first chain 3 ' end poly (c) reaction: with (dT) ₁₈The synthetic first chain cDNA handles (being undertaken by producer's explanation) with RNaseH (TAKARA BIO), carry out purifying (QIAQuick PCR PurificationKit again, Qiagen), then through end deoxyribonucleic acid transferase (TdT, TAKARA BIO) processing adds that at 3 ' end poly (c), its reaction see relevant explanation.

Growth hormone cDNA 5 ' end amplification: first round PCR is a template with the cDNA through poly (c) tailing, increases with universal primer AAP (5 ' GGC CAC GCG TCG ACT AGT ACG GGI IGG GII GGG IIG 3 ') and gene specific primer GSP1 (5 ' AAT CCA GGG ATC TCC TGC 3 '); Second to take turns PCR be template with the product of first round PCR, increases with universal primer AUAP and gene specific primer GSP2 (5 ' TAG GTCTCC ACT TTG TGC 3 '), to obtain growth hormone cDNA 5 ' end.Wherein first round PCR carries out thermal cycling 35 times, and second takes turns PCR carries out thermal cycling 30 times.In addition, twice condition is identical.

4, the comparison of the clone of PCR product, dna sequence analysis and aminoacid sequence

Directly be cloned into pGEM-T easy carrier (Promega) after the PCR product is purified, producer's explanation is seen in its concrete operations.Screening positive clone is transferred to Shanghai associating genome company, adopts carrier primer T7, SP6 and gene specific primer, utilizes the full-automatic sequenator of ABI-377 to carry out two-way order-checking.Hold the sequencing result of RACE to splice RT-PCR and 3 ' end, 5 ', obtain complete cDNA sequence with DNAStar (V.4.01).And carry out the comparative analysis of sequence with DNAStar.Land the NCBI website ( Http:// www.ncbi.nlm.nih.gov/), retrieve nucleotide sequence database (Genbank) and amino acid sequence database respectively, whether former someone never obtains with definite cDNA sequence of cloning, and carries out the sequence similarity search with BlastN and BlastP.

Sequence table

Sequence table one: the full length cDNA sequence of the tethelin of Silurus meridionalis Chen ( Silurus meridionalis ) .＜110〉＜120〉＜160〉2＜210〉1＜211〉1088＜212〉DNA＜213〉 ( Silurus meridionatis )＜220〉＜221〉CDS＜222〉 ( 53 )-( 656 )＜220〉＜221〉Signal peptide＜222〉 ( 53 )-( 119 )＜220〉＜221〉Poly ( A ) Signal＜222〉 ( 1070 )-( 1088 )＜400〉11 aaagtctacc tcgagatttc agcaggatct gagaaagttt cttctgggaa aaatggctag 6061 agtgtgggtg ctgctctccg tggtgctggc gagtttgttc tttagtcagg gcgcgacatt 120121 cgagaaccag cgtctcttca acaacgcagt catccgtgtg caacaccttc atcagctggc 180181 tgccaaaatg atggatgact ttgaggaagc tttgctacct gaagaacgca aacagctgag 240241 caagatcttc ccattgtcct tctgcaattc agactccatc gaagctccat caggcaaaga 300301 cgaaacccag aagagttctg tgttgaagct gctgcacacc tcctaccgtc tgatcgagtc 360361 atgggagttc cccagtaaga acctgggcaa ccccaatcac atctcagaga agctggctga 420421 cctgaaaatg ggcatcggtg tgcttatcga gggatgtctg gatggacaaa ccagcctgga 480481 tgagaacgac tctctggctc cacccttcga ggatttctac cagaccctaa ccgagggaaa 540541 cctgaggaag agcttccgtc tgctctcctg cttcaagaag gacatgcaca aagtggagac 600601 ctatctgagc gtggccaagt gcaggagatc cctggattcc aactgcaccc tgtagagggc 660661 atgatttagc ctcagcctgt gatttgaatt gttttaatag tccaggtgta gctaaattat 720721 taatcctttt cccctggatt tcacttctaa ctttaagtat ttattttctc ctcctccatt 780781 ctcatgtctc ggcaacttta aggttgtaaa atgggactgg tttcacaacg gtgctatgca 840841 aatccagatt tctatcaaat aatgaccagc atatgataac caggatggtg gaagtatacg 900901 ctcctgcctt atgaatatat attatatttt tcatattaga gtctattcta tatactttta 960961 aagtacgaat aataatttgc tatttagctt tgaataattc ctctgtgtaa aaaaaatctc 10201021 tgtctttgtt tttgtggatt tccAataaaC ctaatcttcg cattaaaatc aaaaaaaaaa10801081 aaaaaaa 1088

Sequence table two: by the growth hormone gene coding region deduced amino acid of Silurus meridionalis Chen (Silurus meridionalis).＜210〉2＜211〉200＜212〉PRT＜213〉Silurus meridionalis Chen (Silurus meridionalis)＜400〉1Met Ala Arg Val Trp Val Leu Leu Ser Val Val Leu Ala Ser Leu Phe Phe Ser Gln Gly

-20 -15 -10 -5Ala?Thr?Phe?Glu?Asn?Gln?Arg?Leu?Phe?Asn?Asn?Ala?Val?Ile?Arg?Val?Gln?His?Leu?His

1 5 10 15Gln?Leu?Ala?Ala?Lys?Met?Met?Asp?Asp?Phe?Glu?Glu?Ala?Leu?Leu?Pro?Glu?Glu?Arg?Lys

20 25 30 35Gln?Leu?Ser?Lys?Ile?Phe?Pro?Leu?Ser?Phe?Cys?Asn?Ser?Asp?Ser?Ile?Glu?Ala?Pro?Ser

40 45 50 55Gly?Lys?Asp?Glu?Thr?Gln?Lys?Ser?Ser?Val?Leu?Lys?Leu?Leu?His?Thr?Ser?Tyr?Arg?Leu

60 65 70 75Ile?Glu?Ser?Trp?Glu?Phe?Pro?Ser?Lys?Asn?Leu?Gly?Asn?Pro?Asn?His?Ile?Ser?Glu?Lys

80 85 90 95Lue?Ala?Asp?Leu?Lys?Met?Gly?Ile?Gly?Val?Leu?Ile?Glu?Gly?Cys?Leu?Asp?Gly?Gln?Thr

100 105 110 115Ser?Leu?Asp?Glu?Asn?Asp?Ser?Leu?Ala?Pro?Pro?Phe?Glu?Asp?Phe?Tyr?Gln?Thr?Leu?Thr

120 125 130 135Glu?Gly?Asn?Leu?Arg?Lys?Ser?Phe?Arg?Leu?Leu?Ser?Cys?Phe?Lys?Lys?Asp?Met?His?Lys

140 145 150 155Val?Glu?Thr?Tyr?Leu?Ser?Val?Ala?Lys?Cys?Arg?Arg?Ser?Leu?Asp?Ser?Ash?Cys?Thr?Leu

160 165 170 175-52 AAAGTCTACCTCGAGATTTCAGCAGGATCTGAGAAAGTTTCTTCTGGGAAAA -1

21 A T F E N Q R L F N N A V I R V Q H L H 40121?CAG?CTG?GCT?GCC?AAA?ATG?ATG?GAT?GAC?TTT?GAG?GAA?GCT?TTG?CTA?CCT?GAA?GAA?CGC?AAA 18041 Q L A A K M M D D F E E A L L P E E R K 60181?CAG?CTG?AGC?AAG?ATC?TTC?CCA?TTG?TCC?TTC?TGC?AAT?TCA?GAC?TCC?ATC?GAA?GCT?CCA?TCA 24061 Q L S K I F P L S F C N S D S I E A P S 80241?GGC?AAA?GAC?GAA?ACC?CAG?AAG?AGT?TCT?GTG?TTG?AAG?CTG?CTG?CAC?ACC?TCC?TAC?CGT?CTG 30081 G K D E T Q K S S V L K L L H T S Y R L 100301?ATC?GAG?TCA?TGG?GAG?TTC?CCC?AGT?AAG?AAC?CTG?GGC?AAC?CCC?AAT?CAC?ATC?TCA?GAG?AAG 360101 I E S W E F P S K N L G N P N H I S E K 120361?CTG?GCT?GAC?CTG?AAA?ATG?GGC?ATC?GGT?GTG?CTT?ATC?GAG?GGA?TGT?CTG?GAT?GGA?CAA?ACC 420121 L A D L K M G I G V L I E G C L D G Q T 140421?AGC?CTG?GAT?GAG?AAC?GAC?TCT?CTG?GCT?CCA?CCC?TTC?GAG?GAT?TTC?TAC?CAG?ACC?CTA?ACC 480141 S L D E N D S L A P P F E D F Y Q T L T 160481?GAG?GGA?AAC?CTG?AGG?AAG?AGC?TTC?CGT?CTG?CTC?TCC?TGC?TTC?AAG?AAG?GAC?ATG?CAC?AAA 540161 E G N L R K S F R L L S C F K K D M H K 180541?GTG?GAG?ACC?TAT?CTG?AGC?GTG?GCC?AAG?TGC?AGG?AGA?TCC?CTG?GAT?TCC?AAC?TGC?ACC?CTG 600181 V E T Y L S V A K C R R S L D S N C T L 200601?TAG AGGGCATGATTTAGC?CTCAGCCTGT?GATTTGAATT?GTTTTAATAG?TCCAGGTGTA?GCTAAATTAT 668

*669 TAATCCTTTT?CCCCTGGATT?TCACTTCTAA?CTTTAAGTAT?TTATTTTCTC?CTCCTCCATT 728769 CTCATGTCTC?GGCAACTTTA?AGGTTGTAAA?ATGGGACTGG?TTTCACAACG?GTGCTATGCA 788789 AATCCAGATT?TCTATCAAAT?AATGACCAGC?ATATGATAAC?CAGGATGGTG?GAAGTATACG 848849 CTCCTGCCTT?ATGAATATAT?ATTATATTTT?TCATATTAGA?GTCTATTCTA?TATACTTTTA 908909 AAGTACGAAT?AATAATTTGC?TATTTAGCTT?TGAATAATTC?CTCTGTGTAA?AAAAAATCTC 968969 TGTCTTTGTT?TTTGTGGATT?TCCAATAAAC?CTAATCTTCG?CATTAAAATC?AAAAAAAAAA 10281029 AAAAAAA 1035

Sequence table three: the sequence alignment result who obtains with BlastN and BlastP search in the NCBI website. gi|2147434|pir||I51114growth?hormone-giant?catfish

Length＝200Score＝357bits(917)，Expect＝4e-98Identities＝183/200(91％)，Positives＝185/200(92％)Query：1 MARXXXXXXXXXXXXXXXQGATFENQRLFNNAVIRVQHLHQLAAKMMDDFEEALLPEERK?60

MAR QGATFENQRLFNNAVIRVQHLHQLAAKMMDDFEEALLPEERKSbjct：1 MARVLVVLSVVVASLFFSQGATFENQRLFNNAVIRVQHLHQLAAKMMDDFEEALLPEERK?60Query：61 QLSKIFPLSFCNSDSIEAPSGKDETQKSSVLKLLHTSYRLIESWEFPSKNLGNPNHISEK?120

QLSKIFPLSFCNSDSIEAP+GKDETQKSSVLKLLHTSYRLIESWEFPSKNLGNPNHISEKSbjct：61 QLSKIFPLSFCNSDSIEAPAGKDETQKSSVLKLLHTSYRLIESWEFPSKNLGNPNHISEK?120Query：121?LADLKMGIGVLIEGCLDGQTSLDENDSLAPPFEDFYQTLTEGNLRKSFRLLSCFKKDMHK?180

LADLKMGIGVLIEGCLDGQTSLDENDSLAPPFEDFYQTL+EGNLRKSFRLLSCFKKDMHKSbjct：121?LADLKMGIGVLIEGCLDGQTSLDENDSLAPPFEDFYQTLSEGNLRKSFRLLSCFKKDMHK?180Query：181?VETYLSVAKCRRSLDSNCTL?200

VETYLSVAKCRRSLDSNCTLSbjct：181?VETYLSVAKCRRSLDSNCTL?200 gi|4927276|gb|AAD33070.1|AF147792?1?growth?hormone[Heteropneustes?fossilis]

Length＝200Score＝354bits(909)，Expect＝4e-97Identities＝179/200(89％)，Positives＝184/200(92％)Query：1 MARXXXXXXXXXXXXXXXQGATFENQRLFNNAVIRVQHLHQLAAKMMDDFEEALLPEERK?60

QLSKIFPLSFCNSDSIEAP+GKDETQKSSVLKLLHTSYRLIESWEFPSKNLGNPNHISEKSbjct：61 QLSKIFPLSFCNSDSIEAPAGKDETQKSSVLKLLHTSYRLIESWEFPSKNLGNPNHISEK?120Query：121 LADLKMGIGVLIEGCLDGQTSLDENDSLAPPFEDFYQTLTEGNLRKSFRLLSCFKKDMHK?180

LADLKMGIGVLIEGC+DGQTSLDEND+?APPFEDFYQTL+EGNL+KSFRLLSCFKKDMHKSbjct：121 LADLKMGIGVLIEGCVDGQTSLDENDAFAPPFEDFYQTLSEGNLKKSFRLLSCFKKDMHK?180Query：181 VETYLSVAKCRRSLDSNCTL?200

VETYLSVAKCRRSLDSNCTLSbjct：181 VETYLSVAKCRRSLDSNCTL?200 gi|19071230|gb|AAL84168.1|AF416489?1?growth?hormone?protein[Heteropneustesfossilis]

Length＝200Score＝354bits(909)，Expect＝4e-97Identities＝179/200(89％)，Positives＝183/200(91％)Query：1 MARXXXXXXXXXXXXXXXQGATFENQRLFNNAVIRVQHLHQLAAKMMDDFEEALLPEERK?60

MAR QGATFE?QRLFNNAVIRVQHLHQLAAKMMDDFEEALLPEERKSbjct：1 MARVLVLLSVVVASLFFSQGATFETQRLFNNAVIRVQHLHQLAAKMMDDFEEALLPEERK?60Query：61 QLSKIFPLSFCNSDSIEAPSGKDETQKSSVLKLLHTSYRLIESWEFPSKNLGNPNHISEK?120

LADLKMGIGVLIEGC+DGQTSLDEND+?APPFEDFYQTL+EGNLRKSFRLLSCFKKDMHKSbjct：121?LADLKMGIGVLIEGCVDGQTSLDENDAFAPPFEDFYQTLSEGNLRKSFRLLSCFKKDMHK?180Query：181?VETYLSVAKCRRSLDSNCTL?200

VETYLSVAKCRRSLDSNCTLSbjct：181?VETYLSVAKCRRSLDSNCTL?200 gi|545073|gb|AAC60745.1|?growth?hormone：GH[Ictalurus?punctatus] gi|8745513|gb|AAF78944.1|?growth?hormone[Ictalurus?punctatus]

Length＝200Score＝352bits(904)，Expect＝1e-96Identities＝178/200(89％)，Positives＝183/200(91％)Query：1 MARXXXXXXXXXXXXXXXQGATFENQRLFNNAVIRVQHLHQLAAKMMDDFEEALLPEERK?60

MAR QGATFE+QRLFNNAVIRVQHLHQLAAKMMDDFEEALLPEERKSbjct：1 MARVLVLLSVVVASLLFSQGATFESQRLFNNAVIRVQHLHQLAAKMMDDFEEALLPEERK?60Query：61 QLSKIFPLSFCNSDSIEAPSGKDETQKSSVLKLLHTSYRLIESWEFPSKNLGNPNHISEK?120

QLSKIFPLSFCNSDSIEAP+GKDE?QKSSVLKLLHTSYRLIESWEFPS+NLGNPNHISEKSbjct：61 QLSKIFPLSFCNSDSIEAPAGKDEAQKSSVLKLLHTSYRLIESWEFPSRNLGNPNHISEK?120Query：121?LADLKMGIGVLIEGCLDGQTSLDENDSLAPPFEDFYQTLTEGNLRKSFRLLSCFKKDMHK?180

LADLKMGIGVLIEGC+DGQT?LDENDSLAPPFEDFYQTL+EGNLRKSFRLLSCFKKDMHKSbjct：121?LADLKMGIGVLIEGCVDGQTGLDENDSLAPPFEDFYQTLSEGNLRKSFRLLSCFKKDMHK?180Query：181?VETYLSVAKCRRSLDSNCTL?200

VETYLSVAKCRRSLDSNCTLSbjct：181?VETYLSVAKCRRSLDSNCTL?200 gi|19071226|gb|AAL84166.1|AF416487?1?growth?hormone?protein[Clariasgariepinus] gi|19071228|gb|AAL84167.1|AF416488?1?growth?hormone?protein[Clariasgariepinus]

Length＝200Score＝349bits(896)，Expect＝1e-95Identities＝178/200(89％)，Positives＝182/200(91％)Query：1 MARXXXXXXXXXXXXXXXQGATFENQRLFNNAVIRVQHLHQLAAKMMDDFEEALLPEERK?60

MAR QGATFE?QRLFNNAVIRVQHLHQLAAKMMDDFEEALLPEERKSbjct：1 MARVLVLLSVVVASLFFNQGATFETQRLFNNAVIRVQHLHQLAAKMMDDFEEALLPEERK?60Query：61 QLSKIFPLSFCNSDSIEAPSGKDETQKSSVLKLLHTSYRLIESWEFPSKNLGNPNHISEK?120

LADLKMGIGVLIEG?+DGQTSLDEND+?APPFEDFYQTL+EGNLRKSFRLLSCFKKDMHKSbjct：121?LADLKMGIGVLIEGRVDGQTSLDENDAFAPPFEDFYQTLSEGNLRKSFRLLSCFKKDMHK?180Query：181?VETYLSVAKCRRSLDSNCTL?200

VETYLSVAKCRRSLDSNCTLSbjct：181?VETYLSVAKCRRSLDSNCTL?200 gi|19071222|gb|AAL84164.1|AF416485?1?growth?hormone?protein[Clarias?batrachus] gi|19071224|gb|AAL84165.1|AF416486?1?growth?hormone?protein[Clariasbatrachus]

Length＝200Score＝345bits(884)，Expect＝3e-94Identities＝175/200(87％)，Positives＝179/200(89％)Query：1 MARXXXXXXXXXXXXXXXQGATFENQRLFNNAVIRVQHLHQLAAKMMDDFEEALLPEERK?60

MAR QGAT?E?QRLFNNAVIRVQHLHQLAAKMMDDFEEALLPEERKSbjct：1 MARGLVLLSVVVASLFFSQGATLETQRLFNNAVIRVQHLHQLAAKMMDDFEEALLPEERK?60Query：61 QLSKIFPLSFCNSDSIEAPSGKDETQKSSVLKLLHTSYRLIESWEFPSKNLGNPNHISEK?120

QLSKIFPLSFCNSDSIEAP+GKDETQKSSVLKLLHTSYRLIESWEFPSK+LGNPNHISEKSbjct：61 QLSKIFPLSFCNSDSIEAPAGKDETQKSSVLKLLHTSYRLIESWEFPSKSLGNPNHISEK?120Query：121?LADLKMGIGVLIEGCLDGQTSLDENDSLAPPFEDFYQTLTEGNLRKSFRLLSCFKKDMHK?180

LADLKMGI?VLIEGC?DGQ?SLDEND+?APPFEDFYQTL+EGNLRKSFRLLSCFKKDMHKSbjct：121?LADLKMGISVLIEGCEDGQASLDENDAFAPPFEDFYQTLSEGNLRKSFRLLSCFKKDMHK?180Query：181?VETYLSVAKCRRSLDSNCTL?200

VETYLSVAKCRRSLDSNCTLSbjct：181?VETYLSVAKCRRSLDSNCTL?200 gi|425490|gb|AAB28017.1|?growth?hormone，GH[Ictalurus?punctatus＝catfish，pituitary?glands，

Peptide，178?aa]

Length＝178Score＝341bits(874)，Expect＝4e-93Identities＝170/178(95％)，Positives＝175/178(98％)Query：23 FENQRLFNNAVIRVQHLHQLAAKMMDDFEEALLPEERKQLSKIFPLSFCNSDSIEAPSGK 82

FE+QRLFNNAVIRVQHLHQLAAKMMDDFEEALLPEERKQLSKIFPLSFCNSDSIEAP+GKSbjct：1 FESQRLFNNAVIRVQHLHQLAAKMMDDFEEALLPEERKQLSKIFPLSFCNSDSIEAPAGK 60Query：83 DETQKSSVLKLLHTSYRLIESWEFPSKNLGNPNHISEKLADLKMGIGVLIEGCLDGQTSL 142

DE?QKSSVLKLLHTSYRLIESWEFPS+NLGNPNHISEKLADLKMGIGVLIEGC+DGQT?LSbjct：61 DEAQKSSVLKLLHTSYRLIESWEFPSRNLGNPNHISEKLADLKMGIGVLIEGCVDGQTGL 120Query：143?DENDSLAPPFEDFYQTLTEGNLRKSFRLLSCFKKDMHKVETYLSVAKCRRSLDSNCTL 200

DE?DSLAPPFEDFYQTL+EGNLRKSFRLLSCFKKDMHKVETYLSVAKCRRSLDSNCTLSbjct：121?DEEDSLAPPFEDFYQTLSEGNLRKSFRLLSCFKKDMHKVETYLSVAKCRRSLDSNCTL 178 gi|213566|gb|M63713.1|PNAGH?Pangasius?pangasius?growth?hormone?mRNA，completecds

Length＝603Score＝918bits(463)，Expect＝0.0Identities＝568/603(94％)Strand＝Plus/PlusQuery：1 atggctagagtgtgggtgctgctctccgtggtgctggcgagtttgttctttagtcagggc 60

|||||||||||||?||||?|||||||?||||||?||||||||||||||||||||||?|||5bjct：1 atggctagagtgttggtggtgctctctgtggtggtggcgagtttgttctttagtcaaggc 60Query：61 gcgacattcgagaaccagcgtctcttcaacaacgcagtcatccgtgtgcaacaccttcat 120

||||||||||||||||||||?|||||||||||||||||||||||||||||||||||||||Sbjct：61 gcgacattcgagaaccagcggctcttcaacaacgcagtcatccgtgtgcaacaccttcat 120Query：121?cagctggctgccaaaatgatggatgactttgaggaagctttgctacctgaagaacgcaaa 180

||||||||||||||?||||||||||||||||||||||||?||?||||||||||||||||||Sbjct：121?cagctggctgccaagatgatggatgactttgaggaagctctgttacctgaagaacgcaaa 180Query：181?cagctgagcaagatcttcccattgtccttctgcaattcagactccatcgaagctccatca 240

||||||||||||||?|||||?||||?|||||||||?||?||||||||||||||||| ||Sbjct：181?cagctgagcaagattttccccctgtctttctgcaactcggactccatcgaagctcctgca 240Query：241?ggcaaagacgaaacccagaagagttctgtgttgaagctgctgcacacctcctaccgtctg?300

|||||?|||||?||||||||?||?||||||?||||?||||||||||||||||||||||||?Sbjct：241?ggcaaggacgagacccagaaaagctctgtgctgaaattgctgcacacctcctaccgtctg 300Query：301?atcgagtcatgggagttccccagtaagaacctgggcaaccccaatcacatctcagagaag 360

|||||||||||||||||||||||?||||||||?||||||||||||?||||||||||||||Sbjct：301?atcgagtcatgggagttccccagcaagaacctcggcaaccccaaccacatctcagagaag 360Query：361?ctggctgacctgaaaatgggcatcggtgtgcttatcgagggatgtctggatggacaaacc 420

||||||||||||||||||||||||||?||||||||||||||||||?|||||||||||||| Sbjct：361?ctggctgacctgaaaatgggcatcggcgtgcttatcgagggatgtttggatggacaaacc 420Query：421?agcctggatgagaacgactctctggctccacccttcgaggatttctaccagaccctaacc 480

|||||||||||||||||||||||||||||?||||||||||||||||||||||||?|?|?|Sbjct：421?agcctggatgagaacgactctctggctccgcccttcgaggatttctaccagaccttgagc 480Query：481?gagggaaacctgaggaagagcttccgtctgctctcctgcttcaagaaggacatgcacaaa 540

||||||||||||||||||||||||||||||||?|||||||||||||||||||||||||||Sbjct：481?gagggaaacctgaggaagagcttccgtctgctgtcctgcttcaagaaggacatgcacaaa?540Query：541?gtggagacctatctgagcgtggccaagtgcaggagatccctggattccaactgcaccctg?600

||||||||||||||?|||||||||||||||||||||||||||||||||||||||||||||Sbjct：541?gtggagacctatctcagcgtggccaagtgcaggagatccctggattccaactgcaccctg?600Query：601?tag?603

|||Sbjct：601?tag?603 gi|450294|gb|L27835.1|?PGSGHA?Pangasianodon?gigas?growth?hormone(GH)mRNA，complete?cds

Length＝1176Score＝910bits(459)，Expect＝0.0Identities＝567/603(94％)Strand＝Plus/PlusQuery：1 atggctagagtgtgggtgctgctctccgtggtgctggcgagtttgttctttagtcagggc 60

|||||||||||||?||||?|||||||?||||||?||||||||||||||||||||||?|||Sbjct：59 atggctagagtgttggtggtgctctctgtggtggtggcgagtttgttctttagtcaaggc 118Query：61 gcgacattcgagaaccagcgtctcttcaacaacgcagtcatccgtgtgcaacaccttcat 120

||||||||||||||||||||?||||||||||||||||||||||||||||||||||||||| Sbjct：119?gcgacattcgagaaccagcggctcttcaacaacgcagtcatccgtgtgcaacaccttcat?178Query：121?cagctggctgccaaaatgatggatgactttgaggaagctttgctacctgaagaacgcaaa?180

||||||||||||||?||||||||||||||||||||||||?||?|||||||||||||||||Sbjct：179?cagctggctgccaagatgatggatgactttgaggaagctctgttacctgaagaacgcaaa?238Query：181?cagctgagcaagatcttcccattgtccttctgcaattcagactccatcgaagctccatca?240

||||||||||||||?|||||?||||?|||||||||?||?||||||||||||||||| ||Sbjct：239?cagctgagcaagattttccccctgtctttctgcaactcggactccatcgaagctcctgca?298Query：241?ggcaaagacgaaacccagaagagttctgtgttgaagctgctgcacacctcctaccgtctg?300

|||||?|||||?||||||||?||?||||||?||||?||||||||||||||||||||||||Sbjct：299?ggcaaggacgagacccagaaaagctctgtgctgaaattgctgcacacctcctaccgtctg?358Query：301?atcgagtcatgggagttccccagtaagaacctgggcaaccccaatcacatctcagagaag?360

||||||||?||||||||||||||?||||||||?|||||||||||?|||||||||||||||Sbjct：359?atcgagtcgtgggagttccccagcaagaacctcggcaaccccaaccacatctcagagaag?418Query：361?ctggctgacctgaaaatgggcatcggtgtgcttatcgagggatgtctggatggacaaacc?420

||||||||||||||||||||||||||?||||||||||||||||||?||||||||||||||Sbjct：419?ctggctgacctgaaaatgggcatcggcgtgcttatcgagggatgtttggatggacaaacc?478Query：421?agcctggatgagaacgactctctggctccacccttcgaggatttctaccagaccctaacc?480

|||||||||||||||||||||||||||||?||||||||||||||||||||||||?|?|?|?Sbjct：479?agcctggatgagaacgactctctggctccgcccttcgaggatttctaccagaccttgagc?538Query：481?gagggaaacctgaggaagagcttccgtctgctctcctgcttcaagaaggacatgcacaaa?540

||||||||||||||||||||||||||||||||?|||||||||||||||||||||||||||Sbjct：539?gagggaaacctgaggaagagcttccgtctgctgtcctgcttcaagaaggacatgcacaaa?598Query：541?gtggagacctatctgagcgtggccaagtgcaggagatccctggattccaactgcaccctg?600

||||||||||||||?|||||||||||||||||||||||||||||||||||||||||||||Sbjct：599?gtggagacctatctcagcgtggccaagtgcaggagatccctggattccaactgcaccctg?658Query：601?tag?603

|||Sbjct：659?tag?661 gi|4927275|gb|AF147792.1|?AF147792?Heteropneustes?fossilis?growth?hormone(GH)mRNA，complete?cds

Length＝1132Score＝799bits(403)，Expect＝0.0Identities＝553/603(91％)Strand＝Plus/PlusQuery：1 atggctagagtgtgggtgctgctctccgtggtgctggcgagtttgttctttagtcagggc 60

||||||||||||||||||||?||||||||||||||||||||||||||||||||||||||| Sbjct：119?gcgacattcgagaaccagcggctcttcaacaacgcagtcatccgtgtgcaacaccttcat 178Query：121?cagctggctgccaaaatgatggatgactttgaggaagctttgctacctgaagaacgcaaa 180

||||||||||||||?||||||||||||||||||||||||?||?|||||||||||||||||Sbjct：179?cagctggctgccaagatgatggatgactttgaggaagctctgttacctgaagaacgcaaa 238Query：181?cagctgagcaagatcttcccattgtccttctgcaattcagactccatcgaagctccatca 240

||||||||||||||?|||||?||||?|||||||||?||?|||||?|||||?||||| ||Sbjct：239?cagctgagcaagattttcccgctgtctttctgcaactcggactctatcgaggctccggca 298Query：241?ggcaaagacgaaacccagaagagttctgtgttgaagctgctgcacacctcctaccgtctg 300

|||||?|||||?||||||||?||?||||||?||||?||||||||||||||||||||||||Sbjct：299?ggcaaggacgagacccagaaaagctctgtgctgaaactgctgcacacctcctaccgtctg 358Query：301?atcgagtcatgggagttccccagtaagaacctgggcaaccccaatcacatctcagagaag 360

||||||||?||||||||||||||?||||||||?||||||||?||?||||||||?||||||Sbjct：359?atcgagtcgtgggagttccccagcaagaacctcggcaaccctaaccacatctccgagaag 418Query：361?ctggctgacctgaaaatgggcatcggtgtgcttatcgagggatgtctggatggacaaacc 420

||||||||||||||||||||||||||?|||||||||||||||||?|||||||||||||||Sbjct：419?ctggctgacctgaaaatgggcatcggcgtgcttatcgagggatgcgtggatggacaaacc 478Query：421?agcctggatgagaacgactctctggctccacccttcgaggatttctaccagaccctaacc 480

||||||||?|||||||||?||?|?||?||?||||||||||||||||||||||||||?|?|Sbjct：479?agcctggacgagaacgacgcttttgcgccgcccttcgaggatttctaccagaccctgagc 538Query：481?gagggaaacctgaggaagagcttccgtctgctctcctgcttcaagaaggacatgcacaaa 540

|||||?|||||||?|||?||||||||||||||?||||||||||||||||||||||||||Sbjct：539?gaggggaacctgaagaaaagcttccgtctgctgtcctgcttcaagaaggacatgcacaag 598Query：541?gtggagacctatctgagcgtggccaagtgcaggagatccctggattccaactgcaccctg 600

|||||||?||||||?|||||?|||||||||||||||||||||||||||||||||||||||Sbjct：599?gtggagacttatctaagcgtagccaagtgcaggagatccctggattccaactgcaccctg 658Query：601?tag?603

|||Sbjct：659?tag?661 gi|19071221|gb|AF416485.1|?Clarias?batrachus?growth?hormone?protein?precursorRNA，complete?cds

Length＝663Score＝735bits(371)，Expect＝0.0Identities＝545/603(90％)Strand＝Plus/PlusQuery：1 atggctagagtgtgggtgctgctctccgtggtgctggcgagtttgttctttagtcagggc 60

||||||?|||?||?||||||||||||?||||||?||||||||?|||||||||||||?|||Sbjct：61 atggctcgagggttggtgctgctctctgtggtggtggcgagtctgttctttagtcaaggc 120Query：61 gcgacattcgagaaccagcgtctcttcaacaacgcagtcatccgtgtgcaacaccttcat 120

||||||?|?||||?||||||?|||||||||||?|||||||||||?||||||||||||||Sbjct：121?gcgacacttgagacccagcggctcttcaacaatgcagtcatccgcgtgcaacaccttcac 180Query：121?cagctggctgccaaaatgatggatgactttgaggaagctttgctacctgaagaacgcaaa 180

||?|||||||||||?|||||||||||||||||?|||||||||?|||||||||||||||||Sbjct：181?caactggctgccaagatgatggatgactttgaagaagctttgttacctgaagaacgcaaa 240Query：181?cagctgagcaagatcttcccattgtccttctgcaattcagactccatcgaagctccatca 240

||||||||||||||||||||?||||?|||||||||?||?|||||||||||?||?|| ||?Sbjct：241?cagctgagcaagatcttccccctgtcattctgcaactcggactccatcgaggcgccggca 300Query：241?ggcaaagacgaaacccagaagagttctgtgttgaagctgctgcacacctcctaccgtctg 300

|||||?|||||?||||||||?||?||||||?||||?||||||||||||||||||||||||Sbjct：301?ggcaaggacgagacccagaaaagctctgtgctgaaactgctgcacacctcctaccgtctg 360Query：301?atcgagtcatgggagttccccagtaagaacctgggcaaccccaatcacatctcagagaag 360

|||||||||||||||||||||||?||||?||||||||||||?||?||?|||||?||||||Sbjct：361?atcgagtcatgggagttccccagcaagagcctgggcaacccgaaccatatctctgagaag 420Query：361?ctggctgacctgaaaatgggcatcggtgtgcttatcgagggatgtctggatggacaaacc 420

||||||||||||||||||||||||?|?|||||||||||||||||?||||||||||||?||Sbjct：421?ctggctgacctgaaaatgggcatcagcgtgcttatcgagggatgcgaggatggacaagcc 480Query：421?agcctggatgagaacgactctctggctccacccttcgaggatttctaccagaccctaacc 480

||||||||?|||||||||?| |?|||||?|||||||||||||||||||||||?||?|?|?Sbjct：481?agcctggacgagaacgacgcatttgctccgcccttcgaggatttctaccagactctgagc 540Query：481?gagggaaacctgaggaagagcttccgtctgctctcctgcttcaagaaggacatgcacaaa 540

|||||?||||||||||||||||||||||||||?||||||||||||||||||||||||||| Sbjct：541?gagggcaacctgaggaagagcttccgtctgctgtcctgcttcaagaaggacatgcacaaa 600Query：541?gtggagacctatctgagcgtggccaagtgcaggagatccctggattccaactgcaccctg 600

||||||||||||||?|||||||||||||||||||||||||||||||||||||||||?||| Sbjct：601?gtggagacctatctaagcgtggccaagtgcaggagatccctggattccaactgcactctg 660Query：601?tag?603

|||Sbjct：661?tag?663 gi|19071225|gb|AF416487.1|?Clarias?gariepinus?growth?hormone?protein?precursorRNA，complete?cds

Length＝619Score＝712bits(359)，Expect＝0.0Identities＝542/603(89％)Strand＝Plus/PlusQuery：1 atggctagagtgtgggtgctgctctccgtggtgctggcgagtttgttctttagtcagggc 60

||||||?||||?|?||||||||||||?||||||?||||||||?|||||||||?|||?|||Sbjct：17 atggctcgagttttggtgctgctctctgtggtggtggcgagtctgttctttaatcaaggc 76Query：61 gcgacattcgagaaccagcgtctcttcaacaacgcagtcatccgtgtgcaacaccttcat 120

|||||||||?||||?||||||?|||||||||||||?||||||||||||||||||||||| Sbjct：77 gcgacatttgagacccagcggctcttcaacaacgcggtcatccgtgtgcaacaccttcac 136Query：121 cagctggctgccaaaatgatggatgactttgaggaagctttgctacctgaagaacgcaaa 180

||?|||||||||||?|||||||||||||||||?|||||||||?||||||||||||||||| Sbjct：137 caactggctgccaagatgatggatgactttgaagaagctttgttacctgaagaacgcaaa 196Query：181 cagctgagcaagatcttcccattgtccttctgcaattcagactccatcgaagctccatca 240

||||||||||||||||||||?||||?|||||||||?||?||||||?|||||?|||||?|| Sbjct：197 cagctgagcaagatcttccccctgtcattctgcaactcggactctatcgaggctccggca 256Query：241 ggcaaagacgaaacccagaagagttctgtgttgaagctgctgcacacctcctaccgtctg 300

|||||?|||||?||||||?||?||?|||?||||?|||||||||||||?|||||?||||||Sbjct：257 ggcaaggacgagacccagaaaagctccgtgctgaaactgctgcacacatcctatcgtctg 316Query：301 atcgagtcatgggagttccccagtaagaacctgggcaaccccaatcacatctcagagaag 360

|||||||||||||||||||||||?|||||||||||||||||?||?||?|||||?||?|||Sbjct：317 atcgagtcatgggagttccccagcaagaacctgggcaaccctaaccatatctctgaaaag 376Query：361 ctggctgacctgaaaatgggcatcggtgtgcttatcgagggatgtctggatggacaaacc 420

|||||||||||||||||||||||||||||||||||?||||||?||?||||||||||||||Sbjct：377 ctggctgacctgaaaatgggcatcggtgtgcttattgagggacgtgtggatggacaaacc 436Query：421 agcctggatgagaacgactctctggctccacccttcgaggatttctaccagaccctaacc 480

||||||||?|||||?|||?| |?|||||?||||||||||||||||||||||||||?|?|Sbjct：437 agcctggacgagaatgacgcatttgctccgcccttcgaggatttctaccagaccctgagc 496Query：481 gagggaaacctgaggaagagcttccgtctgctctcctgcttcaagaaggacatgcacaaa 540

||||||?|||?|||||||||||||||||||||?||?|||||||||||?||||||||||||Sbjct：497 gaggggaacttgaggaagagcttccgtctgctgtcttgcttcaagaaagacatgcacaaa 556Query：541 gtggagacctatctgagcgtggccaagtgcaggagatccctggattccaactgcaccctg 600

||||||||?|||||?|||||||||||||||||||||||||||||||||||||||||?|||Sbjct：557 gtggagacttatctcagcgtggccaagtgcaggagatccctggattccaactgcactctg 616Query：601 tag?603

|||Sbjct：617 tag?619

Claims

1, a kind of Silurus meridionalis growth hormone gene, it is characterized in that it is that template is through RT-PCR with mRNA pituitary, obtain first chain of cDNA, again according to the codeword triplet of other known fish growth hormone aminoacid sequence conserved regions LLPEER and the codeword triplet of SNCTL and terminator codon, the design pair of degenerate primers, forward GHFC 5 ' CTG TTR YCT GAD GAM CGC 3 ', reverse GHRA 5 ' TCT ACA GGGTRC AGT TKG 3 ', article one chain with cDNA is that the template clone obtains growth hormone cDNA portion homologous molecule, method through 5 ' RACE and 3 ' RACE obtains Silurus meridionalis growth hormone cDNA full length sequence then, and derive aminoacid sequence, the full length cDNA sequence of the tethelin of its Silurus meridionalis Chen is as follows: 1 aaagtctacc tcgagatttc agcaggatct gagaaagttt cttctgggaa aaatggctag, 6061 agtgtgggtg ctgctctccg tggtgctggc gagtttgttc tttagtcagg gcgcgacatt, 120121 cgagaaccag cgtctcttca acaacgcagt catccgtgtg caacaccttc atcagctggc, 180181 tgccaaaatg atggatgact ttgaggaagc tttgctacct gaagaacgca aacagctgag, 240241 caagatcttc ccattgtcct tctgcaattc agactccatc gaagctccat caggcaaaga, 300301 cgaaacccag aagagttctg tgttgaagct gctgcacacc tcctaccgtc tgatcgagtc, 360361 atgggagttc cccagtaaga acctgggcaa ccccaatcac atctcagaga agctggctga, 420421 cctgaaaatg ggcatcggtg tgcttatcga gggatgtctg gatggacaaa ccagcctgga, 480481 tgagaacgac tctctggctc cacccttcga ggatttctac cagaccctaa ccgagggaaa, 540541 cctgaggaag agcttccgtc tgctctcctg cttcaagaag gacatgcaca aagtggagac, 600601 ctatctgagc gtggccaagt gcaggagatc cctggattcc aactgcaccc tgtagagggc, 660661 atgatttagc ctcagcctgt gatttgaatt gttttaatag tccaggtgta gctaaattat, 720721 taatcctttt cccctggatt tcacttctaa ctttaagtat ttattttctc ctcctccatt, 780781 ctcatgtctc ggcaacttta aggttgtaaa atgggactgg tttcacaacg gtgctatgca, 840841 aatccagatt tctatcaaat aatgaccagc atatgataac caggatggtg gaagtatacg, 900901 ctcctgcctt atgaatatat attatatttt tcatattaga gtctattcta tatactttta, 960961 aagtacgaat aataatttgc tatttagctt tgaataattc ctctgtgtaa aaaaaatctc, 10201021 tgtctttgtt tttgtggatt tcc AataaaCctaatcttcg cattaaaatc aaaaaaaaaa 10801081 aaaaaaa

2, Silurus meridionalis growth hormone gene according to claim 1 is characterized in that the growth hormone gene amino acid sequence coded of Silurus meridionalis Chen is as follows: Met Ala Arg Val Trp Val Leu Leu Ser Val Val Leu Ala Ser Leu Phe Phe Ser Gln Gly

40 45 50 55Gly?Lys?Asp?Glu?Thr?Gln?Lys?Set?Ser?Val?Leu?Lys?Leu?Leu?His?Thr?Ser?Tyr?Arg?Leu

140 145 150 155Val?Glu?Thr?Tyr?Leu?Ser?Val?Ala?Lys?Cys?Arg?Arg?Ser?Leu?Asp?Ser?Asn?Cys?Thr?Leu

160 165 170 175-52 AAAGTCTACCTCGAGATTTCAGCAGGATCTGAGAAAGTTTCTTCTGGGAAAA -1

21 A T F E N Q R L F N N A V I R V Q H L H 40121 CAG?CTG?GCT?GCC?AAA?ATG?ATG?GAT?GAC?TTT?GAG?GAA?GCT?TTG?CTA?CCT?GAA?GAA?CGC?AAA 18041 Q L A A K M M D D F E E A L L P E E R K 60181 CAG?CTG?AGC?AAG?ATC?TTC?CCA?TTG?TCC?TTC?TGC?AAT?TCA?GAC?TCC?ATC?GAA?GCT?CCA?TCA 24061 Q L S K I F P L S F C N S D S I E A P S 80241 GGC?AAA?GAC?GAA?ACC?CAG?AAG?AGT?TCT?GTG?TTG?AAG?CTG?CTG?CAC?ACC?TCC?TAC?CGT?CTG 30081 G K D E T Q K S S V L K L L H T S Y R L 100301 ATC?GAG?TCA?TGG?GAG?TTC?CCC?AGT?AAG?AAC?CTG?GGC?AAC?CCC?AAT?CAC?ATC?TCA?GAG?AAG 360101 I E S W E F P S K N L G N P N H I S E K 120361 CTG?GCT?GAC?CTG?AAA?ATG?GGC?ATC?GGT?GTG?CTT?ATC?GAG?GGA?TGT?CTG?GAT?GGA?CAA?ACC 420121 L A D L K M G I G V L I E G C L D G Q T 140421 AGC?CTG?GAT?GAG?AAC?GAC?TCT?CTG?GCT?CCA?CCC?TTC?GAG?GAT?TTC?TAC?CAG?ACC?CTA?ACC 480141 S L D E N D S L A P P F E D F Y Q T L T 160481 GAG?GGA?AAC?CTG?AGG?AAG?AGC?TTC?CGT?CTG?CTC?TCC?TGC?TTC?AAG?AAG?GAC?ATG?CAC?AAA 540161 E G N L R K S F R L L S C F K K D M H K 180541 GTG?GAG?ACC?TAT?CTG?AGC?GTG?GCC?AAG?TGC?AGG?AGA?TCC?CTG?GAT?TCC?AAC?TGC?ACC?CTG 600181 V E T Y L S V A K C R R S L D S N C T L 200601 TAG AGGGCATGATTTAGC?CTCAGcCTGT?GATTTGAATT?GTTTTAATAG?TCCAGGTGTA?GCTAAATTAT 668

*669 TAATCCTTTT?CCCCTGGATT?TCACTTCTAA?CTTTAAGTAT?TTATTTTCTC?CTCCTCCATT 728769 CTCATGTCTC?GGCAACTTTA?AGGTTGTAAA?ATGGGACTGG?TTTCACAACG?GTGCTATGCA 788789 AATCCAGATT?TCTATCAAAT?AATGACCAGC?ATATGATAAC?CAGGATGGTG?GAAGTATACG 848849 CTCCTGCCTT?ATGAATATAT?ATTATATTTT?TCATATTAGA?GTCTATTCTA?TATACTTTTA 908909 AAGTACGAAT?AATAATTTGC?TATTTAGCTT?TGAATAATTC?CTCTGTGTAA?AAAAAATCTC 968969 TGTCTTTGTT?TTTGTGGATT?TCC AATAAAC?CTAATCTTCG?CATTAAAATC?AAAAAAAAAA 10281029 AAAAAAA 1035