CN103408651B - Cynoglossus semilaevis female specific gene CSW1 and application thereof - Google Patents
Cynoglossus semilaevis female specific gene CSW1 and application thereof Download PDFInfo
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Abstract
The invention relates to a cynoglossus semilaevis female specific protein CSW1, of which the amino acid sequence is SEQ ID NO: 1, and the nucleotide sequence encoding the female specific protein CSW1 is SEQ ID NO: 2. The in-vitro recombinant protein of CSW1 gene can remarkably improve the expression level of female related gene foxl2, and has the biological activity to stimulate female related gene expression. After the CSW1 recombinant expression produce is used as a feed additive, the function of stimulating female gonad development is performed, and the effect of increasing the hen proportion is achieved. Therefore, the cynoglossus semilaevis female specific protein CSW1 has application potentials in the aspects of controlling the gender of cynoglossus semilaevis and increasing the proportion of female fish.
Description
Technical field
The invention belongs to the functional gene screening and application technical field in aquatic living things technology, be specifically related to the female specific gene of a kind of Cynoglossus semilaevis, i.e. the activation analysis of screening, clone, in-vitro recombination expression and the expression product thereof of the female specific gene CSW1 of Cynoglossus semilaevis.
Background technology
Cynoglossus semilaevis (Cynogloss μ s semilaevis) is the large-scale ground fish of a kind of coastal waters warm water, and its delicious meat, nutritious, is liked by human consumer deeply, is one of leading fish of China's sea farming.But female, the male individual growth speed difference of Cynoglossus semilaevis is very large, female than the fast 2-4 of male growth times.Due to male poor growth, the individual reason such as little, causing has increased the aquaculture cost of Cynoglossus semilaevis, has reduced cultured output, has had a strong impact on the popularization of semi-smooth tongue sole offspring breed and the development of aquaculture industry.Thereby carry out the Screening and Identification of Cynoglossus semilaevis male and female specific gene and the analysis and research of function thereof be disclose Cynoglossus semilaevis Sex Determination Mechanism, explore male and female growth differences molecule mechanism, set up the vital task of Sex Control.
In growth velocity, there are gender difference in many fish.For example, the male individual growth of tilapia is faster more than 30% than female individuals; Oncorhynchi female individuals is grown fast more than 30% than male.Equally, the growth of the female individuals of many seawater fishes in bothid and true plaice is also fast many than male, and for example, the female individuals of the fishes in bothid and true plaice such as Cynoglossus semilaevis, lefteye flounder and verasper moseri is grown more than fast 30-100% than male.The countries such as the U.S., Japan, Germany, France, Canada are numerous and confused throws huge fund and take Oncorhynchi, tilapia and blue or green Medaka etc. fish sex specific gene screens and research (Lee et al., 2004 of sex determination mechanism for material is carried out; Devlin and Nagahama, 2002; Mats μ da et al., 2002; Nanda et al., 2002), result of study, at Nature, publishes thesis many pieces on the international top publication such as PNAS.Be the most successfully wherein Japanese scientist take blue or green Medaka as material screening arrives the special functional gene DMY of the male Sex determination gene Y chromosome of blue or green Medaka, paper is delivered (Matsuda et al., 2002) on Nature.But, research subsequently shows, DMY only finds in blue or green Medaka and the very near blue or green Medaka of the back of a bow of another kind of sibship, and in other sibship Medaka section fish far away and other fish, has no the male Sex determination gene of DMY (Matsuda et al., 2003; Volff et al., 2003; Kondo et al., 2003).Thereby, up to now, only only a few fish such as blue or green Medaka and hunchbacked blue or green Medaka, be cloned into male Sex determination gene (DMY), and other fish, there is not yet relevant sex determining gene clone's successful report.In addition, the sex of above-mentioned fish is most, and for XY determines type, milter is XY type, and raun is XX type, and Cynoglossus semilaevis is ZW sex determination type, and female have special W karyomit(e), and male sex chromosome homotype (ZZ) (Zhou Liqing etc., 2005).And about fish female special or determine that screening and the clone, the particularly screening of the female specific gene of Cynoglossus semilaevis of gene and clone there is not yet report at present both at home and abroad.Therefore, fish sex is special/determine gene, particularly Cynoglossus semilaevis is female special or determine that the screening of gene and the research of clone and sex determination molecular mechanism are urgency important scientific issues to be captured both at home and abroad at present, is also the important foundation of setting up Cynoglossus semilaevis sex control and complete female rearing of fingerling.
Summary of the invention
The object of this invention is to provide the female specific gene CSW1 of a kind of Cynoglossus semilaevis, and this gene is carried out to in-vitro recombination expression, obtain recombination expression product, identify the biological activity of recombination expression product and the function of gene, for Cynoglossus semilaevis sex control and complete female seed development provide genetic resources and technological method.
One aspect of the invention relates to Cynoglossus semilaevis Female-specific protein, and its aminoacid sequence is SEQ ID NO:1;
The nucleotides sequence of above-mentioned Female-specific protein CSW1 of encoding is classified SEQ ID NO:2 as.
Another aspect of the present invention relates to the recombinant plasmid for recombinant expressed CSW1 albumen, and in described recombinant plasmid, having inserted sequence is the DNA fragmentation of SEQ ID NO:3.
Another aspect of the present invention relates to CSW1 albumen in Cynoglossus semilaevis sex control and improves the application in female fry ratio.
The albumen of said gene coding is as fodder additives, for stimulating the female gonad development of Cynoglossus semilaevis.
Can obviously the raise expression level of female genes involved foxl2 of the vitro recombination albumen of CSW1 gene of the present invention, has the biological activity that stimulates female related gene expression; There is the biological activity that reduces male genes involved SOX9 expression level simultaneously.After using CSW1 recombination expression product as fodder additives, will there is the effect that stimulates female gonad development, there is the effect that improves raun ratio, therefore, aspect Cynoglossus semilaevis sex control and the female fry ratio of raising, there is application potential.
Accompanying drawing explanation:
Fig. 1: the expression level analysis of CSW1 gene in Cynoglossus semilaevis different tissues.A: sxemiquantitative pcr analysis; B: in good time quantitative PCR analysis.
Fig. 2: the expression level analysis of CSW1 gene in the female fish ovaries of Cynoglossus semilaevis different development stage and milter spermary;
Fig. 3: the recombinant expression vector plasmid figure of Cynoglossus semilaevis CSW1 gene;
Fig. 4: containing CSW1 recombinant protein electrophorogram after the total protein in the recombination bacillus coli culture supernatant of Cynoglossus semilaevis CSW1 gene and purifying;
M wherein: standard protein marker; 1, the CSW1 recombinant protein electrophorogram after purifying, 2, CSW1 recombinant expression vector pet-32-CSW1 tropina electrophorogram after IPTG induction, 3, empty carrier pet-32a tropina electrophorogram after IPTG induction;
Fig. 5: after Cynoglossus semilaevis recombinant C SW1 protein injection foxl2 gene at 0 hour, 6 hours, 12 hours, 24 hours, 36 hours, 48 hours, the expression analysis of 72 hours and 96 hours;
Fig. 6: after Cynoglossus semilaevis recombinant C SW1 protein injection sox9 gene at 0 hour, 6 hours, 12 hours, 24 hours, 36 hours, 48 hours, the expression analysis of 72 hours and 96 hours.
Embodiment
Below in conjunction with accompanying drawing example, method of the present invention is described further.But example only limits to explanation, is not limited to this.The experimental technique of unreceipted actual conditions in the following example, conventionally condition routinely, condition described in the < < molecular cloning experiment guide > > writing as J. Pehanorm Brooker (Sambrook) etc., or the condition of advising according to manufacturer operation.
One, clone and the sequence thereof of the female specific gene CSW1 of Cynoglossus semilaevis
, milter female to Cynoglossus semilaevis carried out respectively genome sequencing, pass through bioinformatic analysis, find to there is a special gene C SW1 in ZW female individuals genome, adopt cDNA end rapid amplifying (RACE) technology, from Cynoglossus semilaevis ovary, be cloned into the cDNA of female specific gene CSW1, its full length sequence is SEQ ID NO:2, and the aminoacid sequence of coding is SEQ ID NO:1.
The cDNA sequence total length of the female specific gene CSW1 of Cynoglossus semilaevis is 1541bp, the open reading frame (ORF region) that comprises 495bp, 165 amino acid of encoding, 5 ' non-coding head of district 360bp, 3 ' non-coding head of district 686bp, has polyadenylic acid tailing signal and polyadenylic acid tail.
Concrete steps are as follows:
1, the screening of the female specific gene of Cynoglossus semilaevis and clone
Female special gene sequence in patent of the present invention derives from Cynoglossus semilaevis gene order-checking project, to Cynoglossus semilaevis female individuals and male, adopt SOLEXA sequencing technologies to carry out respectively genome sequencing and assembling, adopt bioinformatics method to compare to the raun genome sequence and the milter genome sequence that obtain, therefrom filter out the female special candidate gene of Cynoglossus semilaevis; To these female special candidate gene design primers, utilize these special primers to carry out respectively pcr amplification to the female milter genomic dna of Cynoglossus semilaevis, find that 10 candidate genes exist in raun genomic dna, and increase not out in milter genome.By these 10 unnamed genes, it is female gene group specific gene.
2, the sequence of the female specific gene of Cynoglossus semilaevis
Next, analyze the encoder block of these 10 genome specific genes, the primer of design amplification cDNA fragment, adopt these primer pair rauns and milter sexual gland cDNA to increase, the gene that specifically expressing in raun sexual gland is not substantially expressed in milter sexual gland is defined as female specific expression gene.By these methods, we have found 1 gene of specifically expressing in ZW raun sexual gland, called after CSW1.Adopt cDNA end rapid amplifying (RACE) technology, the specific PCR primer of employing CSW1 gene (
CSW1-5 ' GSP:GCTCCAGAATCCGCCTGATCCTCAAG, CSW1-3 ' GSP:TGTATCATCCACGGCAACGACATGGATC), from Cynoglossus semilaevis ovary, be cloned into the full-length cDNA of female specific gene CSW1 gene, its full length sequence is SEQ ID NO:2, and the aminoacid sequence of its proteins encoded is SEQ ID NO:1.
CDNA sequence obtains and takes cDNA RLM-RACE (RACE), and method is according to SMARTerTM RACE cDNA Amplification Kit (Clontech), and its concrete operations condition is as follows:
The system that 3 ' RACE is used and reaction conditions:
50 μ l reaction systems
5μl?10X?BD?Advantage?2?PCR?Bμffer
1μl?dNTP?Mix?(10?mM)
5μl?μPM
1μl?GSP
2μl?3'-RACE-Ready?cDNA
1μl50X?BD?Advantage?2?Polymerase?Mix
35μl?PCR-grade?water
Reaction conditions is as follows:
94 ℃ of 30S, 5 circulations of 72 ℃ of 3min, 94 ℃ of 30S, 70 ℃ of 30S, 5 circulations of 72 ℃ of 3min, 94 ℃ of 30S, 68 ℃ of 30S, 25 circulations of 72 ℃ of 3min
5 ' RACE system used and reaction conditions:
50 μ l reaction systems
5μl?10X?BD?Advantage?2?PCR?Bμffer
1μl?dNTP?Mix?(10?mM)
5μl?μPM
1μl?GSP
2μl?5'-RACE-Ready?cDNA
1μl50X?BD?Advantage?2?Polymerase?Mix
35μl?PCR-grade?water
Reaction conditions is as follows:
94 ℃ of 30S, 5 circulations of 72 ℃ of 3min, 94 ℃ of 30S, 70 ℃ of 30S, 5 circulations of 72 ℃ of 3min, 94 ℃ of 30S, 68 ℃ of 30S, 25 circulations of 72 ℃ of 3min.
The PCR product of 3 ' RACE and 5 ' RACE is detected with 1.5% agarose gel electrophoresis, with glue, reclaim recovery and the purifying that test kit (Omega) carries out PCR product, be connected with pMD-18T carrier (Takara) again, select positive colony and extract plasmid, 3730DNA Analyzer(ABI) order-checking, the sequence recording is analyzed splicing through CLUSTER and is obtained full length sequence.
The cDNA sequence total length of the female specific gene CSW1 of Cynoglossus semilaevis is 1541bp, the open reading frame (ORF region) that comprises 495bp, 165 amino acid of encoding, 5 ' non-coding head of district 360bp, 3 ' non-coding head of district 686bp, has polyadenylic acid tailing signal and polyadenylic acid tail.
Two, the female specific gene CSW1 of Cynoglossus semilaevis is at the expression pattern of different tissues and different development stage
1, the female specific gene CSW1 of Cynoglossus semilaevis is at the expression pattern of different tissues: adopt sxemiquantitative and in good time quantitative PCR technique analyzed the expression of CSW1 gene in Cynoglossus semilaevis different tissues (heart, liver, the gill, skin, blood, kidney, intestines, brain, spleen, muscle, hypophysis, ovary, spermary and pseudo-milter spermary), find that CSW1 expression level in female fish ovaries is the highest, in each tissue such as other tissue of raun and pseudo-milter spermary, express all seldom, expression level in milter spermary is very low, almost detects not out.Prove thus, CSW1 is the gene of Cynoglossus semilaevis raun sexual gland specifically expressing.The evaluation of raun and pseudo-milter genetic sex is carried out (Chen S L et al., 2012, Marine Biotechnology, 14 (1): 120-128.) according to the method for having reported.CSW1 gene carries out according to ordinary method in the sxemiquantitative PCR method of the detection of expression of different tissues, and the primer is CSW1-S (ATCATCCACGGCAACGACAT) and CSW1-A (TCTTTTTTGCGACACATTCCAC).And the concrete steps of quantitative PCR analysis are in good time:
(1). extraction and the reverse transcription of the total RNA of Cynoglossus semilaevis different tissues: the heart, liver, the gill, skin, blood, kidney, intestines, brain, spleen, muscle, hypophysis, ovary, spermary and the pseudo-milter spermary tissue of getting the Cynoglossus semilaevis raun of 250-500 grammes per square metre, with RNA, extract test kit and extract total RNA, adopt ordinary method to become cDNA by M-MLV ThermoScript II (Takara) reverse transcription.
(2). real-time fluorescence quantitative PCR reaction conditions
According to Cynoglossus semilaevis CSW1 gene cDNA sequence design real-time fluorescence quantitative PCR primer CSW1-S, CSW1-A (CSW1-S:ATCATCCACGGCAACGACAT, CSW1-A:TCTTTTTTGCGACACATTCCAC), to Cynoglossus semilaevis raun different tissues (heart, liver, the gill, skin, blood, kidney, intestines, brain, spleen, muscle, hypophysis, ovary) and milter spermary, in pseudo-milter spermary, the expression of CSW1 is analyzed.
Real-time fluorescence quantitative PCR reaction system and reaction conditions:
20 μ l reaction systems
10μl?SYBR?Taq
0.4μl?primer?F
0.4μl?Primer?R
0.4μl?Rox?RD?II
0.5μl?cDNA(600ng/μl)
8.3μl?H
20
Find that CSW1 expression level in ovary is the highest, substantially do not express or express and seldom (see Figure 1A and 1B) in other tissue of raun and each tissue such as milter spermary and pseudo-milter spermary, therefore, CSW1 is Cynoglossus semilaevis raun sexual gland specific expression gene.
2, the female specific gene CSW1 of Cynoglossus semilaevis is in the expression of Cynoglossus semilaevis different development stage: adopt real time quantitative PCR method to detect the female specific gene CSW1 of Cynoglossus semilaevis at Cynoglossus semilaevis raun and milter different development stage (4d, 62d, 106d, 5m, 8m, 10m, 1y, 2y) the expression in sexual gland, find that CSW1 gene just starts obvious expression in the ovary of 62 days, and expression in milter spermary, do not detected, subsequently, in the ovary of 106 days, the expression of CSW1 gene obviously raises, and expression in spermary, do not detected yet, the expression amount of the CSW1 gene from 106 days to 1 age fish (340-360 days) presents the trend that increases in time and increase, to one age fish expression amount the highest, two fish in age (650-720 days) expression amounts are in a slight decrease.In spermary, all very low at the expression level of each stage CSW1, even almost can't detect expression, and different steps there was no significant difference.Prove thus, CSW1 gene plays an important role in Cynoglossus semilaevis raun Gonad Differentiation and growth course, and substantially there is no what effect in milter gonad development process.
The evaluation of female milter genetic sex is carried out (Chen S L et al., 2012, Marine Biotechnology, 14 (1): 120-128.) according to the method for having reported.CSW1 gene in the concrete steps of Cynoglossus semilaevis different development stage detection of expression is:
(1) extraction and the reverse transcription of the total RNA of Cynoglossus semilaevis different development stage raun sexual gland: get the fry of different development stage or the ovaries of adult fish such as Cynoglossus semilaevis 4d, 62d, 106d, 5m, 8m, 10m, 1y, 2y, with RNA, extract test kit and extract Total RNA, M-MLV ThermoScript II (Takara) reverse transcription becomes cDNA.
(2) real-time fluorescence quantitative PCR reaction conditions:
CDNA to different development stage (4d, 62d, 106d, 5m, 8m, 10m, 1y, 2y) raun sexual gland carries out real-time fluorescence quantitative PCR analysis, and reaction system and reaction conditions are as follows:
20 μ l reaction systems
10μl?SYBR?Taq
0.4μl?primer?F
0.4μl?Primer?R
0.4μl?Rox?RD?II
0.5μl?cDNA(600ng/μl)
8.3μl?H
20。
Result shows that CSW1 gene is along with the carrying out of fish bulk-growth and female gonad development, in ovary, the expression level of CSW1 raise gradually from 4 days, 62 days, 106 days, 5 months, 8 months and 1 year, when 1 age, reach the highest, expression amount (Fig. 2) in a slight decrease when two ages.
3, the genetic sex of Cynoglossus semilaevis fry and adult fish is identified:
From Cynoglossus semilaevis fry or adult fish, take the tail fin tissue of 0.1g size, according to conventional phenol chloroform method, extract genomic dna, adopt subsequently the cynoglossus semilaevis gunther sex-linked microsatellite marker of the old pine forest of Inst of Huanghai Sea Marine Products, Chinese Academy of Aquatic Product Science laboratory foundation and genetic sex identification method (the Chen S L et al. of foundation, 2012, Marine Biotechnology, 14:120-128) genetic sex of carrying out Cynoglossus semilaevis fry and adult fish identifies.
Three, the structure of the female specific gene CSW1 of Cynoglossus semilaevis recombinant expression vector and in the separation and purification of colibacillary recombinant expressed and expression product
Adopt conventional round pcr, the increased open reading frame ORF region of CSW1 gene, and be cloned on pET-32a (+) expression vector, build CSW1 prokaryotic expression carrier, this expression vector has been transformed in e. coli bl21, carried out the in-vitro recombination expression of CSW1 gene, obtained recombination expression product, recombination expression product, after the His-tag of GE company affinity chromatography column purification, has obtained the CSW1 recombinant protein of purifying, and recombinant protein molecular weight is 40kd left and right.The concrete scheme of realizing above-mentioned target is as follows:
1, the construction process of CSW1 recombinant expression vector:
PET-32a (+) carrier sequence and CSW1 gene ORF region sequence are carried out to restriction enzyme site analysis, and design band restriction enzyme site special primer PET-32-CSW1S and PET-32-CSW1A, sequence is as follows: (PET-32-CSW1S:GGC
gATATCaTGGATATTTTGAGAGACGACGAAGAG,
PET-32-CSW1A:CCG
GAATTCTCATTGCTTGTCTCGTCTTTTGTCC)
Upstream 5 ' end is containing EcoRV restriction enzyme site, and downstream 3 ' end is containing EcoRI restriction enzyme site.The ovarian cdna of take carries out PCR reaction as template, the CSW1 coding region of increasing.By being connected with cloning vector pMD18-T after PCR product purification, positive colony is checked order.By order-checking correct cloned plasmids and PET-32a(+) with EcoRI and EcoRV, carry out double digestion simultaneously, agarose electrophoresis reclaims CSW1 fragment and expression vector plasmid, according to ligase enzyme specification sheets, with T4DNA ligase enzyme, connect, build recombinant expression plasmid PET-32a-CSW1(Fig. 3), transform intestinal bacteria TOP10 competence, detection positive colony is checked order, the correct clone that checks order extracts plasmid.Again PET-32a-CSW1 plasmid transform is expressed to competence BL21 (DE3) bacterium, add simultaneously 30% glycerine in 2-3 milliliter competence bacteria culture in-80 ℃ of freezing preservations as conservation bacterium.
2, the abduction delivering of recombinant protein and the analysis of expression product
Get 5 μ l conservation coli strains and join (containing 100 μ g/mL penbritins) in the LB substratum that 1mL is fresh, 37 ℃, 200r/min incubated overnight, the bacterium of incubated overnight 1% is inoculated into (containing 100 μ g/mL penbritins) in the LB substratum that 10mL is fresh, 37 ℃, when 250r/min is cultured to OD600 and reaches 0.6-0.8, in control group (PET-32a) and experimental group (PET-32a-CSW1) bacterium liquid, add IPTG induction, making IPTG final concentration is 1mmol/L, 28 ℃, 200r/min continues to cultivate 6h, 0, 1, 2, 3, 4, 5 and 6h sample respectively 1mL, by the sample gathering at 4 ℃, centrifugal 5min under 12000r/min condition, PBS damping fluid (the NaCl 137mmol/L of 200 μ L for bacterium mud, KCl 2.7mmol/L, Na2HPO4 10mmol/L, KH2PO4 2mmol/L) wash three times, the centrifugal 2min of 12000r/min after each rinsing, add the PBS of 30 μ L to mix, adding isopyknic 2x sample-loading buffer (1mol/L pH 6.8 Tris-HCl, 1% tetrabromophenol sulfonphthalein, 0.154gDTT, 10%SDS, 10% glycerine) process, boiling water bath 5min cracking thalline, collect cracking bacterium liquid sample and carry out 12%SDS-PAGE electrophoresis detection, discovery has the protein band that 1 molecular weight is 38.75KD (Fig. 4) at CSW1 recombinant expression vector pet-32-CSW1 in the tropina after IPTG induction, in empty carrier pet-32a control group without this protein band (Fig. 4).
3. the separation and purification of recombinant protein
Above-mentioned conservation bacterium a large amount of (250mL) under 37 ℃ of conditions is cultured to cell density, and to reach OD600 be 0.6, after adding IPTG final concentration to be 28 ℃ of induction 6h of 1mmol/L, 4 ℃ of centrifugal 5min of 12000r/min collect thalline, according to 1g wet thallus/10mL lysis buffer (20mM/L Na3PO4, 0.5M NaCl, 20mM/L imidazoles, DNaseI 10 μ, 200 μ L 50mM/L, 200 μ l 10mg/L N,O-Diacetylmuramidases) the resuspended thalline of ratio, ultrasonication under condition of ice bath, 4 ℃, the centrifugal 10min of 12000r/min collects supernatant, supernatant is filtered with the strainer of 0.45 μ L, the supernatant filtering is collected standby.Application His Trap HP(GE company) affinity column carries out separation and purification, and concrete operations are as follows:
First, with 5mL distillation, wash 1mL pillar once, subsequently 5ml binding buffer liquid (20mM/L Na3PO4,0.5M NaCl, 20mM/L imidazoles) wash pillar, again the pretreated supernatant of collecting is crossed to pillar, flow velocity is 1-2mL/min, with 5mL binding buffer liquid, washes pillar, use again elution buffer (the 20mM/L Na3PO4 of 5mL, 0.5M NaCl, 500mM/L imidazoles) wash-out once, finally with the binding buffer liquid regeneration pillar of 5mL.Collection recombinant protein sample of purifying after affinity chromatography carries out 12%SDS-PAGE electrophoresis detection, and observing recombinant protein molecular weight is 40KD left and right (Fig. 4).
4. determination of protein concentration
The albumen of the BCA protein quantification test kit that adopts Beijing Tian Gen biochemical technology company limited after to purifying carries out quantitative analysis, and concrete operations are carried out to specifications.
Four, the bioactive mensuration of Cynoglossus semilaevis CSW1 recombination expression product and application thereof
The recombinant expressed purified product of CSW1 is expelled to the Cynoglossus semilaevis milter spermary of 100-200 grammes per square metre, detected the impact of recombinant C SW1 albumen on sexual gland foxl2 and two kinds of Sexual-related gene expression doses of sox9, find after injection CSW1 recombination expression product in 6-36 hour, the expression level of female genes involved foxl2 progressively raises, at 36 hours, reach the highest, decline gradually subsequently, to 96 hours, return to normal level (Fig. 5).In contrast, the expression level of male genes involved sox9 starts progressively to decline for 12 hours from injecting, and reaches minimumly to 48 hours, raises gradually subsequently, to 96 hours, recovers normal level (Fig. 6).
Concrete detection method is as follows: the concrete scheme of realizing above-mentioned target is:
Get 100-200 grammes per square metre one age Cynoglossus semilaevis milter 40 tails, wherein 4 tails are got sexual gland, as 0 hour contrast.Get 18 tail fish injection recombinant C SW1 albumen (150 μ l, 1 μ g/ μ l), the lost live-weight group of another 18 endnote CSW1 albumen (boiling water bath 5min, 150 μ l, 1 μ g/ μ l) as a control group.Experimental group and control group be 6h after injection respectively, 12h, and 24h, 36h, 48h, 72h, 96h gets sexual gland and drops into rapidly in liquid nitrogen, proceeds to subsequently-80 ℃ and saves backup, and each organizes each time point 3 tails of sampling.According to the Cynoglossus semilaevis Sexual-related gene Foxl2(GQ402462 reporting in NCBI) and the sequences Design quantification PCR primer such as Sox9a(GQ402461), the sequence of each primer is as follows:
Foxl2-S：TGGTTGGAAGTGCGTGGG，
Foxl2-A：GAGAGGAAGGGCAACTACTGGA；
Sox9a-S：CGATTCCCCTGCGTCCA，
Sox9a-A:?GCTTCAGTTCGGCTTTATTGG.
Sexual gland real-time quantitative PCR after injection recombinant protein is detected to each changes in gene expression.Each gene different time expression amount is carried out to independent sample T check, with SPSS 17 statistical softwares, carry out significant difference analysis, will have the group of significant difference to mark (Fig. 6) with *.
Result shows, can obviously the raise expression level of female genes involved foxl2 of the vitro recombination albumen of CSW1 gene of the present invention, there is the biological activity that stimulates female related gene expression, reduce in addition the biological activity of male genes involved SOX9 expression level simultaneously.Therefore, after using CSW1 recombination expression product as fodder additives, by thering is the effect that stimulates the female gonad development of Cynoglossus semilaevis, produce the effect that improves raun ratio.Therefore, the female specific gene of Cynoglossus semilaevis of the present invention's screening and recombinant expression protein thereof have application potential aspect Cynoglossus semilaevis sex control and the female fry ratio of raising.
Claims (5)
1. a Cynoglossus semilaevis Female-specific protein, its aminoacid sequence is SEQ ID NO:1.
2. the female specific gene of Cynoglossus semilaevis, the encoding gene that described gene is Female-specific protein claimed in claim 1.
3. gene as claimed in claim 2, its nucleotides sequence is classified SEQ ID NO:2 as.
4. a recombinant plasmid, described recombinant plasmid is used for expressing albumen claimed in claim 1.
5. recombinant plasmid as claimed in claim 4, is characterized in that, in described recombinant plasmid, having inserted sequence is the DNA fragmentation of SEQ ID NO:3.
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| CN101225437A (en) * | 2007-12-01 | 2008-07-23 | 中国水产科学研究院黄海水产研究所 | Tongue sole molecular marker auxiliary sex control method |
| CN101240348A (en) * | 2008-01-23 | 2008-08-13 | 中国水产科学研究院黄海水产研究所 | Rapid identification method for half-smooth tongue-sole genetic sex |
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| 半滑舌鳎养殖群体中自然性逆转伪雄鱼的发现;季相山等;《水产学报》;20100228;第34卷(第02期);全文 * |
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| 季相山等.半滑舌鳎养殖群体中自然性逆转伪雄鱼的发现.《水产学报》.2010,第34卷(第02期), |
| 邓思平等.半滑舌鳎的性腺分化和温度对性别决定的影响.《中国水产科学》.2007,第14卷(第05期), |
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