CN1840711A - End-point detection kit and method for content of poisonous substance in fresh water fish - Google Patents

Abstract

The disclosed terminal detection reagent box for poison content in freshwater fish (silver carp, grass carp, crucian, etc) comprises any one or more combined or all sGST detoxificated gene expression detection primer, DNA polymerase lymerase, PCR reaction buffer liquid, dNTP solution, and the beta-actin mRNA expression detection primer of freshwater fish as outside reference. This invention can determine the edible safety simply.

Description

The end point determination test kit and the detection method of toxic content in a kind of cultured freshwater fish body

Technical field

Present technique belongs to biological technology application, is specifically related to the end point determination test kit and the detection method of toxic content in a kind of cultured freshwater fish body.

Background technology

1. the source of freshwater aquiculture toxicity

The fresh water body eutrophication causes harmful blue-green alga bloom to take place again and again, and may produce the national healthy cyanophycean toxin of serious harm.Wherein microcystic aeruginosa Microcystis aeruginosa wawter bloom is a kind of algal bloom that may produce poison wide, longer duration that distributes in countries in the world freshwater lake, the pond, the toxin of its generation be called Microcystin (microcystin, MC).The agent structure of Microcystin is a ring seven peptide, has more than 60 kind of isomer, and what toxicity was bigger is LR, YR, RR type, and L, Y, R represent leucine, tyrosine, arginine respectively.These toxin have similar biological function, can cause hepatocellular damage, and severe patient also can cause hepatic entorrhagia, causes animal dead, and wherein microcapsule algae toxin is a kind of ring-type seven peptide hepatotoxin, and are the most common and toxicity is high.Because Microcystin is very low by the membrane permeation ability of striding of simple diffusion, must be that bile acid transport system on the liver plasma membrane is transported to hepatic parenchymal cells by intermediate carrier, so liver becomes the intoxicating target organ of algae toxin.This toxin is in the special accumulation of liver, and the activity of arrestin phosphatase 1 (PP1) and phosphoprotein phosphatase 2A (PP2A), the peroxophosphoric acidization that causes multiple proteins in the cell, break the balance of intracellular protein phosphorylation/dephosphorylation, and by this biochemical effect of the further amplification of cell signal system, cause the disorder of a series of biochemical reactions in the cell, finally cause hepatocellular injury, even the acute death incident takes place.The short function of tumor of Microcystin is also realized in this way.Long-term drinking or edible water or the fishery products that contain Microcystin may cause liver cancer, and particularly under the situation that has hepatitis virus and aflatoxin, the possibility of this initiation liver cancer is higher.Therefore, the pollution of solution water body Microcystin is national healthy particularly important to China.

Microcystin mainly by following approach in the aquatic organism inner accumulated: (1) organism is by body surface contact algae toxin, as waterplant and fish-egg; (2) organism is directly drunk the water of algae endotoxin contamination or is foodstuff with the blue-green alga bloom, and this approach is also referred to as biological concentrate (bioconcentration), as zooplankton, shellfish and some algophagous fish; (3) organism is positioned at the upstream of food chain, with one-level human consumers such as zooplankton, shellfish and some algophagous fish is food, these hydrobionts are by food chain accumulation algae toxin, and this approach is also referred to as biological amplify (biomagnification), as some predacious fish etc.When hydrobiont absorbed poison from environment by all means, toxin just took place in the intravital accumulation of organism thereupon.Studies show that different hydrobionts are to the ability of aggregation and the purification required time difference of Microcystin.Waterplant is the primary producer in the aquatic ecosystem, directly and the Microcystin in the environment contact, different waterplant are to the ability of aggregation of algae toxin difference to some extent, and are relevant with the receptivity of its surface-area and contratoxin.In addition, toxin absorption back required time of removing in the waterplant body also is not quite similar.Some algae, for example elongated synechococcus has the ability that purifies toxin more by force, and toxin was handled after one day, and the algae content of toxins in the substratum has only 21.7% of starting point concentration; Handle after 6 days, the toxin in the substratum only remains 8.18%.But generally, compare with hydrocoles, the gathering of waterplant contratoxin is less, purifies also slower.

Shellfish and crayfish etc. have shell class invertebrates slow than vertebratess such as fish to the removing of algae toxin, and the effect of enrichment toxin is arranged.Compare with shellfish, fish are lower and removing speed is very fast to the accumulated amount of Microcystin.Fill out hello rainbow trout (Oncorhynchus mykiss) with Microcystis aeruginosa under the experiment condition, toxin is assembled rapidly in its liver after one hour, and major part is excreted in subsequently 24 hours.Because freshwater fish possesses rapid absorption and removes the ability of Microcystin, illustrate and have flourishing Microcystin detoxification system in its body, at present viewpoint is thought freshwater fish liver sGST, be that freshwater fish Microcystin detoxifying enzymes plays a crucial role in this detoxification process, but the rarely seen so far report of research of relevant freshwater fish Microcystin detoxifying enzymes gene.

In addition, because the use of relevant agricultural chemicals, make poisonous substance such as agricultural chemicals in cultured freshwater fish body inner accumulated.But the long-term edible fishery products teratogenesis that contains these medicines, carcinogenic causes serious threat to human health.Oneself causes the extensive concern of countries in the world public health system to each toxoid to threat that human health caused, and the long-term edible fishery products that contain toxin can cause multiple diseases such as liver cancer.Discover that the main foster fresh-water fishes in pond such as tilapia, silver carp, flathead all have the very strong effect of ingesting to blue-green algaes such as Microcystis aeruginosas, add different source of pollution, so should carry out safety detection to freshwater fish toxicity content such as tilapias as early as possible.

2. detoxifying enzymes gene---glutathione S-transferase gene

Glutathione S-transferase (glutathione S-transferase, GST, EC2.5.1.18) being prevalent in the various organisms, is one group of important component part by detoxifcation enzyme system a plurality of genes encodings, the multiple endogenous or external source toxicant of metabolism, belongs to II phase metabolic enzyme system.This enzyme has cytosol and film in conjunction with two kinds of forms, based on cytosol GST (sGST).All contain different types of sGST in each tissue of vertebrates, its content and activity also have nothing in common with each other, and be wherein the highest with content in the liver.SGST exists with same or heterodimer form, main catalysis number of chemical material comprises the various toxic metabolites that medicine, chemotherapeutics, carcinogens and oxidative stress produce etc., with reduced glutathion (glutathione, GSH) (SH) combination of sulfydryl, make these electrophilic hydrophobic compounds become hydrophilic material, be easy to from bile or urine, drain.Some sGST also has the activity of peroxidase and isomerase, can remove the effect of lipid free radical and anti peroxidation of lipid, thereby alleviates the degree of injury of DNA.Therefore, sGST avoids playing an important role in attack of acute toxicity chemical substance and the inhibition cell carcinogenesis at metabolism, the protection cell of body toxic compounds.

Freshwater fish sGST gene is mainly used in the detoxification of natural poisonous substance such as Microcystin under native state, and is culturing under the pollutional condition, then is mainly used in the detoxification of artificial poisonous substance such as agricultural chemicals.SGST Gene Handling institute Toxic is (the 2nd o'clock phase) common adduction toxin expelling process after the oxidation, hydrolysis detoxification process in early stage (the 1st o'clock phase), also therefore is called as the 2nd o'clock phase detoxifying enzymes.Because in Microcystin detoxification metabolic process, the adduction toxin expelling process of the 2nd o'clock phase has unique keying action, so the freshwater fish sGST gene Microcystin detoxifying enzymes gene that is otherwise known as.

According to different standardss such as gene structure, aminoacid sequence, substrate specificity, chemical affinity and dynamic behaviors, Mammals sGST can be divided into 8 classes: a (alpha), μ (mu), P (pi), s (sigma), θ (theta), ω (omega), κ (kappa) and ζ (zeta).In same class, the amino acid sequence homology of different sGST reaches 40% at least, and between the inhomogeneous sGST, amino acid sequence homology is less than 30%.All kinds of sGST are divided into different subclass by sequence similarity and immunological cross-reaction, and the expression level of different subclass has tissue specificity.SGST is to the very high specificity that is combined with of GSH, but to the specificity of second electrophilic substrates significant difference between inhomogeneity and in the same class.A, μ, p and θ class sGST gene are obviously different on its size, intron/exons structure, studies show that θ class isozyme gsh is different from a, μ, p and s class in conjunction with the amino acid at center.In addition, the θ zymoid is evolved prior to other enzymes.Isoformgene has the trend of cluster, has type specificity bunch as human sGST gene.Up to the present, 4 kinds of sGST genes, promptly GSTM1, GSTM3, GSTP1 and GSTT1 have confirmed to have polymorphism.The polymorphism of gene can cause the change of sGST enzymic activity, and causes between individuality the difference to the potential carcinogen metabolic capacity.

3. fishery products toxicity residue detection analytical procedure

The residue detection analytical procedure at first is that objectionable impurities is extracted from fishery products, separated, and utilizes plant and instrument to carry out qualitative and quantitative analysis then.Fishery products toxicity residue detection analytical procedure mainly contains liquid phase chromatography and immunization etc. at present.

High performance liquid chromatography (HPLC) is a kind of method that a kind of sensitivity is higher, reliability is stronger, and this method good reproducibility, speed are fast, and many extremely difficult isolating determinands are analyzed, and the HPLC method is all adopted in the analysis of present most of fishery products drug residue.But HPLC method detection limit is higher, is 5～10 μ g/kg, can not reach in the world the minimum of the residual requirement of fishery products limited the quantity of, and as the content of paraxin in the fishery products, European Union requires less than 0.1 μ g/kg.In addition, because of complicated component in the animal body, some impurity interference measurements, when using the detection of HPLC method, must be carefully careful for sample pretreatment, measure accuracy and sensitivity to improve.

The immuno analytical method immunoassay is to grow up in recent years based on the specificity of antigen and antibody, the novel analytical technology of reversibility association reaction.Immune response has very high selectivity and susceptibility, therefore, immuno analytical method no matter as the detection means of residue of veterinary drug analysis still be the sample clean method can both make analytic process particularly pre-treatment step simplify greatly.As relatively independent detection method, promptly, comprise enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), solid-phase immunity transmitter etc. based on the immunoassay of competition binding analysis principle.Use at present euzymelinked immunosorbent assay (ELISA) (ELISA) the most generally, have easy and simple to handle, highly sensitive, sample capacity big, instrumentation degree and the low advantage of analysis cost, is one of optimal residual screening analytical procedure at present.Nearly all important fishery products drug residue has all been set up or has been attempted to set up the ELISA detection method at present, as paraxin, tsiklomitsin, Streptomycin sulphate, stilboestrol etc.The shortcoming of euzymelinked immunosorbent assay (ELISA) is that influence factor is more, is prone to false positive results.

In sum, because the fishing pharmacopoeia class of violating a ban is various, each fishing medicine all will detect with ad hoc approach, and it is very expensive to detect all fishing expensess for medicine, and the fresh-water fishes price general charged is not high, so generally can only carry out the conventional sense of part fishing medicine.Though also spending huge fund every year, country use chemical means that part fishing medicine is carried out conventional sense, often owing to certain fishing medicine of omission produces serious consequence.The eel of in July, 2005 China's outlet Japan is not because malachite green belongs to the conventional sense project and omission, cause the eel outlet to stop production with sold inside the country, not only the eel industry is caused crushing blow, and involve whole freshwater fish culturing industry, to such an extent as to appearance Hong Kong resident in a period of time dare not eat the panic phenomenon of China's Mainland cultured freshwater fish.Therefore, provide a kind of can be easy, accurately toxic content in the cultured fishes body to be carried out development and supervision that the detection method of qualitative, quantitative cultures cause to China freshwater fish be very necessary.

Summary of the invention

The objective of the invention is to overcome the defective of the technical existence of poisonous substance content detection in existing China freshwater fish body, a kind of test kit that can carry out qualitative and quantitative analysis to the different strains of multiple fish, in-house toxic content quickly and accurately is provided.

Another object of the present invention provides the method for utilizing the mentioned reagent box to detect toxic content in the fish body.

The present invention determines whether to contain in the fish body poisonous substance and edible safety problems thereof such as Microcystin by measuring fish body detoxifying enzymes genetic expression state.The present invention needn't adopt different pharmaceutical special detection method blindly to inspect by random samples whether to contain certain medicine, finally all will cause one solubility glutathione-S-transferase (sGST) genetic expression of fish liver detoxifying enzymes and be based on different poisonous substances, detect this index of sGST gene expression dose by fixing means and can reflect that organism is subjected to the pollution condition of different poisonous substances, and easy thus and determine cultured freshwater fish edible safety problem reliably.Though the present invention can not determine the concrete kind of polluting poisonous substance etc., definitely guaranteeing to be examined qualified fish, not to be in contaminated state also safe to eat.If necessary, then can on purpose be adopted the conventional chemical means, possible poisonous substance is further detected one by one to determine to pollute the kind of poisonous substance for examining defective fish.

The said fish of the present invention are freshwater fishes, preferably the main foster freshwater fishes in pond such as tilapia, silver carp, bighead, mandarin fish, grass carp, crucian, dace.And disclose silver carp (Hypophthalmichthysmolitrix), bighead (Aristichthys nobilis), tilapia (Oreochromis niloticus), dace (Cirrhina molitorella), crucian (Carassius auratu), grass carp (Ctenopharyngodon idella), mandarin fish freshwater fish liver detoxifying enzymes---the cDNA sequence and 5 ' the flank regulating and controlling sequences of solubility glutathione-S-transferase (sGST) gene such as (Siniperca chuatsi) first, and designed detection primer in the test kit of the present invention with this.

Below technology disclosed by the invention is further detailed:

1. to the clone of the main foster freshwater fish detoxifying enzymes gene in ponds such as silver carp, bighead, tilapia, dace, crucian, grass carp, mandarin fish.

Clone's fish detoxication enzyme gene is normally used functional gene cloning process: at first, conservative region design degenerated primer according to the aminoacid sequence of a certain specific gene of known vertebrates, utilize the RT-PCR technology to obtain the core segment of this gene, to be connected with the T carrier after this segment separation and purification, transformed into escherichia coli, the picking white colony obtains positive colony through PCR reaction evaluation, send the bacterium liquid of positive colony to go order-checking, thereby obtain the pulsating sequence of cDNA core of goal gene; 3 ' the terminal and 5 ' end that next utilizes 3 '-RACE and 5 '-RACE technology amplification cDNA carries out sequence assembly with cDNA core segment, 3 ' end and 5 ' end, thereby obtains full length cDNA sequence; Obtain the introne DNA sequence by genome PCR at last, further clone the 5 ' flanking sequence (containing promotor and other cis-regulating element) and the 3 ' flanking sequence of this gene by the genomic walking technology, thereby obtain the genomic dna complete sequence of this gene.

As above said functional gene cloning process, the conventional molecule clone technology that relates to comprises the extraction of DNA, RNA, sepharose is connected with polyacrylamide gel electrophoresis, dna fragmentation, the equal reference of restriction enzyme reaction " molecular cloning laboratory manual " (Msniatis etc., cold spring harbor laboratory publishes, cold spring port, New York, the U.S., 1989).Archaeal dna polymerase chain reaction (PCR), reverse transcription PCR reaction and nest-type PRC react equal reference " round pcr experiment guide " (Carl W.Dieffenbach etc., cold spring harbor laboratory publishes, cold spring port, New York, the U.S., 1995).It is synthetic that needed oligonucleotide primer of dna sequencing, DNA cloning and reverse transcription PCR such as oligo (dT) 18 etc. can transfer to special mechanism.The used intestinal bacteria of the present invention be JM109 available from U.S. GIBCO company, pMD 18-T carrier is available from Japanese TaKaRa company.In addition, the extraction of the recovery of the extraction of plasmid DNA and purifying, dna fragmentation, RNA and purifying etc. also can adopt the test kit of functional body's production and operate with reference to the schedule of operation that provides.

In the embodiment that the present invention provides, separate liver organization from silver carp, bighead, tilapia, dace, crucian, grass carp, mandarin fish at first respectively, carry out the extraction and purification of total RNA; Be template with silver carp, bighead, tilapia, dace, crucian, grass carp, mandarin fish liver total RNA respectively again, oligo (dT) 18 is the reverse transcription primer, synthetic cDNA first chain.

According to the conservative region design degenerated primer of known vertebrates sGST aminoacid sequence, silver carp, bighead, tilapia sGST cDNA core segment that the clone obtains design 5 ' RACE reverse transcription primer again.The principle of 5 ' RACE is seen accompanying drawing 1, and working method is as follows: at first with the synthetic cDNA template of reverse transcription primer, then digest to produce strand cDNA with the RNA enzyme, connect the generation pcr template with the T4 ligase enzyme again.Carry out two-wheeled PCR reaction with nested PCR primer respectively.

The principle of 3 ' RACE is seen accompanying drawing 2, and working method is as follows: with purpose mRNA is template, uses OligodT-3 ' joint primer (3sites Adaptor Primer) to carry out reverse transcription reaction, synthetic cDNA template.Use contains restriction enzyme site, and upstream Auele Specific Primer and 3 ' the joint primer of KpnI, XbaI, BamH I carry out the PCR reaction as shown in drawings.Select suitable carrier cloning PCR product for use and carry out dna sequencing.

The PCR product is through 2% agarose electrophoresis, and purifying reclaims rear clone to pMD 18-T carrier, and transformed competence colibacillus E.coli JM109 utilizes the forward and reverse primer of M13, obtains positive colony by the PCR reaction detection, and positive colony is checked order by specialized company.Carry out sequential analysis with DNA analysis software vector NTI suite 6.0.

2. be outer reference with beta-actin, to the detoxifying enzymes gene mRNA relatively level detect.

Present technique is on the basis of the new sequence operative of freshwater fish liver Microcystin detoxifying enzymes gene cDNA such as success clone tilapia, silver carp, bighead, mandarin fish, grass carp, crucian, dace that takes the lead in, further under the pond culture working condition, beta-actin with freshwater fish is outer reference, with the RT-PCR method relative level of mRNA of different varieties, strain pond culture tilapia liver and muscle sGST detoxifying enzymes gene relatively.

Detection method comprises: careful separation goes out to detect the liver of fish, and muscle tissue is drawn materials from the back, is used to extract total RNA after weighing rapidly.Total RNA extracts and cDNA synthesizes ditto described.The PCR reaction system of sGST and beta-actin is identical with reaction conditions, its PCR product behind 2% agarose gel electrophoresis and ethidium bromide staining with gel imaging system and related software (AlphaImaget, Alpha Inotech, USA) analyze, the result is with ratio (%) expression of sGST with the RT-PCR product brightness of beta-actin mRNA.

Adopt 10.0 pairs of fish liver sGSTmRNA relative expression horizontal average differences not of the same race of statistical analysis software SPSS to carry out statistical study.If P＜0.05, the difference of mean value is promptly thought significantly.

3. the preparation of cultured fishes end point determination test kit

This detection kit is to be prepared from according to following detection technique: contained toxic content height in the cultured freshwater fish body, then relative expression's level of sGST mRNA is just high, so with the expression of the beta-actin mRNA of freshwater fish as outer reference, expression level to sGST mRNA detects, compare with contrast, just can know in the fish body whether conformance with standard of contained poisonous substance.

According to aforesaid detection technique, detect step and comprise:

(1) liver total RNA of extraction and purification cultured freshwater fish as the reverse transcription primer, adopts the synthetic cDNA of reverse transcription method with oligo (dT) 18;

(2) be template with (1) synthetic cDNA, get and detect primer, DNA polymerase, PCR reaction buffer, dNTP, sterile purified water etc. with the sGST detoxifying enzymes genetic expression of the corresponding fingerling of this template and mix in right amount, carry out the PCR reaction, reaction conditions is: 94 ℃ of pre-sex change 3～6min at first, 94 ℃ of 30sec～1min then, 55～65 ℃ of 30sec～1min, 72 ℃ of 30sec～1min, totally 25～32 circulations, last 72 ℃ are extended 5～10min;

(3) be template with (1) synthetic cDNA, beta-actin mRNA detection of expression primer, DNA polymerase, PCR reaction buffer, dNTP, sterile purified water mixes, and carries out the PCR reaction, and reaction system and reaction conditions are with (2);

(4) after PCR reaction was finished, the PCR product of (2) and (3) of getting equivalent was through 1～2% agarose gel electrophoresis that contains ethidium bromide, and brightness detects comparison to product, determines the expression conditions of sGST detoxifying enzymes gene.

According to aforesaid detection step, detection kit should contain following component: the DNA polymerase that adopts when being used for pcr amplification reaction is as rTaq commonly used, with the matching used Mg that contains of this enzyme ²⁺Reaction buffer (PCR Buffer), and dNTP damping fluid (2.5mM), the upstream and downstream that crucial is is used to detect relative expression's level of freshwater fish sGSTmRNA detects primer; In addition, also need the outer reference of upstream and downstream primer when detecting of the beta-actin mRNA detection of expression of freshwater fish, and the sGST cDNA that is used for Control cDNA freshwater fish is as positive reference substance.

Here relative expression's level detection primer of said cultured freshwater fish sGST mRNA, be meant the detection primer of silver carp, bighead, tilapia, dace, mandarin fish, grass carp, crucian, according to different needs, can be these several fish any or any two or combination even comprise whole detection primers of these fish at random more than any two; In addition, these primers all are that the cDNA sequence of these fish sGST disclosed according to the present invention is designed, totally 7 kinds of cDNA, its cDNA sequence is respectively shown in SEQ ID NO:1, SEQID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ IDNO:13.

In addition, because the higher homology of cDNA sequence of the sGST of silver carp, bighead, grass carp, therefore, the sGST of these three kinds of fish detects primer can be according to the homologous region design universal primer of cDNA sequence.The cDNA sequence homology of the sGST of silver carp, bighead, grass carp, mandarin fish, tilapia, dace, crucian is relatively seen accompanying drawing 3.The design and the application of universal primer see specific embodiment for details.

In addition, the selected DNA polymerase that is used for pcr amplification reaction in the detection kit disclosed by the invention can be the DNA polymerase of arbitrary company, and the PCR reaction buffer is this arbitrary company and the supporting reaction buffer of this enzyme.DNA polymerase commonly used at present has many kinds such as rTaq, Pfu, Taq Plus, HotStartTaq, Taq Platinum or Pyrobest etc., and in specific embodiments of the invention, that select for use is rTaq.

In addition, the used dNTP solution that is used for pcr amplification reaction in the detection kit disclosed by the invention can be the dNTP solution of arbitrary company, contains dATP, dCTP, dGTP and four kinds of components of dTTP, in the specific embodiment that the present invention provides, the concentration of dNTP solution is 2.5mM.

In addition, the outer reference of the upstream and downstream primer of the beta-actin mRNA detection of expression of the cultured freshwater fish that this test kit provides when detecting, mainly be for the target RNA that is used for extracting from the fish liver cell quantitatively, to avoid the quantitative error of template, amplification efficiency heterogeneity in application of sample error and each reaction system, the error that the temperature head between each hole etc. causes can also detect the integrity of template, especially in RT-PCR, can also detect that template is whether negative to be expressed etc.Because the beta-actin of freshwater fish has higher conservative property, can be that different fish are special so beta-actin mRNA provided by the present invention detects primer, also can be multiple fish are general.In specific embodiment provided by the present invention, silver carp, mandarin fish, tilapia, dace are that general beta-actin detects primer.

In addition, detection kit provided by the present invention also provides the sGST cDNA of freshwater fish as the positive control template.Get this template and relevant detection primer, under certain PCR reaction system, can amplify corresponding positive PCR product, RT-PCR amplified production with testing sample carries out agarose gel electrophoresis, can be by the position of more positive PCR product, find the position of purpose band fast, reach purpose qualitatively.

In addition, detection kit provided by the present invention can also comprise and be used for cDNA synthetic oligo (dT) 18 universal primers and ThermoScript II, reaction buffer.Its ThermoScript II is the ThermoScript II of arbitrary company that can adopt at present, and reaction buffer is this arbitrary company and the supporting reaction buffer of ThermoScript II, and oligo (dT) 18 universal primers can be transferred to relevant speciality company and synthesize.ThermoScript II at present commonly used has the AMV that the AMV that derives from the avian myeloblastosis virus extract such as companies such as Takara and Promega produce and derives from the Mo-MLV of murine leukemia virus such as the Stratascript of Stratagene company, the MLV series of the Suprscript of Life Technologies company and Suprscript II, invitrogen Corporation's Super ScriptIII and Promega.

Adopt test kit disclosed in this invention, the reaction system of suggestion is

dd H 2O 10 * PCR damping fluid (contains Mg 2+) dNTP (2.5mM) upstream primer downstream primer cDNA rTaq enzyme	36.75μl 5μl 4μl 1μl 1μl 1μl 0.25μl
		Total	50μl

The pcr amplification reaction condition: 94 ℃ of pre-sex change 3～6min, 94 ℃ of 30sec～1min then, 55～65 ℃ of 30sec～1min, 72 ℃ of 30sec～1min, totally 25～32 circulations, last 72 ℃ are extended 5～10min;

The PCR product is through analyzing with gel imaging system and related software behind electrophoresis on 1～2% sepharose (method is identical with test two), and the result is with ratio (%) expression of testing gene with the RT-PCR product brightness of beta-actin mRNA.

Compared with prior art, the present invention has following beneficial effect: the present invention determines whether to contain in the fish body poisonous substance and edible safety problems thereof such as Microcystin by measuring fish body detoxifying enzymes gene expression dose.The present invention needn't adopt different pharmaceutical special detection method blindly to inspect by random samples whether to contain certain medicine, finally all will cause the fish liver detoxifying enzymes and be based on different poisonous substances---solubility glutathione-S-transferase (sGST) genetic expression, detect the sGST gene expression dose by fixing means, thereby the intravital content of toxins of cultured freshwater fish is carried out qualitative, quantitative, for the cultured freshwater fish edible safety provides safe, easy, reliable index.Though the present invention can not determine the concrete kind of polluting poisonous substance etc., definitely guaranteeing to be examined qualified fish, not to be in contaminated state also safe to eat.If necessary, then can on purpose be adopted the conventional chemical means, possible poisonous substance is further detected one by one to determine to pollute the kind of poisonous substance for examining defective fish.

Description of drawings

Fig. 1 is 5 '-RACE method principle schematic;

Fig. 2 is 3 '-RACE method principle schematic;

Fig. 3 is a freshwater fish sGST amino acid identity comparison diagram;

Fig. 4 is the expression of different fish sGST;

The RT-PCR that different fish sGST (a) of Fig. 5 and beta-actin (b) mRNA express analyzes: M, marker; 1, silver carp; 2, bighead; 3, grass carp; 4, crucian; 5, dace; 6, tilapia;

Fig. 6 tilapia carries out abdominal injection PBS, LPS, MC-LR, MC-LR+LPS respectively, specific expressed GST of liver organization behind the 24h (glutathione-S-transferring enzyme) and beta-actin expression amount per-cent; PBS, LPS, MC-LR+LPS treatment group all have significant difference with the MC-LR treatment group; But there is not significant difference between PBS, LPS, the MC-LR+LPS treatment group;

The RT-PCR that the different toxin of Fig. 7 tilapia are handled sGST (A) and beta-actin (B) mRNA expression analyzes: M, marker; 1, control group; 2, abdominal injection LPS; 3, abdominal injection MC-LR; 4, abdominal injection MC-LR+LPS.

Embodiment

The successful clone of the new sequences of liver Microcystin detoxifying enzymes gene cDNA such as embodiment 1 silver carp, bighead, grass carp, crucian, dace, tilapia, mandarin fish

Silver carp, bighead, grass carp, crucian, dace, tilapia show hilllock reservoir and near waters thereof and catch in Boluo County, mandarin fish is buied in Stone steles market, Guangzhou.

(1) total RNA extraction and cDNA first chain is synthetic

Separate liver organization from silver carp, bighead, grass carp, crucian, dace, tilapia, mandarin fish respectively, the extraction and purification of total RNA is undertaken by the SV Total RNA Isolation System test kit recommend method of Promega company.Synthetic RNA LA PCRTMKit (AMV) the Ver.1.1 test kit that uses TaKaRa company of cDNA first chain, be template with silver carp, bighead, grass carp, crucian, dace, tilapia, mandarin fish liver total RNA respectively, oligo (dT) 18 is the reverse transcription primer, and operation is undertaken by the test kit recommend method.

(2) the pulsating clone of cultured freshwater fish such as silver carp, bighead Microcystin detoxifying enzymes gene cDNA core

According to 3 degenerated primers of conservative region design of known vertebrates sGST aminoacid sequence, sGST01F, sGST02F, sGST03R, primer sequence sees Table 1.SGST01F and sGST03R are used to clone silver carp, crucian sGST cDNA core fragment; SGST02F and sGST03R are used to clone bighead, grass carp, dace, tilapia, mandarin fish sGST cDNA core fragment.The pcr amplification condition is: 94 ℃ of pre-sex change 3min, and 94 ℃ of 1min, 40 ℃ of 1min, 72 ℃ of 1min, totally 30 circulations, last 72 ℃ are extended 5min.

(3) the detoxifying enzymes gene 5 ' end cDNA of silver carp, bighead, tilapia etc. amplification

The silver carp, bighead, the tilapia sGST cDNA core segment that obtain according to the clone, design 5 ' RACE reverse transcription primer SCRT (silver carp), BCRT (bighead), TRT (tilapia) and nested PCR primer SCS1/SCA1 and SCS2/SCA2 (silver carp), BCS1/BCA1 and BCS2/BCA2 (bighead), TS1/TA1 and TS2/TA2 (tilapia), primer sequence sees Table 1.

The operation of 5 ' RACE is undertaken by the test kit recommend method.At first, then digest to produce strand cDNA, connect the generation pcr template with the T4 ligase enzyme again with the RNA enzyme with the synthetic cDNA template of reverse transcription primer (SCRT, BCRT, TRT or TSRT).Be that primer carries out two-wheeled PCR reaction with nested PCR primer SCS1/SCA1 (silver carp), BCS1/BCA1 (bighead), TS1/TA1 (tilapia) with SCS2/SCA2 (silver carp), BCS2/BCA2 (bighead), TS2/TA2 (tilapia) respectively.First run PCR reaction conditions is: 94 ℃ of pre-sex change 3min, and 94 ℃ of 1min thereupon, 55 ℃ of 1min, 72 ℃ of 1min, 25 circulations, last 72 ℃ are extended 5min; Second takes turns the PCR reaction conditions is: 94 ℃ of pre-sex change 3min, and 94 ℃ of 1min thereupon, 60 ℃ of 1min, 72 ℃ of 1min, 30 circulations, last 72 ℃ are extended 5min.

(4) detoxifying enzymes gene 3 ' the end cDNA amplification of silver carp, bighead, tilapia etc.

The operation reference reagent box recommend method of 3 ' RACE.At first the oligodT-3sites Adaptor primer that provides with test kit is that primer carries out reverse transcription reaction, and SCS2 (silver carp), BCS2 (bighead), the TS2 (tilapia) (table 1) of 3sitesAdaptor primer that provides with test kit and 5 ' RACE are that primer carries out PCR reaction first then.The PCR reaction conditions is identical with 5 ' RACE first run PCR, but cycle index is 30 times.The nest-type PRC the primer is 3sites Adaptor primer and SCS3 (silver carp), BCS3 (bighead), TS3 (tilapia), and primer sequence sees Table 1, and it is identical that PCR reaction conditions and 5 ' RACE second take turns PCR.

(5) clone of PCR product and sequential analysis

The PCR product is through 2% agarose electrophoresis purifying, H.Q.﹠amp; Q.Gel Extraction Kit II (U-gene) reclaims rear clone to pMD 18-T carrier (TaKaRa), transformed competence colibacillus E.coli JM109, utilize the forward and reverse primer of M13, obtain positive colony by the PCR reaction detection, positive colony is checked order by Bo Ya company.Carry out sequential analysis with DNA analysis software vector NTI suite 6.0.

The sGST sequence of the silver carp that obtains, bighead, tilapia, dace, crucian, grass carp, mandarin fish is asked for an interview sequence table; Figure of description 3 is asked for an interview in the sequence homology comparison of silver carp, bighead, tilapia, dace, crucian, grass carp, mandarin fish.

Table 1 silver carp, bighead, grass carp, crucian, dace, tilapia, mandarin fish sGST gene degenerated primer

Reach silver carp, bighead tilapia 5 ', 3 ' RACE PCR primer

The primer title	Primer sequence
		sGST01F sGST02F sGST03R TRT TS1 TA1 TS2 TA2 TS3 SCRT SCS1 SCA1 SCS2 SCA2	5’-TACTTCAATGGCAGAGG(C/G)AA(A/G)ATGGA(A/G)-3’ 5’-TC(T/C)TTTGAGAAAATGGA(G/A)CA(C/T)AA-3’ 5’-CCCTC(A/G)AACATGCG(C/T)TG(G/A)TACAT-3’ 5’-(P)CACAGGAAGGTAGCG-3’ 5’-CATGATACTTCCCTTCACC-3’ 5’-TTCCATGAGGTCTGTCAA-3’ 5’-GGACAACATTCAGAGCAA-3’ 5’-CCTCAGAGTACATGTTGA-3’ 5’-AGGGCAGGATGACACAA-3’ 5’-(P)AACACAGGCAGGAAG-3’ 5’-ACAGAGGGTACCATTGATA-3’ 5’-AGCCCGTTCTTTAAGGTC-3’ 5’-TTGAGCCACCTGAGACCA-3’ 5’-AACTCGACTCCAGCTGCG-3’

SCS3 BCRT BCS1 BCA1 BCS2 BCA2 BCS3

5’-AGGGCAGGATGACACAA-3’ 5’-(P)AACACAGGCAGGAAG-3’ 5’-GGATCTCATCATAATGTCTG-3’ 5’-CTCTGCATACATGTCAATC-3’ 5’-GCAGAAACAGCTCGGTAA-3’ 5’-AGCCCGTTCTTTAAGGTC-3’ 5’-GCCTTACCAAGAATCAGCA-3’

The preparation of the end point determination test kit of embodiment 2 freshwater fish edible safeties

(1), test kit is formed:

rTaq(5U/μl)

10×PCR Buffer(Mg ²⁺plus)

dNTP(2.5mM)

Control cDNA (silver carp, bighead, grass carp, mandarin fish, tilapia, dace, crucian be totally 7 kinds of cDNA)

Primer (totally 12 of various fresh-water fishes primers)

GST01F：5’-GACCTTAAAGAACGGGC-3’

GST02R：5’-GGAACTTGCTGATTCTTGG-3’

GST03F：5’-AAGGCCTCAAAGACCGTG-3’

GST04R：5’-CAGGAACCTGTTGATGGC-3’

GST05F：5’-AGGATCCCAAAGAACGAG-3’

GST06R：5’-CAGAAACCTGCTGATGGC-3’

GST07F：5’-AAGAAAGGGCTCTGATTG-3’

GST08R：5’-GCTGGAGGAACTTGCTGA-3’

GST09R：5’-GAAACTTGCTGATTGTTGG-3’

ACT01F：5’-CGTGACATCAAGGAGAAGC-3’

ACT02R：5’-TCTGCTGGAAGGTGGACAG-3’

ACT03R：5’-TCTGCTGGAAGATAGACAG-3’

Wherein: GST01F and GST02R are the sGST gene universal primer of silver carp, bighead, grass carp, and the clip size that amplifies is 334bp;

GST03F and GST04R are the sGST primer of mandarin fish, and the clip size that amplifies is 332bp;

GST05F and GST06R are the sGST primer of tilapia, and the clip size that amplifies is 332bp;

GST07F and GST08R are the sGST primer of dace, and the clip size that amplifies is 333bp;

GST01F and GST09R are the sGST primer of crucian, and the clip size that amplifies is 334bp;

ACT01F and ACT02R are the general beta-actin primer of silver carp, mandarin fish, tilapia, dace, and the clip size that amplifies is 436bp;

ACT01F and ACT03R are the beta-actin primer of crucian primer, and the clip size that amplifies is 436bp.

(2) test kit operation instructions:

The PCR reaction system of suggestion is shown in the table 2:

Table 2

dd H 2O 10×PCR Buffer(Mg 2+Plus) dNTP (2.5mM) Primer 1 Primer 2 cDNA rTaq enzymes (TaKaRa)	36.75μl 5μl 4μl 1μl 1μl 1μl 0.25μl
		Total	50μl

The pcr amplification reaction condition of suggestion:

94 ℃ of pre-sex change 3min are 94 ℃ of sex change 1min then, 55 ℃ of annealing 1min, and 72 ℃ are extended 1min, carry out 30 circulating reactions altogether; 72 ℃ are extended 5min again after the loop ends.

The PCR product through behind electrophoresis on 2% sepharose with gel imaging system and software kit (AlphaImaget, Alpha Inotech USA) analyzes, the result is with ratio (%) expression of testing gene and the RT-PCR product brightness of beta-actin mRNA.

The application of embodiment 3 detection kit aspect freshwater fish liver detoxifying enzymes mrna expression mensuration

Total RNA of silver carp, bighead, grass carp, crucian, dace, tilapia liver organization extracts and cDNA synthesizes with described in the embodiment 1.Get embodiment 2 described test kits, use special primer GST01F and GST02R amplification silver carp, bighead, grass carp sGST cDNA fragment (334bp); GST01F and GST09R amplification crucian sGST cDNA fragment (334bp); GST07F and GST08R amplification dace sGST cDNA fragment (333bp); GST05F and GST06R amplification tilapia sGST cDNA fragment (333bp).With special primer ACT01F and ACT02R amplification silver carp, bighead, grass carp, dace, tilapia beta-actin cDNA fragment (436bp); With ACT01F and ACT03R amplification crucian beta-actin cDNA fragment (436bp).

The PCR reaction conditions is 94 ℃ of pre-sex change 3min, 94 ℃ of 1min, and 55 ℃ of 1min, 72 ℃ of 1min, totally 30 circulations, last 72 ℃ are extended 5min.Termination reaction in the exponential growth phase.The PCR product behind 2% agarose gel electrophoresis and ethidium bromide staining with gel imaging system and related software (AlphaImaget, Alpha Inotech, USA) analyze, the result is with ratio (%) expression of sGST with the RT-PCR product brightness of beta-actin mRNA.

Adopt 10.0 pairs of fish liver sGST mRNA relative expression horizontal average differences not of the same race of statistical analysis software SPSS to carry out statistical study.If P＜0.05, the difference of mean value is promptly thought significantly.

Correlated results is asked for an interview Figure of description 4 and 5.

The application of embodiment 4 detection kit in the tilapia viral infection test

Raise and train in the laboratory the economic fish of 3 above feedings of week eat and carry out artificial toxin expelling tilapia, 20 trail machine is divided into 4 groups, every group 5 tail is used for viral infection test.Tilapia contaminating mode behind the artificial breeding toxin expelling adopts abdominal injection, and phosphoric acid buffer PBS is a solvent, and the MCLR purified product is diluted to corresponding concentration.Control group injected with phosphate buffer solution PBS, experimental group 1 injection MC-LR, experimental group 2 injection LPS, experimental group 3 injection MC-LR+LPS, toxin all injects the abdominal cavity through the abdomeinal fin below.Frost anesthesia behind the 24h separates liver organization.The synthetic method of total RNA extraction and cDNA first chain as described in example 1 above.

Get the detection kit described in the embodiment 2, with the sGST cDNA fragment of a 332bp of GST05F-GST06R sGST gene specific primer amplification.β 2 Actin muscle cDNA fragments with a 436bp of ACT01F-ACT02R primer amplification.

The PCR reaction conditions is 94 ℃ of pre-sex change 3min, 94 ℃ of 1min, and 55 ℃ of 1min, 72 ℃ of 1min, totally 30 circulations, last 72 ℃ are extended 5min.Termination reaction in the exponential growth phase.The PCR product behind 2% agarose gel electrophoresis and ethidium bromide staining with gel imaging system and related software (AlphaImaget, Alpha Inotech, USA) analyze, the result is with ratio (%) expression of sGST with the RT-PCR product brightness of beta-actin mRNA.

Adopt statistical analysis software SPSS 1.0.The gene induced front and back of tilapia liver sGST mRNA relative expression horizontal average difference is carried out statistical study.If P＜0.05, the difference of mean value is promptly thought significantly.

Tilapia carries out abdominal injection PBS (phosphoric acid buffer), LPS (lipopolysaccharides), MC-LR (the LR type of Microcystin), MC-LR+LPS respectively, and the specific expressed GST of liver organization behind the 24h (glutathione-S-transferring enzyme) and the result of beta-actin expression amount per-cent show: PBS, LPS, MC-LR+LPS treatment group all have significant difference with the MC-LR treatment group.Tilapia sGST expression of gene level is compared (figure-11,12), find that tilapia sGST expression of gene level changes before and after different treatment, sGST its expression amount after toxin-induced be significantly improved (P＜0.05) wherein, behind the tilapia abdominal injection MC-LR 24h, the specific expressed sGST amount of liver organization rises approximately than untreated control group sGST expression level and doubles.The proof toxin-induced can increase sGST and express.

Correlated results is asked for an interview Figure of description 6 and 7.

SGSTcDNA complete sequence of silver carp, bighead, tilapia, dace, crucian, grass carp, mandarin fish or partial sequence .ST25

SEQUENCE LISTING

＜110〉Ji'nan University

＜120〉the end point determination test kit and the detection method of toxic content in a kind of cultured freshwater fish body

＜130〉sGSTcDNA complete sequence or the partial sequence of silver carp, bighead, tilapia, dace, crucian, grass carp, mandarin fish

<160>14

<170>PatentIn version 3.2

<210>1

<211>920

<212>DNA

＜213〉Hypophthalmichthys molitrix (silver carp)

<220>

<221>CDS

<222>(75)..(746)

<400>1

acacacattg catttctgca cacactgtct gctgcacagc aaagagtttt gaagtgtcgc 60

attattttgt gaaa atg tcc ggg aaa gtt gtg ttg cat tac ttc aat gga 110

Met Ser Gly Lys Val Val Leu His Tyr Phe Asn Gly

1 5 10

aga ggg aag atg gag tcg atc cga tgg ctg ttg gcc gca gct gga gtc 158

Arg Gly Lys Met Glu Ser Ile Arg Trp Leu Leu Ala Ala Ala Gly Val

15 20 25

gag ttt gag gag gtg ttt ctg acc aaa agg gag cat tat gat aaa ctg 206

Glu Phe Glu Glu Val Phe Leu Thr Lys Arg Glu His Tyr Asp Lys Leu

30 35 40

ctg aat gat gga gcc ctg atg ttt cag cag gtg cct ttg gtt gaa ata 254

Leu Asn Asp Gly Ala Leu Met Phe Gln Gln Val Pro Leu Val Glu Ile

45 50 55 60

gat ggg atg cag ctt gtg cag tca agg gct atc ttg aat tac atc gct 302

Asp Gly Met Gln Leu Val Gln Ser Arg Ala Ile Leu Asn Tyr Ile Ala

65 70 75

gga aaa tac aat ctc tat gga aaa gac ctt aaa gaa cgg gct ttg att 350

Gly Lys Tyr Asn Leu Tyr Gly Lys Asp Leu Lys Glu Arg Ala Leu Ile

80 85 90

gac atg tat aca gag ggt acc att gat ata atg gat ctt atc atg cat 398

Asp Met Tyr Thr Glu Gly Thr Ile Asp Ile Met Asp Leu Ile Met His

95 100 105

ttt cct gtt gag cca cct gag acc aag cag aaa cag ctc ggt aat att 446

Phe Pro Val Glu Pro Pro Glu Thr Lys Gln Lys Gln Leu Gly Asn Ile

110 115 120

gag caa aag gca aaa gag cgc ttc ctg cct gtg ttt gaa aag gct ctt 494

Glu Gln Lys Ala Lys Glu Arg Phe Leu Pro Val Phe Glu Lys Ala Leu

125 130 135 140

gca aac tct caa ttc ctg gtg gga aac cag ttg agc cgc gct gat gtt 542

Ala Asn Ser Gln Phe Leu Val Gly Asn Gln Leu Ser Arg Ala Asp Val

145 150 155

cac ctt ctg gaa gtc act ctg atg ctg cag gag tta ctc cct aca ata 590

His Leu Leu Glu Val Thr Leu Met Leu Gln Glu Leu Leu Pro Thr Ile

160 165 170

ctg tca acc ttt ccc aaa atc cag gcg ttc cag gaa aga atg aag gcc 638

Leu Ser Thr Phe Pro Lys Ile Gln Ala Phe Gln Glu Arg Met Lys Ala

175 180 185

tta cca aga atc agc aag ttc ctg cag ccc ggc agt gca aga aaa cct 686

Leu Pro Arg Ile Ser Lys Phe Leu Gln Pro Gly Ser Ala Arg Lys Pro

190 195 200

cct cct gat gag gag gtg atg aaa acc gtg aag gag gtg ttg agc cac 734

Pro Pro Asp Glu Glu Val Met Lys Thr Val Lys Glu Val Leu Ser His

205 210 215 220

ctc ttt aag tag aagaaccatg gaaaaccagt tcttcagaac attttaatga 786

Leu Phe Lys

tgctaatcac taattgattt agaagctttc ttattaaaac taaaacagat ttatttggaa 846

tgtaaaagat acagtttgga taaagtgata atattgtgat atgaaaaaaa aaaaaaaaaa 906

aaaaaaaaaa aaaa 920

<210>2

<211>223

<212>PRT

＜213〉Hypophthalmichthys molitrix (silver carp)

<400>2

Met Ser Gly Lys Val Val Leu His Tyr Phe Asn Gly Arg Gly Lys Met

1 5 10 15

Glu Ser Ile Arg Trp Leu Leu Ala Ala Ala Gly Val Glu Phe Glu Glu

SGSTcDNA complete sequence of silver carp, bighead, tilapia, dace, crucian, grass carp, mandarin fish or partial sequence .ST25

20 25 30

Val Phe Leu Thr Lys Arg Glu His Tyr Asp Lys Leu Leu Asn Asp Gly

35 40 45

Ala Leu Met Phe Gln Gln Val Pro Leu Val Glu Ile Asp Gly Met Gln

50 55 60

Leu Val Gln Ser Arg Ala Ile Leu Asn Tyr Ile Ala Gly Lys Tyr Asn

65 70 75 80

Leu Tyr Gly Lys Asp Leu Lys Glu Arg Ala Leu Ile Asp Met Tyr Thr

85 90 95

Glu Gly Thr Ile Asp Ile Met Asp Leu Ile Met His Phe Pro Val Glu

100 105 110

Pro Pro Glu Thr Lys Gln Lys Gln Leu Gly Asn Ile Glu Gln Lys Ala

115 120 125

Lys Glu Arg Phe Leu Pro Val Phe Glu Lys Ala Leu Ala Asn Ser Gln

130 135 140

Phe Leu Val Gly Asn Gln Leu Ser Arg Ala Asp Val His Leu Leu Glu

145 150 155 160

Val Thr Leu Met Leu Gln Glu Leu Leu Pro Thr Ile Leu Ser Thr Phe

165 170 175

Pro Lys Ile Gln Ala Phe Gln Glu Arg Met Lys Ala Leu Pro Arg Ile

180 185 190

Ser Lys Phe Leu Gln Pro Gly Ser Ala Arg Lys Pro Pro Pro Asp Glu

195 200 205

Glu Val Met Lys Thr Val Lys Glu Val Leu Ser His Leu Phe Lys

210 215 220

<210>3

<211>978

<212>DNA

＜213〉Aristichthys nobilis (bighead)

<220>

<221>CDS

<222>(158)..(820)

<400>3

gcagaaacag ctcggtaata ttgagcaaaa ggcaaaagag cgctggtacc ctctgcacac 60

acacacacac actgctgcac agcaaagagt ttcgcttcat tttgagaaca tttattttat 120

tttttaagtg tcgctttatt ttgtgaaaat gtctggg aaa gtt gtg ttg cat tac 175

Lys Val Val Leu His Tyr

1 5

ttc aat gga aga ggg aaa atg gag tcg gtc cga tgg ctt ttg gcc gca 223

Phe Asn Gly Arg Gly Lys Met Glu Ser Val Arg Trp Leu Leu Ala Ala

10 15 20

gct gga gtc gag ttt gag gag gtg ttt ctg acc aaa agg gag cat ttt 271

Ala Gly Val Glu Phe Glu Glu Val Phe Leu Thr Lys Arg Glu His Phe

25 30 35

gat aaa ctg ctg aat gat gga gcc ctg atg ttt cag cag gtg cct ttg 319

Asp Lys Leu Leu Asn Asp Gly Ala Leu Met Phe Gln Gln Val Pro Leu

40 45 50

gtt gaa ata gat ggg atg cag ctt gtg cag tca agg gct atc ttg aat 367

Val Glu Ile Asp Gly Met Gln Leu Val Gln Ser Arg Ala Ile Leu Asn

55 60 65 70

tac atc gct gga aaa tac aat ctc tat gga aaa gac ctt aaa gaa cgg 415

Tyr Ile Ala Gly Lys Tyr Asn Leu Tyr Gly Lys Asp Leu Lys Glu Arg

75 80 85

gct ttg att gac atg tat gca gag ggt acc agc gat cta atg gat ctc 463

Ala Leu Ile Asp Met Tyr Ala Glu Gly Thr Ser Asp Leu Met Asp Leu

90 95 100

atc ata atg tct gtt ttc gct cca ccc gaa aac aag cag aaa cag ctc 511

Ile Ile Met Ser Val Phe Ala Pro Pro Glu Asn Lys Gln Lys Gln Leu

105 110 115

ggt aat att gag caa aag gca aaa gag cgc ttc ctg cct gtg ttt gaa 559

Gly Asn Ile Glu Gln Lys Ala Lys Glu Arg Phe Leu Pro Val Phe Glu

120 125 130

aag ggt ctt gca aac tct caa ttc ctg gtg gga aac cag ttg agc cgc 607

Lys Gly Leu Ala Asn Ser Gln Phe Leu Val Gly Asn Gln Leu Ser Arg

135 140 145 150

gct gat gtt cac ctt ctg gag gtc act ctg atg ctg cag gag tta ttc 655

Ala Asp Val His Leu Leu Glu Val Thr Leu Met Leu Gln Glu Leu Phe

155 160 165

cct aca ata ctg tca acc ttt ccc aaa atc cag gcg ttc cag gaa aga 703

Pro Thr Ile Leu Ser Thr Phe Pro Lys Ile Gln Ala Phe Gln Glu Arg

170 175 180

atg aag gcc tta cca aga atc agc aag ttc ctg cag ccc ggc agt gca 751

Met Lys Ala Leu Pro Arg Ile Ser Lys Phe Leu Gln Pro Gly Ser Ala

SGSTcDNA complete sequence of silver carp, bighead, tilapia, dace, crucian, grass carp, mandarin fish or partial sequence .ST25

185 190 195

aga aaa cct cct cct gat gag gtg tac gtg aaa act gtg aag gag gtg 799

Arg Lys Pro Pro Pro Asp Glu Val Tyr Val Lys Thr Val Lys Glu Val

200 205 210

ttg agc cac ctc ttt aag tag aaggaccact taaaaacatt tagatgatgt 850

Leu Ser His Leu Phe Lys

215 220

taatcactaa ttaatttaca agcttcctta ttaaaactga ggcactaaaa tggacagatt 910

tatttggaat gtacaagata ctgtctggat aaagtaataa tattgtgata tgcaaaaaaa 970

aaaaaaaa 978

<210>4

<211>220

<212>PRT

＜213〉Aristichthys nobilis (bighead)

<400>4

Lys Val Val Leu His Tyr Phe Asn Gly Arg Gly Lys Met Glu Ser Val

1 5 10 15

Arg Trp Leu Leu Ala Ala Ala Gly Val Glu Phe Glu Glu Val Phe Leu

20 25 30

Thr Lys Arg Glu His Phe Asp Lys Leu Leu Asn Asp Gly Ala Leu Met

35 40 45

Phe Gln Gln Val Pro Leu Val Glu Ile Asp Gly Met Gln Leu Val Gln

50 55 60

Ser Arg Ala Ile Leu Asn Tyr Ile Ala Gly Lys Tyr Asn Leu Tyr Gly

65 70 75 80

Lys Asp Leu Lys Glu Arg Ala Leu Ile Asp Met Tyr Ala Glu Gly Thr

85 90 95

Ser Asp Leu Met Asp Leu Ile Ile Met Ser Val Phe Ala Pro Pro Glu

100 105 110

Asn Lys Gln Lys Gln Leu Gly Asn Ile Glu Gln Lys Ala Lys Glu Arg

115 120 125

Phe Leu Pro Val Phe Glu Lys Gly Leu Ala Asn Ser Gln Phe Leu Val

130 135 140

Gly Asn Gln Leu Ser Arg Ala Asp Val His Leu Leu Glu Val Thr Leu

145 150 155 160

Met Leu Gln Glu Leu Phe Pro Thr Ile Leu Ser Thr Phe Pro Lys Ile

165 170 175

Gln Ala Phe Gln Glu Arg Met Lys Ala Leu Pro Arg Ile Ser Lys Phe

180 185 190

Leu Gln Pro Gly Ser Ala Arg Lys Pro Pro Pro Asp Glu Val Tyr Val

195 200 205

Lys Thr Val Lys Glu Val Leu Ser His Leu Phe Lys

210 215 220

<210>5

<211>887

<212>DNA

＜213〉Oreochromis nilotica (tilapia)

<220>

<221>CDS

<222>(25)..(720)

<400>5

aaaaagaaaa cagcaacaaa agcc atg tct gaa aaa cct gtg ctg tac tat 51

Met Ser Glu Lys Pro Val Leu Tyr Tyr

1 5

ttt aat ggg aga ggg aag atg gag tca atc cgc tgg ctt tta act gtt 99

Phe Asn Gly Arg Gly Lys Met Glu Ser Ile Arg Trp Leu Leu Thr Val

10 15 20 25

gct gaa gtc gag ttt gat gaa gtg ctt ctg aca act cgg gag cag tat 147

Ala Glu Val Glu Phe Asp Glu Val Leu Leu Thr Thr Arg Glu Gln Tyr

30 35 40

gaa aaa ctc ctg aat gat ggg gcg ctc atg ttt caa cag gtc cct ttg 195

Glu Lys Leu Leu Asn Asp Gly Ala Leu Met Phe Gln Gln Val Pro Leu

45 50 55

gtg gaa atg gat ggc atg aag ctc att cag aca aaa gca atc ctg aat 243

Val Glu Met Asp Gly Met Lys Leu Ile Gln Thr Lys Ala Ile Leu Asn

60 65 70

tac atc gca gag aaa tac atc ctg aac tac atc gca ggg aag tat aat 291

Tyr Ile Ala Glu Lys Tyr Ile Leu Asn Tyr Ile Ala Gly Lys Tyr Asn

75 80 85

ctg cat gca aag gat ccc aaa gaa cga gta atg atc aac atg tac tct 339

Leu His Ala Lys Asp Pro Lys Glu Arg Val Met Ile Asn Met Tyr Ser

SGSTcDNA complete sequence of silver carp, bighead, tilapia, dace, crucian, grass carp, mandarin fish or partial sequence .ST25

90 95 100 105

gag gga ttg aca gac ctc atg gaa atg atc atg ata ctt ccc ttc acc 387

Glu Gly Leu Thr Asp Leu Met Glu Met Ile Met Ile Leu Pro Phe Thr

110 115 120

cca gat ccc aaa ccc aaa ctg gac aac att cag agc aaa gca aag gag 435

Pro Asp Pro Lys Pro Lys Leu Asp Asn Ile Gln Ser Lys Ala Lys Glu

125 130 135

cgc tac ctt cct gtg tat gaa aag gct ctg act gga ccc gtg tac ctg 483

Arg Tyr Leu Pro Val Tyr Glu Lys Ala Leu Thr Gly Pro Val Tyr Leu

140 145 150

gtg gga ggt aaa cta agc ctt gct gat gtg ctg ctt gtt gaa tgc acc 531

Val Gly Gly Lys Leu Ser Leu Ala Asp Val Leu Leu Val Glu Cys Thr

155 160 165

ctg atg ctg gag gag aaa ttt cca gac att ctg aaa gac ttc ccc aat 579

Leu Met Leu Glu Glu Lys Phe Pro Asp Ile Leu Lys Asp Phe Pro Asn

170 175 180 185

atc aag tcc ttt cag ggc agg atg aca caa atc ccc gcc atc agc agg 627

Ile Lys Ser Phe Gln Gly Arg Met Thr Gln Ile Pro Ala Ile Ser Arg

190 195 200

ttt ctg cag ccg ggc agc aag agg aag cca gcg cca gat gaa aaa tac 675

Phe Leu Gln Pro Gly Ser Lys Arg Lys Pro Ala Pro Asp Glu Lys Tyr

205 210 215

ttg aaa aat gtt gtg gaa gtc cta aac ctc aag ttg cca ctt taa 720

Leu Lys Asn Val Val Glu Val Leu Asn Leu Lys Leu Pro Leu

220 225 230

gaaacactac aaaagatctg ttacattcct cattaggcaa cgtgattatg atcagttaca 780

tctggaggca tacgtgaaga taaacgctgc actaacacaa caacatggaa agaacttctg 840

taatctgctt taccaataaa cacatttgac acaaaaaaaa aaaaaaa 887

<210>6

<211>231

<212>PRT

＜213〉Oreochromis nilotica (tilapia)

<400>6

Met Ser Glu Lys Pro Val Leu Tyr Tyr Phe Asn Gly Arg Gly Lys Met

1 5 10 15

Glu Ser Ile Arg Trp Lou Leu Thr Val Ala Glu Val Glu Phe Asp Glu

20 25 30

Val Leu Leu Thr Thr Arg Glu Gln Tyr Glu Lys Leu Leu Asn Asp Gly

35 40 45

Ala Leu Met Phe Gln Gln Val Pro Leu Val Glu Met Asp Gly Met Lys

50 55 60

Leu Ile Gln Thr Lys Ala Ile Leu Asn Tyr Ile Ala Glu Lys Tyr Ile

65 70 75 80

Leu Asn Tyr Ile Ala Gly Lys Tyr Asn Leu His Ala Lys Asp Pro Lys

85 90 95

Glu Arg Val Met Ile Asn Met Tyr Ser Glu Gly Leu Thr Asp Leu Met

100 105 110

Glu Met Ile Met Ile Leu Pro Phe Thr Pro Asp Pro Lys Pro Lys Leu

115 120 125

Asp Asn Ile Gln Ser Lys Ala Lys Glu Arg Tyr Leu Pro Val Tyr Glu

130 135 140

Lys Ala Leu Thr Gly Pro Val Tyr Leu Val Gly Gly Lys Leu Ser Leu

145 150 155 160

Ala Asp Val Leu Leu Val Glu Cys Thr Leu Met Leu Glu Glu Lys Phe

165 170 175

Pro Asp Ile Leu Lys Asp Phe Pro Asn Ile Lys Ser Phe Gln Gly Arg

180 185 190

Met Thr Gln Ile Pro Ala Ile Ser Arg Phe Leu Gln Pro Gly Ser Lys

195 200 205

Arg Lys Pro Ala Pro Asp Glu Lys Tyr Leu Lys Asn Val Val Glu Val

210 215 220

Leu Asn Leu Lys Leu Pro Leu

225 230

<210>7

<211>405

<212>DNA

＜213〉Cirrhinus molitorella (dace)

<220>

<221>CDS

<222>(1)..(405)

<400>7

SGSTcDNA complete sequence of silver carp, bighead, tilapia, dace, crucian, grass carp, mandarin fish or partial sequence .ST25

atc ctg aac tac atc gca gga aag tac aat cta tat gga aaa gac ctt 48

Ile Leu Asn Tyr Ile Ala Gly Lys Tyr Asn Leu Tyr Gly Lys Asp Leu

1 5 10 15

aaa gaa agg gct ctg att gac atg tat aca gag ggt gcc atc gat cta 96

Lys Glu Arg Ala Leu Ile Asp Met Tyr Thr Glu Gly Ala Ile Asp Leu

20 25 30

atg gat cag att ata acg ctt cct ttt aca cca cct gsa aac aaa cag 144

Met Asp Gln Ile Ile Thr Leu Pro Phe Thr Pro Pro Glu Asn Lys Gln

35 40 45

aaa caa ctc agt aac ata gag gaa aag gca aaa gat cgc tet tta cct 192

Lys Gln Leu Ser Asn Ile Glu Glu Lys Ala Lys Asp Arg Tyr Leu Pro

50 55 60

gca ttt gaa aag ggt ctt aaa aac tct cag ttc ctt gtg gga aac cag 240

Ala Phe Glu Lys Gly Leu Lys Asn Ser Gln Phe Leu Val Gly Asn Gln

65 70 75 80

ttg agc cgt gca gac gtt cac ctt ctt gaa gtc att ctg atg ttg cag 288

Leu Ser Arg Ala Asp Val His Leu Leu Glu Val Ile Leu Met Leu Gln

85 90 95

gag tta ttc cca aca ata ctc tca acc ttt ccc aaa atc cag gca ttt 336

Glu Leu Phe Pro Thr Ile Leu Ser Thr Phe Pro Lys Ile Gln Ala Phe

100 105 110

caa gag aaa atg aag gcc ttg cca aca gtc agc aag ttc ctc cag cca 384

Gln Glu Lys Met Lys Ala Leu Pro Thr Val Ser Lys Phe Leu Gln Pro

115 120 125

ggc agc gct agg aaa cct cca 405

Gly Ser Ala Arg Lys Pro Pro

130 135

<210>8

<211>135

<212>PRT

＜213〉Cirrhinus molitorella (dace)

<400>8

Ile Leu Asn Tyr Ile Ala Gly Lys Tyr Asn Leu Tyr Gly Lys Asp Leu

1 5 10 15

Lys Glu Arg Ala Leu Ile Asp Met Tyr Thr Glu Gly Ala Ile Asp Leu

20 25 30

Met Asp Gln Ile Ile Thr Leu Pro Phe Thr Pro Pro Glu Asn Lys Gln

35 40 45

Lys Gln Leu Ser Asn Ile Glu Glu Lys Ala Lys Asp Arg Tyr Leu Pro

50 55 60

Ala Phe Glu Lys Gly Leu Lys Asn Ser Gln Phe Leu Val Gly Asn Gln

65 70 75 80

Leu Ser Arg Ala Asp Val His Leu Leu Glu Val Ile Leu Met Leu Gln

85 90 95

Glu Leu Phe Pro Thr Ile Leu Ser Thr Phe Pro Lys Ile Gln Ala Phe

100 105 110

Gln Glu Lys Met Lys Ala Leu Pro Thr Val Ser Lys Phe Leu Gln Pro

115 120 125

Gly Ser Ala Arg Lys Pro Pro

130 135

<210>9

<211>591

<212>DNA

＜213〉Carassius auratu (crucian)

<220>

<221>CDS

<222>(1)..(591)

<400>9

tac ttc aat ggc aga ggg aag atg ggg tcg atc cga tgg ctg ctg gct 48

Tyr Phe Asn Gly Arg Gly Lys Met Gly Ser Ile Arg Trp Leu Leu Ala

1 5 10 15

gca gct gga gtc cag ttt gag gaa gtg ttt ttg acc aaa aga gag gag 96

Ala Ala Gly Val Gln Phe Glu Glu Val Phe Leu Thr Lys Arg Glu Glu

20 25 30

tat gaa aag ctg ctc aaa gat gga gcc ctg atg ttc cag cag gtg cct 144

Tyr Glu Lys Leu Leu Lys Asp Gly Ala Leu Met Phe Gln Gln Val Pro

35 40 45

ttg gtg gaa atc gac ggg atg cag ctt gtg cag tcc aag gcc atc ctg 192

Leu Val Glu Ile Asp Gly Met Gln Leu Val Gln Ser Lys Ala Ile Leu

50 55 60

aac tac atc gct gga aaa tac aat cta tat gga caa gac ctt aaa gaa 240

SGSTcDNA complete sequence of silver carp, bighead, tilapia, dace, crucian, grass carp, mandarin fish or partial sequence .ST25

Asn Tyr Ile Ala Gly Lys Tyr Asn Leu Tyr Gly Gln Asp Leu Lys Glu

65 70 75 80

cgg gct atg atc gac atg tat tca gag gga atc ggt gat ctc atg gac 288

Arg Ala Met Ile Asp Met Tyr Ser Glu Gly Ile Gly Asp Leu Met Asp

85 90 95

ttg att ata atg tat cct ttc aaa ccg cct gaa aac aaa ccg gca cac 336

Leu Ile Ile Met Tyr Pro Phe Lys Pro Pro Glu Asn Lys Pro Ala His

100 105 110

ctc aat act ata gag cag aag gcc aaa gat cgc ttt ctt cct gtg ttt 384

Leu Asn Thr Ile Glu Gln Lys Ala Lys Asp Arg Phe Leu Pro Val Phe

115 120 125

gaa agg ggt ctt gcg aac tct cag ttc ctg gtg gga aac cag ctg agc 432

Glu Arg Gly Leu Ala Asn Ser Gln Phe Leu Val Gly Asn Gln Leu Ser

130 135 140

cgt gct gac gtt cat ctt ccc gag gtc act ctg atg ctg cag gag ctg 480

Arg Ala Asp Val His Leu Pro Glu Val Thr Leu Met Leu Gln Glu Leu

145 150 155 160

ctc ccc acc att ctt tca acc ttt ccc aaa atc cag gcg ttt caa gat 528

Leu Pro Thr Ile Leu Ser Thr Phe Pro Lys Ile Gln Ala Phe Gln Asp

165 170 175

aaa atg aag gcc ttg cca aca atc agc aag ttt ctc cag cca ggc agc 576

Lys Met Lys Ala Leu Pro Thr Ile Ser Lys Phe Leu Gln Pro Gly Ser

180 185 190

gct agg aaa cct cca 591

Ala Arg Lys Pro Pro

195

<210>10

<211>197

<212>PRT

＜213〉Carassius auratu (crucian)

<400>10

Tyr Phe Asn Gly Arg Gly Lys Met Gly Ser Ile Arg Trp Leu Leu Ala

1 5 10 15

Ala Ala Gly Val Gln Phe Glu Glu Val Phe Leu Thr Lys Arg Glu Glu

20 25 30

Tyr Glu Lys Leu Leu Lys Asp Gly Ala Leu Met Phe Gln Gln Val Pro

35 40 45

Leu Val Glu Ile Asp Gly Met Gln Leu Val Gln Ser Lys Ala Ile Leu

50 55 60

Asn Tyr Ile Ala Gly Lys Tyr Asn Leu Tyr Gly Gln Asp Leu Lys Glu

65 70 75 80

Arg Ala Met Ile Asp Met Tyr Ser Glu Gly Ile Gly Asp Leu Met Asp

85 90 95

Leu Ile Ile Met Tyr Pro Phe Lys Pro Pro Glu Asn Lys Pro Ala His

100 105 110

Leu Asn Thr Ile Glu Gln Lys Ala Lys Asp Arg Phe Leu Pro Val Phe

115 120 125

Glu Arg Gly Leu Ala Asn Ser Gln Phe Leu Val Gly Asn Gln Leu Ser

130 135 140

Arg Ala Asp Val His Leu Pro Glu Val Thr Leu Met Leu Gln Glu Leu

145 150 155 160

Leu Pro Thr Ile Leu Ser Thr Phe Pro Lys Ile Gln Ala Phe Gln Asp

165 170 175

Lys Met Lys Ala Leu Pro Thr Ile Ser Lys Phe Leu Gln Pro Gly Ser

180 185 190

Ala Arg Lys Pro Pro

195

<210>11

<211>405

<212>DNA

＜213〉Ctenopharyngodon idella (grass carp)

<220>

<221>CDS

<222>(1)..(405)

<400>11

atc ctg aac tac atc gca gga aag tac aac ctc tat gga aaa gac ctt 48

Ile Leu Asn Tyr Ile Ala Gly Lys Tyr Asn Leu Tyr Gly Lys Asp Leu

1 5 10 15

aaa gaa cgg gct ttg att gac atg tat aca gag ggt acc ctc gat cta 96

Lys Glu Arg Ala Leu Ile Asp Met Tyr Thr Glu Gly Thr Leu Asp Leu

20 25 30

SGSTcDNA complete sequence of silver carp, bighead, tilapia, dace, crucian, grass carp, mandarin fish or partial sequence .ST25

atg gat att atc atg ctg tgg gct att gag aca ccc gaa aac aaa cag 144

Met Asp Ile Ile Met Leu Trp Ala Ile Glu Thr Pro Glu Asn Lys Gln

35 40 45

aaa cag ctc agt aat ata gag caa aag gca aaa gag cgc ttc ctt cct 192

Lys Gln Leu Ser Asn Ile Glu Gln Lys Ala Lys Glu Arg Phe Leu Pro

50 55 60

gtg ttt gaa aag ggt ctt gca aac tct gat ttc ctg gtg gga aac cag 240

Val Phe Glu Lys Gly Leu Ala Asn Ser Asp Phe Leu Val Gly Asn Gln

65 70 75 80

ttg agc cgt gct gac gtt cac ctt ctg gaa gcc act ctg acg ctg cag 288

Leu Ser Arg Ala Asp Val His Leu Leu Glu Ala Thr Leu Thr Leu Gln

85 90 95

gag ata tta cct aca ata ctg tca acc ttt ccc aaa atc cag gcg ttt 336

Glu Ile Leu Pro Thr Ile Leu Ser Thr Phe Pro Lys Ile Gln Ala Phe

100 105 110

cag gaa cga atg aag gct tta cca aga atc agc aag ttc ctg cag cct 384

Gln Glu Arg Met Lys Ala Leu Pro Arg Ile Ser Lys Phe Leu Gln Pro

115 120 125

ggc agc gct agg aaa cct cca 405

Gly Ser Ala Arg Lys Pro Pro

130 135

<210>12

<211>135

<212>PRT

＜213〉Ctenopharyngodon idella (grass carp)

<400>12

Ile Leu Asn Tyr Ile Ala Gly Lys Tyr Asn Leu Tyr Gly Lys Asp Leu

1 5 10 15

Lys Glu Arg Ala Leu Ile Asp Met Tyr Thr Glu Gly Thr Leu Asp Leu

20 25 30

Met Asp Ile Ile Met Leu Trp Ala Ile Glu Thr Pro Glu Asn Lys Gln

35 40 45

Lys Gln Leu Ser Asn Ile Glu Gln Lys Ala Lys Glu Arg Phe Leu Pro

50 55 60

Val Phe Glu Lys Gly Leu Ala Asn Ser Asp Phe Leu Val Gly Asn Gln

65 70 75 80

Leu Ser Arg Ala Asp Val His Leu Leu Glu Ala Thr Leu Thr Leu Gln

85 90 95

Glu Ile Leu Pro Thr Ile Leu Ser Thr Phe Pro Lys Ile Gln Ala Phe

100 105 110

Gln Glu Arg Met Lys Ala Leu Pro Arg Ile Ser Lys Phe Leu Gln Pro

115 120 125

Gly Ser Ala Arg Lys Pro Pro

130 135

<210>13

<211>399

<212>DNA

＜213〉Siniperca chuatsi (mandarin fish)

<220>

<221>CDS

<222>(1)..(399)

<400>13

atc ctg aac tac atc gca ggg aag tat aac ctt cat gga aaa ggc ctc 48

Ile Leu Asn Tyr Ile Ala Gly Lys Tyr Asn Leu His Gly Lys Gly Leu

1 5 10 15

aaa gac cgt gtc atg atc aat ata tac tca gag gga gtg atg gat ctc 96

Lys Asp Arg Val Met Ile Asn Ile Tyr Ser Glu Gly Val Met Asp Leu

20 25 30

atg gaa atg atc atg atg ttg ccc ttc agc cca gat cca aaa gaa aaa 144

Met Glu Met Ile Met Met Leu Pro Phe Ser Pro Asp Pro Lys Glu Lys

35 40 45

ctg gat act att caa act aaa gca aaa gat cgc tac ctt cct gtg ttt 192

Leu Asp Thr Ile Gln Thr Lys Ala Lys Asp Arg Tyr Leu Pro Val Phe

50 55 60

gaa aag gcg ctg tct ggg caa ata tac ctg gtt gga ggt aaa cta agt 240

Glu Lys Ala Leu Ser Gly Gln Ile Tyr Leu Val Gly Gly Lys Leu Ser

65 70 75 80

tgt gca gac gtg cag ctg ctt gaa tgc acc ctg atg ttg gag gag aaa 288

Cys Ala Asp Val Gln Leu Leu Glu Cys Thr Leu Met Leu Glu Glu Lys

85 90 95

ttt act gga atc ctc tct gag ttt ccc aac gtc aag tct ttc cag ggc 336

Phe Thr Gly Ile Leu Ser Glu Phe Pro Asn Val Lys Ser Phe Gln Gly

SGSTcDNA complete sequence of silver carp, bighead, tilapia, dace, crucian, grass carp, mandarin fish or partial sequence .ST25

100 105 110

agg atg atc cag att cct gcc atc aac agg ttc ctg cag cca ggc agc 384

Arg Met Ile Gln Ile Pro Ala Ile Asn Arg Phe Leu Gln Pro Gly Ser

115 120 125

gct agg aaa cct cca 399

Ala Arg Lys Pro Pro

130

<210>14

<211>133

<212>PRT

＜213〉Siniperca chuatsi (mandarin fish)

<400>14

Ile Leu Asn Tyr Ile Ala Gly Lys Tyr Asn Leu His Gly Lys Gly Leu

1 5 10 15

Lys Asp Arg Val Met Ile Asn Ile Tyr Ser Glu Gly Val Met Asp Leu

20 25 30

Met Glu Met Ile Met Met Leu Pro Phe Ser Pro Asp Pro Lys Glu Lys

35 40 45

Leu Asp Thr Ile Gln Thr Lys Ala Lys Asp Arg Tyr Leu Pro Val Phe

50 55 60

Glu Lys Ala Leu Ser Gly Gln Ile Tyr Leu Val Gly Gly Lys Leu Ser

65 70 75 80

Cys Ala Asp Val Gln Leu Leu Glu Cys Thr Leu Met Leu Glu Glu Lys

85 90 95

Phe Thr Gly Ile Leu Ser Glu Phe Pro Asn Val Lys Ser Phe Gln Gly

100 105 110

Arg Met Ile Gln Ile Pro Ala Ile Asn Arg Phe Leu Gln Pro Gly Ser

115 120 125

Ala Arg Lys Pro Pro

130

Claims (9)

1, the end point determination test kit of toxic content in a kind of cultured freshwater fish body comprises the genetic expression of cultured freshwater fish sGST detoxifying enzymes and detects primer, is used for the DNA polymerase of pcr amplification, PCR reaction buffer, dNTP solution.

2, test kit as claimed in claim 1, it is characterized in that described cultured freshwater fish sGST detoxifying enzymes genetic expression detect primer be in silver carp, bighead, tilapia, dace, crucian, grass carp, the mandarin fish any or any two or more than any two at random combination so that whole detoxifying enzymes gene specifics detects primer.

3, test kit as claimed in claim 2, it is characterized in that it is that sGST detoxifying enzymes gene cDNA sequence with silver carp, bighead, tilapia, dace, crucian, grass carp, mandarin fish is a basic design that described cultured freshwater fish sGST detoxifying enzymes genetic expression detects primer, its cDNA sequence is respectively shown in SEQ ID NO:1, SEQID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ IDNO:13.

4, test kit as claimed in claim 1 is characterized in that the described DNA polymerase that is used for pcr amplification is rTaq, Pfu, Taq Plus, HotStart Taq, Taq Platinum or Pyrobest; Described PCR reaction buffer is and the supporting reaction buffer of this DNA polymerase.

5, test kit as claimed in claim 1, the concentration that it is characterized in that described dNTP solution is 2.5mM.

6, test kit as claimed in claim 1 is characterized in that also including the beta-actin mRNA detection of expression primer as the freshwater aquiculture Chelonian of outer reference.

7, claim 1 or 6 described test kits is characterized in that also comprising and are used for cDNA synthetic oligo (dT) 18 universal primers and ThermoScript II, reaction buffer.

8, test kit as claimed in claim 7, it is characterized in that described ThermoScript II is AMV, Stratascript, Suprscript and Suprscript II, SuperScriptIII or M-MLV, described reaction buffer are and the supporting reaction buffer of this ThermoScript II.

9, the detection method of the described test kit of a kind of claim 7 the steps include:

(1) liver total RNA of extraction and purification cultured freshwater fish as the reverse transcription primer, adopts the synthetic cDNA of reverse transcription method with oligo (dT) 18;

(2) be template with (1) synthetic cDNA, get and detect primer, DNA polymerase, PCR reaction buffer, dNTP, sterile purified water etc. with the sGST detoxifying enzymes genetic expression of the corresponding fingerling of this template and mix in right amount, carry out the PCR reaction, reaction conditions is: 94 ℃ of pre-sex change 3～6min at first, 94 ℃ of 30sec～1min then, 55～65 ℃ of 30sec～1min, 72 ℃ of 30sec～1min, totally 25～32 circulations, last 72 ℃ are extended 5～10min;

(3) be template with (1) synthetic cDNA, beta-actin mRNA detection of expression primer, DNA polymerase, PCR reaction buffer, dNTP, sterile purified water mixes, and carries out the PCR reaction, and reaction system and reaction conditions are with (2);

(4) after PCR reaction was finished, the PCR product of (2) and (3) of getting equivalent was through 1～2% agarose gel electrophoresis that contains ethidium bromide, and brightness detects comparison to product, determines the expression conditions of sGST detoxifying enzymes gene.

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