CN110714026B

CN110714026B - Construction method and application of type II diabetic zebra fish model

Info

Publication number: CN110714026B
Application number: CN201911005040.4A
Authority: CN
Inventors: 薛钰; 彭伟; 许璟瑾; 雷妙玉; 潘裕添
Original assignee: Minnan Normal University
Current assignee: Minnan Normal University
Priority date: 2019-10-22
Filing date: 2019-10-22
Publication date: 2022-10-04
Anticipated expiration: 2039-10-22
Also published as: CN110714026A

Abstract

The invention discloses a construction method and application of a zebra fish model with type II diabetes, and relates to the technical field of medicines. Which comprises the following steps: constructing transgenic zebra fish; (2) adopting a medicament to model the transgenic zebra fish; in the process of constructing the transgenic zebra fish in the step (1), firstly constructing an expression vector, then mixing the expression vector with Tol2mRNA, and microinjecting the mixture into the zebra fish embryo; the expression vector comprises a pdx1 gene promoter, a gene for coding fluorescent protein and an nsfb gene; wherein the medicament adopted in the step (2) is a compound containing a nitro group. The invention constructs a transgenic strain of pancreatic tissue specific expression fluorescent protein and nitroreductase by utilizing a pdx1promoter, and can specifically kill pancreatic tissue cells by inducing the strain by using a compound containing a nitro group, thereby simulating type II diabetes.

Description

Construction method and application of type II diabetic zebra fish model

Technical Field

The invention relates to the technical field of medicines, and particularly relates to a construction method and application of a zebra fish model with type II diabetes.

Background

Diabetes Mellitus (DM), characterized by damage to islet beta cells, is a chronic, lifelong metabolic disease that compromises human health. Diabetes is caused by blood sugar rise caused by abnormal glucose metabolism due to the fact that insulin signal channels in a body are damaged or the sensitivity of insulin targets is reduced, and diabetes is accompanied by complications such as cardiovascular diseases and the like, such as diabetic nephropathy, nerve chronic damage and retinopathy, and along with the continuous increase of the prevalence rate of diabetes, the diabetes and related organic lesions become main health problems which disturb the life of people.

Lower insulin levels or higher blood glucose concentrations are the most obvious indications for diabetic patients, insulin acts to lower blood glucose, and islet beta cells are the only cells that synthesize and release insulin, so the developmental status of the pancreas is critical to blood glucose regulation. The invention is especially provided for the research of the pathogenesis of diabetes and the development of hypoglycemic drugs.

Disclosure of Invention

The invention aims to provide a construction method and application of a zebra fish model with type II diabetes to solve the technical problems.

The invention is realized by the following steps:

a construction method of a zebra fish model with type II diabetes comprises the following steps:

(1) Constructing transgenic zebra fish;

(2) Carrying out molding on the transgenic zebra fish by adopting a medicament;

in the process of constructing the transgenic zebra fish in the step (1), firstly constructing an expression vector, then mixing the expression vector with Tol2mRNA, and microinjecting the mixture into the zebra fish embryo; the expression vector comprises a pdx1 gene promoter, a gene for coding fluorescent protein and an nsfb gene;

wherein the medicament adopted in the step (2) is a compound containing a nitro group.

The method firstly constructs the transgenic zebra fish strain, then molds the constructed transgenic zebra fish with the medicine, and finally obtains the zebra fish model with type II diabetes.

In the process of constructing the transgenic zebra fish, the construction of an expression vector is carried out firstly. The expression vector comprises a pdx1 gene promoter, a pdx1 gene encodes a pancreatic duodenal homeobox protein 1 (pancrea and duodenum homeobox1, pdx 1) also called an insulin promoter 1, pdx1 gene is a gene related to diabetes, particularly related to the susceptibility increase of type II diabetes, and pdx1 is mostly expressed in beta cells of insulin endocrine and delta cells secreting somatostatin, has the function of regulating pancreatic development and differentiation, and the expression level of the pdx1 gene is important for maintaining normal blood sugar homeostasis. Homozygous mutation of the pdx1 gene in mice is lethal, and heterozygous mutation is manifested as hyperglycemia; knock-out of pdx1 in zebrafish results in a significant reduction of exocrine and endocrine gland cells, knock-out of pdx1 will trigger a defect in pancreatic development, loss of insulin secretion leading to diabetic symptoms (Wang X,2018 keller M p,2018 yoshida t,2010 gu g,2002 brissova M,2002 linking Y e,2017 ahlgren u, 1998. The pdx1 gene promoter is inserted into the vector, so that the constructed transgenic zebra fish can specifically express nitroreductase and red fluorescent protein in pancreas, and the pancreas tissue can be specifically killed when metronidazole substrate is added for treatment, thereby inducing type II diabetes.

The nsfb gene encodes a protein called NTR, the nsfb gene is Nitroreductase B (nifsb) gene, which is derived from Escherichia coli B, and Nitroreductase (NTR) protein encoded by the Nitroreductase B gene can bind to a compound containing a nitro group, such as Metronidazole (MTZ), to reduce Metronidazole to methazolyl, which is toxic to cells. The substance can bind to DNA double strand effectively, inhibit synthesis of cell nucleic acid, interfere growth of cell, and ultimately cause cell death. The expression vector is provided with the nsfB gene segment, so that the transgenic strain can normally express NTR protein, and pancreatic tissue cells can be effectively and specifically killed under the induction of metronidazole subsequently.

Other nitroimidazoles are also capable of reacting chemically with nitroreductase, preferably MTZ.

Furthermore, the expression vector also comprises a gene for coding the fluorescent protein, and the gene for coding the fluorescent protein can express the fluorescent protein, when the drug is induced, pancreatic tissue cells are specifically killed, the fluorescence is weakened, and diabetic symptoms such as blood sugar increase and insulin secretion disorder are caused, so that the zebra fish type II diabetes is simulated. The disease model can be widely used for screening, developing and applying hypoglycemic drugs and has important significance.

Tol2_ mRNA was used to increase transposition efficiency for insertion of exogenous expression vectors into the genome.

In a preferred embodiment of the present invention, the pdx1 gene has a promoter sequence shown in SEQ ID No.6, and the nsfb gene has a nucleotide sequence shown in SEQ ID No. 2; the sequence of the NTR protein coded by the nsfb gene is shown in SEQ ID NO. 8; the gene for coding the fluorescent protein is any one of mCherry gene, orange fluorescent protein gene or yellow fluorescent protein gene;

preferably, the gene encoding the fluorescent protein is mCherry gene, and the protein sequence encoded by the mCherry gene is shown in SEQ ID No. 7; the nucleotide sequence of the mCherry gene is shown as SEQ ID NO. 3.

The gene for coding the fluorescent protein can be adjusted according to needs, and is preferably an mCherry reporter gene which can express the red fluorescent protein, when the drug is induced, pancreatic tissue cells are specifically killed, red fluorescence is weakened, and diabetic symptoms such as blood sugar increase and insulin secretion disorder are caused, so that the zebra fish type II diabetes is simulated. The disease model can be widely used for screening, developing and applying hypoglycemic drugs and has important significance.

In a preferred embodiment of the present invention, the nucleotide sequence of the expression vector is shown in SEQ ID NO. 1.

In a preferred embodiment of the present invention, the method for constructing the expression vector comprises the following steps: firstly constructing a pSTEos-pdx1 intermediate vector and amplifying an mCherry-NTR insert, then carrying out double enzyme digestion on the pSTEos-pdx1 intermediate vector and the mChery-NTR insert, recovering and connecting to construct the expression vector Tol2-pdx 1-mChery-NTR.

Both pSTEos and mCherry-NTR template plasmids were offered in the Meng Anming laboratory. The mCherry-NTR insert can be obtained by amplifying or enzyme cutting pST-mylz2-mCherry-NTR plasmid. The pST-mylz2-mCherry-NTR plasmid was given by Meng Anming the laboratory.

In a preferred embodiment of the present invention, the pSTEos-pdx1 intermediate vector and the mCherry-NTR fragment are digested simultaneously with XbaI and BamHI restriction enzymes. And carrying out enzyme digestion on the pSTEos-pdx1 intermediate vector by adopting double enzyme digestion to obtain a pST-pdx1 intermediate vector with enzyme digestion sites at two ends, carrying out double enzyme digestion on the mCherry-NTR fragment by adopting double enzyme digestion, recovering glue, and connecting the fragment and the vector to obtain the final expression vector Tol2-pdx 1-mChery-NTR.

In a preferred embodiment of the present invention, the method for constructing the expression vector further comprises constructing a pSTEos-pdx1 intermediate vector, wherein the construction of the pSTEos-pdx1 intermediate vector comprises the following steps: amplifying a pdx1promoter fragment by using a zebra fish genome as a template and a first primer and a second primer, performing double enzyme digestion on the amplified pdx1promoter fragment and a pSTEos vector, and after enzyme digestion recovery, connecting to obtain a pSTEos-pdx1 intermediate vector;

preferably, the nucleotide sequence of the first primer is shown as SEQ ID NO.4, and the nucleotide sequence of the second primer is shown as SEQ ID NO. 5;

preferably, the amplified pdx1promoter fragment and pSTEos vector are digested simultaneously with BglII and BamHI restriction enzymes.

In a preferred embodiment of the application of the invention, the medicine is metronidazole, and the action concentration of the metronidazole is 8-10mM;

preferably, the concentration of metronidazole used is 8mM.

When the action concentration of metronidazole is 8mM, the red fluorescence is obviously weakened, and the survival condition of the transgenic zebra fish is good.

In the preferred embodiment of the invention, the zebra fish embryo is injected at the unicellular stage, the injection amount is 1-2nL, the fluorescent expression is observed when the zebra fish embryo develops to 72hpf, the success rate of co-injecting the expression vector and the Tol2mRNA to obtain the transgenic zebra fish is higher, and the effect is best when the injection amount is 1-2nL.

An application of the zebra fish model with type II diabetes mellitus obtained by the construction method in screening of medicines for treating type II diabetes mellitus.

In a preferred embodiment of the present invention, the method for screening a drug for treating type ii diabetes comprises the following steps:

if the red fluorescence of the experimental group is enhanced, the blood sugar concentration is reduced, and the insulin concentration is increased compared with the MTZ-induced type II diabetes model group after the medicine is applied, the medicine is judged to have the effect of treating type II diabetes;

and if the red fluorescence of the experimental group is kept unchanged and the blood glucose concentration and the insulin concentration are not obviously changed compared with the MTZ-induced type II diabetes model group after the medicine is applied, judging that the medicine has no efficacy of treating type II diabetes.

Preferably, the new drug is a new drug for treating type II diabetes;

preferably, the new drug is a drug for promoting pancreatic tissue regeneration. The construction method of the zebra fish model provided by the invention can be used for constructing the zebra fish type II diabetes model and can be used for screening, developing and applying hypoglycemic drugs.

The invention has the following beneficial effects:

the invention provides a construction method of a zebra fish model with type II diabetes, which takes zebra fish as a model animal, connects a pdx1 gene promoter with a gene coding fluorescent protein and an nsfb gene to construct transgenic zebra fish, and the pdx1promoter fragment can ensure that the constructed transgenic zebra fish can specifically express the fluorescent protein and NTR in pancreatic tissues, can effectively and specifically kill pancreatic tissue cells under the induction of a compound containing a nitro group, embodies the reduction of fluorescence and causes diabetes symptoms such as blood sugar increase, insulin secretion disorder and the like so as to simulate the zebra fish type II diabetes. The method for constructing the zebra fish model with type II diabetes can be used for screening, developing and applying hypoglycemic drugs, and has important significance.

Drawings

In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required in the embodiments will be briefly described below, it should be understood that the following drawings only illustrate some embodiments of the present invention and therefore should not be considered as limiting the scope, and those skilled in the art can also obtain other related drawings based on the drawings without inventive efforts.

FIG. 1 is a Tol2-pdx1-mCherry-NTR vector map (map A), a Tg (pdx 1: mCherry-NTR) transgenic zebra fish line screening map (map B), fluorescent expression of different families of transgenic fish and in situ hybridization co-localization results (map C);

FIG. 2 is a graph of the change in pancreatic fluorescence after the addition of MTZ to transgenic zebrafish (Tg (pdx 1: mCherry-NTR) in the 120hpf period observed after 48h of 72hpf period control group (B) and 8mM MTZ treatment group (C), B1-C1 are partial enlargements of B-C graphs, D is statistical analysis data of relative fluorescence intensity;. P <0.05,. P <0.01,. P < 0.001);

FIG. 3 is a graph showing the glucose concentration and insulin concentration in (pdx 1-mCherry-NTR) transgenic zebra fish.

Detailed Description

In order to make the objects, technical solutions and advantages of the embodiments of the present invention more apparent, the technical solutions of the embodiments of the present invention will be clearly and completely described below. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.

The features and properties of the present invention are described in further detail below with reference to examples.

Example 1

The embodiment provides a construction method of a zebra fish model with type II diabetes. The method comprises the following steps:

(1) Constructing a transgenic zebra fish strain.

Firstly constructing a Tol2-pdx1-mCherry-NTR plasmid, and amplifying a 2596bp fragment of a pdx1 gene promoter by PCR by using a zebra fish genome as a template. The pdx1promoter-F-BglII in Table 1 as the first primer and pdx1promoter-R-BamHI in Table 1 as the second primer were digested simultaneously with BglII and BamHI restriction enzymes, respectively, and the pdx1 gene promoter fragment and pSTEos vector were digested and recovered with a DNA recovery kit (purchased from Tiangen), followed by ligation to construct pSTEos-pdx1 intermediate vector. The pSTEos-pdx1 intermediate vector and the pST-mylz2-mCherry-NTR plasmid (Meng Anming, which is a gift from the laboratory) were digested simultaneously with XbaI and BamHI restriction enzymes, and then the fragments were recovered, ligated to construct the Tol2-pdx 1-mChery-NTR expression vector and verified by sequencing (the primer sequences are shown in Table 1). A vector diagram of the Tol2-pdx1-mCherry-NTR expression vector is shown in FIG. 1, panel A.

TABLE 1 pdx1promoter primer sequences

Before microinjection, the concentrations of Tol2-pdx1-mCherry-NTR plasmid and Tol2 transposase mRNA are measured by a spectrophotometer and are respectively adjusted to be 75 ng/muL and 150 ng/muL, the mixture is mixed in equal volume and is microinjected to zebrafish embryos at the single cell stage, the injection part is positioned at the junction of the yolk sac and the embryo body, and each embryo is injected with about 1-2nL.

And (3) marking the zebra fish hatched by the embryo injected with the Tol2-pdx1-mCherry-NTR plasmid as an F0 generation, selecting the embryo with red fluorescence in the pancreatic tissue under a fluorescence microscope (Nikang) when the F0 generation develops to 72 hours, feeding the embryo to the sexual maturity zebra fish, and carrying out test cross with the Tg (ins: eGFP) zebra fish. The Tg zebra fish is eGFP-transferred zebra fish, the F1 embryo develops for 72h, and the embryo with pancreas specific red fluorescence is selected under a fluorescence microscope for continuous culture. The F1 generation can be stably inherited, and embryos generated after sexual maturation can be used for experimental research. The cultured F1 generation can be self-crossed or cross-crossed with the required strain, and F2 generation with corresponding red fluorescence is screened and cultured to grow up.

The zebra fish in the embodiment and the following embodiments are all cultured in a circulating aquaculture system, the water temperature of adult zebra fish is controlled to be 26-28 ℃, the pH value is kept to be 6.8-7.4, and the illumination is strictly controlled to be 14-hour photoperiod and 10-hour dark period. Adult fish are fed with brine shrimp regularly, three times a day. The young fish is fed with paramecium when the young fish grows to 5 days (5 dpf) after fertilization, and the young fish starts to feed brine shrimp 15 days (15 dpf) after fertilization.

(2) The transgenic zebra fish is modeled by adopting medicines.

Taking a culture dish as an experimental container, culturing embryos generated by F1 generation sexual maturity for 12 hours, then placing the embryos in Holfreter water containing 0.03g/L PTU for culture, picking out juvenile fish with pancreatic tissues expressing red fluorescence when the embryos develop to 72hpf (72 hours after fertilization), adding MTZ solution (metronidazole solution, product number M1547) with the concentration of 8mM for treatment, and observing the change condition of the red fluorescence and the concentrations of glucose and insulin in the tissue fluid of the juvenile fish every 12 hours, thereby completing the construction of the zebra fish model with type II diabetes.

Experimental example 1

In this example, screening of stably inherited transgenic line Tg (pdx 1: mCherry-NTR) was carried out.

The Tg (pdx 1: mCherry-NTR) transgenic zebrafish is initially formed in 72hpf pancreatic tissue, red fluorescence begins to be expressed, the development condition of the pancreatic tissue is known according to the position and the fluorescence intensity of the fluorescence expression, the transgenic zebrafish with the pancreatic tissue specifically expressing the red fluorescence is obtained by screening with a fluorescence microscope, and the specific screening result is as follows.

And (3) crossing the Tg (pdx 1: mCherry-NTR) F0-generation zebra fish with the zebra fish strain Tg (ins: eGFP) one by one to obtain F1 generation with inconsistent fluorescence expression intensity as shown in a C diagram in figure 1, thereby distinguishing and numbering different families, and carrying out side cross breeding on the different F0 families to obtain the F1 generation. In this experiment, 5 families of Tg (pdx 1: mCherry-NTR) 1# F1, tg (pdx 1: mCherry-NTR) 2# F1, tg (pdx 1: mCherry-NTR) 3# F1, tg (pdx 1: mCherry-NTR) 5# F1 and Tg (pdx 1: mCherry-NTR) 7# F1 were obtained.

Selfing the 1# and 7# F1 generations, and screening to obtain homozygous F2 generations.

Cultured F1 # 3 and F1 # 5 were cross-crossed with the strain Tg (ins: eGFP) of zebrafish, F2 generations having both red fluorescence and green fluorescence were selected and grown up by culture.

Experimental example 2

In this example, 5 transgenic fish of the pedigree in example 1 were subjected to fluorescence expression detection and in situ hybridization co-localization experiments. The method specifically comprises the steps of crossing each Tg (pdx 1: mCherry-NTR) F0 generation transgenic zebra fish strip by strip with a wild type strain, collecting embryos 10min after fertilization in the next morning, selecting the embryos which are normally developed, cleaning, culturing in Holfreter water, picking out the embryos with pancreas tissues expressing red fluorescence by using a fluorescence microscope to take pictures when the embryos are developed to 72h, and detecting the space-time expression condition of the pdx1 gene by using an in-situ hybridization technology. Detecting the space-time expression of zebra fish pdx1 gene mRNA by whole embryo in situ hybridization technology, and positioning the position of pancreatic tissues, wherein the specific steps of in situ hybridization are shown in a reference document [ Hwang G T.molecules,2018; dobrzycki T, biology Open,2018 ].

As shown in FIG. 1C, the fluorescence intensities of pancreatic tissues of different families were different and the expression sites were very specific. The fluorescence intensity of 5# and 7# families transgenic zebra fish is strongest, the fluorescence intensity of 3# family is second, the fluorescence intensity of 2# family is weaker, and the fluorescence intensity of 1# family is weakest. Observing the result of in situ hybridization, it was found that the pdx1 gene was expressed at a pancreatic tissue site, and the site of in situ hybridization staining was consistent with the pancreatic tissue red fluorescence expression site observed by a fluorescence microscope.

Experimental example 3

This example gives an example of the optimum concentration of MTZ solution. Specifically, taking a culture dish as an experimental container, culturing embryos generated by sexual maturation of an F1 generation in Holfreter water containing 0.03g/L PTU for 12h, picking juvenile fish with pancreas tissues expressing red fluorescence when the juvenile fish grows to 72hpf (72 h after fertilization), randomly dividing the juvenile fish into five groups, adding MTZ (metronidazole solution, product number M1547) with gradient concentration of 0mM, 5mM, 8mM, 10mM and 12mM into 2-thousandth of DMSO in each group, respectively, observing the change condition of red fluorescence every 12h, picking dead embryos, replacing medicines and mixing uniformly to ensure that active ingredients are distributed uniformly. And (3) culturing the juvenile fish in a constant-temperature incubator at 28 ℃, photographing under a fluorescence microscope after 48 hours, counting the quantity of reduced fluorescence, and determining the optimal action concentration of MTZ (pdx 1: mCherry-NTR) in-vivo induced Tg (Tg-NTR) transgenic zebra fish for constructing a type II diabetes model.

As shown in Table 2, it is understood from Table 2 that the percentage of decrease in pancreatic fluorescence of juvenile fish exposed to 12mM MTZ was the highest and 100% higher in juvenile fish treated with the drug 48 hours, while the percentage of decrease in juvenile fish exposed to 8mM MTZ was 93.56%, 82.59% and 32% in 5mM MTZ, respectively, compared with the fluorescence of the control group. The death rate of juvenile fish in the 12mM MTZ group is the fastest during the administration period, the death rate reaches 100 percent after continuous administration for 48 hours, and the next experiment cannot be carried out. Juvenile fish began to die massively 24h after treatment with the 10mM MTZ group. The 8mM MTZ group survived well, and therefore, it can be seen that 8mM is the optimal concentration for constructing a type II diabetes model by MTZ induced Tg (pdx 1-mCherry-NTR) transgenic zebrafish.

TABLE 2 construction of diabetes model by treating zebra fish with MTZ of different concentrations

Experimental example 4

72hpf embryos with pancreatic red fluorescence were picked under a fluorescence microscope, a control group and an 8mM MTZ group were set, and fluorescence was observed when the embryos were cultured to 120hpf, as shown in FIG. 2, the red fluorescence of Tg (pdx 1-mCherry-NTR) transgenic juvenile fish was expressed in pancreatic sites, the pancreatic fluorescence of control juvenile fish was strongly expressed (B panel, B1 panel in FIG. 2), and the pancreatic fluorescence of juvenile fish was reduced after being induced by 8mmol/L MTZ (C panel, C1 panel in FIG. 2). The fluorescence expression intensity is compared quantitatively by software, and the fluorescence expression intensity of MTZ group is obviously lower than that of the control group. The construction method of the zebra fish model with type II diabetes, provided by the invention, can kill pancreatic histiocytes of transgenic zebra fish under the induction of MTZ, so that the fluorescence is weakened.

The results of the experimental example show that the red fluorescence expression specificity of the zebra fish pancreatic tissue is weakened after 48 hours of treatment by 8mM MTZ, the glucose concentration of juvenile fish tissue fluid and adult fish blood is obviously increased, and the insulin concentration is obviously reduced, so that the construction success of the zebra fish type II diabetes model is shown, different transgenic zebra fish families are successfully screened and obtained, and more ways are provided for the subsequent screening and the research of novel medicines for treating type II diabetes.

Experimental example 5

In the experimental example, the concentrations of glucose, insulin and adult fish blood insulin in tissue fluid of each group of juvenile fish in the experimental example 1 are detected by a double-antibody one-step sandwich enzyme-linked immunosorbent assay (ELISA). The blood sugar concentration of adult fish is detected by a blood glucose and blood ketone instrument by adopting an eye socket blood sampling method. The method for measuring the content of the insulin is basically the same as the operation steps for measuring the Glucose concentration of the interstitial fluid, and the difference is mainly that a selected plate strip and an enzyme-linked reagent are adopted, glucose (Glucose) antibodies are coated in advance in micropores of a Glucose ELISA detection plate strip, and Insulin (INS) antibodies are coated in advance in micropores of an insulin ELISA detection plate strip. The concentration of the glucose in the tissue fluid of the juvenile fish is detected by a fish glucose ELISA detection kit (the product number is 6141071221), and the insulin concentration in the tissue fluid of the juvenile fish and the insulin concentration in the blood of the adult fish are detected by a fish insulin ELISA detection kit (the product number is 6141091419).

The specific ELISA operating method is as follows:

(1) After the F1 generation transgenic strain zebra fish embryo is fertilized, the embryo is molded by MTZ for 72h, then the grouping and administration strategy (shown in A picture in figure 3) which is the same as the grouping and administration strategy is adopted, when the drug is added for 120h, ten juvenile fishes are collected into 1.5mLEP tubes by each treatment group, 2 parallel samples in each group are washed twice by Holfreter and then washed twice by PBS solution, the solution in the tubes is sucked away, the juvenile fishes are quickly fixed by liquid nitrogen and are placed at-80 ℃ for storage.

(2) Taking out the juvenile fish to be detected from a refrigerator at minus 80 ℃, adding sterile 100ul PBS solution into an EP tube, mashing the juvenile fish by using an electric tissue grinder, putting the juvenile fish on ice to prevent protein denaturation caused by high temperature, shaking for 10s, stopping for 10s, repeating for many times to fully crack embryos until no tissue mass is visible to naked eyes. The lysate is centrifuged at 3000r/min for 10min, and the supernatant is stored at-20 ℃ for use.

(3) The fish insulin and fish glucose ELISA kits were stored at 4 ℃ and allowed to equilibrate for 20 minutes at room temperature before use. When the concentrated washing solution has crystals, the crystals can be heated in a water bath to be completely dissolved for use.

(4) And arranging a standard substance hole and a sample hole, wherein the S0 standard substance with the concentration of 0 can be used as a negative control, and 50 mu L of standard substances with different concentrations are added into the standard substance hole.

(5) Sample Kong Xianjia sample to be tested 10 μ L, then sample diluent 40 μ L.

(6) 100 μ L of detection antibody labeled with horseradish peroxidase (HRP) was added to each of the standard wells and the sample wells, the wells were sealed with a sealing plate, and the plate was incubated in a 37 ℃ water bath or incubator for 60min.

(7) Discarding liquid, patting dry on absorbent paper, filling each hole with cleaning solution, standing for 1min, throwing off the cleaning solution, patting dry on absorbent paper, and repeating the washing for 5 times.

(8) 50 μ L of each substrate A, B was added to each well, the wells were sealed with sealing plates, and incubated at 37 ℃ in the dark for 15min.

(9) Adding stop solution 50 μ L into each well, measuring absorbance (OD value) of each well at 25 deg.C and 450nm with enzyme labeling instrument within 15min.

(10) In Excel, a linear regression curve of a standard substance is drawn by taking the concentration of the standard substance as an abscissa and the corresponding OD value as an ordinate, and the concentration value of each sample can be calculated according to a curve equation only when the R value is more than 0.9900.

As shown in FIG. 3, compared with the control group, the tissue fluid (B panel in FIG. 3) and the blood glucose (D panel in FIG. 3) of adult zebra fish after 48h treatment with 8mM MTZ have a very significant increase and the corresponding insulin concentration has a very significant decrease (C panel and E panel in FIG. 3), which indicates that the Tg (pdx 1-mCherry-NTR) transgenic zebra fish in example 1 of the present invention can be used for constructing a zebra fish type II diabetes model, as can be seen from FIG. 3.

The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

SEQUENCE LISTING

<110> university of Master Minnan

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acactcaaac tggaccgtgt atcttgctat cactggcttt gtcttaatct gctttaccaa 2220

tcaaaactca aatcggtctg aataacgttt taaaatcctc caaatacaaa agaaaagtaa 2280

tgccaaaaat gtgtttccaa agaaccttca actgatctat tcttaattct aataaaatat 2340

tgtttcttag tgtaaacttt ttttgttatt aaaaccttat aatgaatcgt gtttaacgaa 2400

actgttccaa tgttagtctt cttgagctat ttaattcatt tattttttag gagtcatgtt 2460

gagaagaaga caatttaaaa cactcacgta aaccttattt tatttgcttt ttatatttaa 2520

tatgtcgaag caccttggtt ttttcttttg ttttgccgtt taagtggtca atagtgacag 2580

cgtttccccc ggagagtcaa gagactattc cgattcacct gtctttgcag tacccaagaa 2640

ctgcaggtgt agcacataca gtcgcaggta aacagaacat gtttgccaag ggcattgatt 2700

tgagcgtggt cagttctgtt caattattgt caaagcgcgg agcagtcgaa aggaaacatg 2760

aacagaatct actgccagtt gacagctttg tgtaaacagt gaaaccaatg gtgcggcagg 2820

gtcacatggt cacgatggca cacctatgag gatccatggt gagcaagggc gaggaggaca 2880

acatggccat catcaaggag ttcatgcgct tcaaggtgca catggagggc tccgtgaacg 2940

gccacgagtt cgagatcgag ggcgagggcg agggccgccc ctacgagggc acccagaccg 3000

ccaagctgaa ggtgaccaag ggcggccccc tgcccttcgc ctgggacatc ctgtcccctc 3060

agttcatgta cggctccaag gcctacgtga agcaccccgc cgacatcccc gactacttga 3120

agctgtcctt ccccgagggc ttcaagtggg agcgcgtgat gaacttcgag gacggcggcg 3180

tggtgaccgt gacccaggac tcctccctgc aggacggcga gttcatctac aaggtgaagc 3240

tgcgcggcac caacttcccc tccgacggcc ccgtaatgca gaagaagacc atgggctggg 3300

aggcctcctc cgagcggatg taccccgagg acggcgccct gaagggcgag atcaagcaga 3360

ggctgaagct gaaggacggc ggccactacg acgccgaggt caagaccacc tacaaggcca 3420

agaagcccgt gcagctgccc ggcgcctaca acgtcaacat caagctggac atcacctccc 3480

acaacgagga ctacaccatc gtggaacagt acgagcgcgc cgagggccgc cactccaccg 3540

gcggcatgga cgagctgtac aagtccggac tcagatctcg agctcaagct tcgaattcag 3600

gcggcggcgc cgccatggac atcatcagcg tggctctgaa gaggcactcc accaaggctt 3660

tcgacgcttc caagaaactg acccctgaac aggccgagca gatcaagacc ctgctccagt 3720

acagccctag ctccaccaac agccagcctt ggcacttcat cgtggctagc accgaggaag 3780

gcaaagctag ggtggctaag agcgccgctg gcaactacgt gttcaacgag aggaagatgc 3840

tggatgctag ccacgtggtg gtgttctgcg ctaagaccgc catggacgat gtgtggctga 3900

agctggtggt ggatcaggaa gatgctgatg gcaggttcgc tacccctgaa gctaaggccg 3960

ctaacgacaa gggcaggaag ttcttcgccg acatgcacag gaaggatctg cacgatgatg 4020

ctgagtggat ggccaagcag gtgtacctga acgtgggcaa cttcctgctc ggcgtggctg 4080

ccctgggcct cgatgctgtg cccatcgaag gcttcgatgc tgctatcctg gatgccgagt 4140

tcggcctgaa ggagaaaggc tacaccagcc tggtggtggt gcctgtgggc caccacagcg 4200

tggaggactt caacgctacc ctgcctaaga gcaggctgcc ccagaacatc accttaaccg 4260

aagtgtaatc tagatcataa tcagccatac cacatttgta gaggttttac ttgctttaaa 4320

aaacctccca cacctccccc tgaacctgaa acataaaatg aatgcaattg ttgttgttaa 4380

cttgtttatt gcagcttata atggttacaa ataaagcaat agcatcacaa atttcacaaa 4440

taaagcattt ttttcactgc attctagttg tggtttgtcc aaactcatca atgtatctta 4500

aagccgggcc caagtgatct ccaaaaaata agtacttttt gactgtaaat aaaattgtaa 4560

ggagtaaaaa gtactttttt ttctaaaaaa atgtaattaa gtaaaagtaa aagtattgat 4620

ttttaattgt actcaagtaa agtaaaaatc cccaaaaata atacttaagt acagtaatca 4680

agtaaaatta ctcaagtact ttacacctct ggttcttgac cccctacctt cagcaagccc 4740

agcagatcca ctagggccgc caccgcggtg gagctccagc ttttgttccc tttagtgagg 4800

gttaattgcg cgctagggat aacagggtaa tcgcgcttgg cgtaatcatg gtcatagctg 4860

tttcctgtgt gaaattgtta tccgctcaca attccacaca acatacgagc cggaagcata 4920

aagtgtaaag cctggggtgc ctaatgagtg agctaactca cattaattgc gttgcgctca 4980

ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc attaatgaat cggccaacgc 5040

gcggggagag gcggtttgcg tattgggcgc tcttccgctt cctcgctcac tgactcgctg 5100

cgctcggtcg ttcggctgcg gcgagcggta tcagctcact caaaggcggt aatacggtta 5160

tccacagaat caggggataa cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc 5220

aggaaccgta aaaaggccgc gttgctggcg tttttccata ggctccgccc ccctgacgag 5280

catcacaaaa atcgacgctc aagtcagagg tggcgaaacc cgacaggact ataaagatac 5340

caggcgtttc cccctggaag ctccctcgtg cgctctcctg ttccgaccct gccgcttacc 5400

ggatacctgt ccgcctttct cccttcggga agcgtggcgc tttctcatag ctcacgctgt 5460

aggtatctca gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc 5520

gttcagcccg accgctgcgc cttatccggt aactatcgtc ttgagtccaa cccggtaaga 5580

cacgacttat cgccactggc agcagccact ggtaacagga ttagcagagc gaggtatgta 5640

ggcggtgcta cagagttctt gaagtggtgg cctaactacg gctacactag aaggacagta 5700

tttggtatct gcgctctgct gaagccagtt accttcggaa aaagagttgg tagctcttga 5760

tccggcaaac aaaccaccgc tggtagcggt ggtttttttg tttgcaagca gcagattacg 5820

cgcagaaaaa aaggatctca agaagatcct ttgatctttt ctacggggtc tgacgctcag 5880

tggaacgaaa actcacgtta agggattttg gtcatgagat tatcaaaaag gatcttcacc 5940

tagatccttt taaattaaaa atgaagtttt aaatcaatct aaagtatata tgagtaaact 6000

tggtctgaca gttaccaatg cttaatcagt gaggcaccta tctcagcgat ctgtctattt 6060

cgttcatcca tagttgcctg actccccgtc gtgtagataa ctacgatacg ggagggctta 6120

ccatctggcc ccagtgctgc aatgataccg cgagacccac gctcaccggc tccagattta 6180

tcagcaataa accagccagc cggaagggcc gagcgcagaa gtggtcctgc aactttatcc 6240

gcctccatcc agtctattaa ttgttgccgg gaagctagag taagtagttc gccagttaat 6300

agtttgcgca acgttgttgc cattgctaca ggcatcgtgg tgtcacgctc gtcgtttggt 6360

atggcttcat tcagctccgg ttcccaacga tcaaggcgag ttacatgatc ccccatgttg 6420

tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg tcagaagtaa gttggccgca 6480

gtgttatcac tcatggttat ggcagcactg cataattctc ttactgtcat gccatccgta 6540

agatgctttt ctgtgactgg tgagtactca accaagtcat tctgagaata gtgtatgcgg 6600

cgaccgagtt gctcttgccc ggcgtcaata cgggataata ccgcgccaca tagcagaact 6660

ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa aactctcaag gatcttaccg 6720

ctgttgagat ccagttcgat gtaacccact cgtgcaccca actgatcttc agcatctttt 6780

actttcacca gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga 6840

ataagggcga cacggaaatg ttgaatactc atactcttcc tttttcaata ttattgaagc 6900

atttatcagg gttattgtct catgagcgga tacatatttg aatgtattta gaaaaataaa 6960

caaatagggg ttccgcgcac atttccccga aaagtgccac ctaaattgta agcgttaata 7020

ttttgttaaa attcgcgtta aatttttgtt aaatcagctc attttttaac caataggccg 7080

aaatcggcaa aatcccttat aaatcaaaag aatagaccga gatagggttg agtgttgttc 7140

cagtttggaa caagagtcca ctattaaaga acgtggactc caacgtcaaa gggcgaaaaa 7200

ccgtctatca gggcgatggc ccactacgtg aaccatcacc ctaatcaagt tttttggggt 7260

cgaggtgccg taaagcacta aatcggaacc ctaaagggag cccccgattt agagcttgac 7320

ggggaaagcc ggcgaacgtg gcgagaaagg aagggaagaa agcgaaagga gcgggcgcta 7380

gggcgctggc aagtgtagcg gtcacgctgc gcgtaaccac cacacccgcc gcgcttaatg 7440

cgccgctaca gggcgcgtcc cattcgccat tcaggctgcg caactgttgg gaagggcgat 7500

cggtgcgggc ctcttcgcta ttacgccagc tggcgaaagg gggatgtgct gcaaggcgat 7560

taagttgggt aacgccaggg ttttcccagt cacgacgttg taaaacgacg gccagtgagc 7620

gcgc 7624

<210> 2

<211> 654

<212> DNA

<213> Artificial sequence

<400> 2

atggacatca tcagcgtggc tctgaagagg cactccacca aggctttcga cgcttccaag 60

aaactgaccc ctgaacaggc cgagcagatc aagaccctgc tccagtacag ccctagctcc 120

accaacagcc agccttggca cttcatcgtg gctagcaccg aggaaggcaa agctagggtg 180

gctaagagcg ccgctggcaa ctacgtgttc aacgagagga agatgctgga tgctagccac 240

gtggtggtgt tctgcgctaa gaccgccatg gacgatgtgt ggctgaagct ggtggtggat 300

caggaagatg ctgatggcag gttcgctacc cctgaagcta aggccgctaa cgacaagggc 360

aggaagttct tcgccgacat gcacaggaag gatctgcacg atgatgctga gtggatggcc 420

aagcaggtgt acctgaacgt gggcaacttc ctgctcggcg tggctgccct gggcctcgat 480

gctgtgccca tcgaaggctt cgatgctgct atcctggatg ccgagttcgg cctgaaggag 540

aaaggctaca ccagcctggt ggtggtgcct gtgggccacc acagcgtgga ggacttcaac 600

gctaccctgc ctaagagcag gctgccccag aacatcacct taaccgaagt gtaa 654

<210> 3

<211> 708

<212> DNA

<213> Artificial sequence

<400> 3

atggtgagca agggcgagga ggacaacatg gccatcatca aggagttcat gcgcttcaag 60

gtgcacatgg agggctccgt gaacggccac gagttcgaga tcgagggcga gggcgagggc 120

cgcccctacg agggcaccca gaccgccaag ctgaaggtga ccaagggcgg ccccctgccc 180

ttcgcctggg acatcctgtc ccctcagttc atgtacggct ccaaggccta cgtgaagcac 240

cccgccgaca tccccgacta cttgaagctg tccttccccg agggcttcaa gtgggagcgc 300

gtgatgaact tcgaggacgg cggcgtggtg accgtgaccc aggactcctc cctgcaggac 360

ggcgagttca tctacaaggt gaagctgcgc ggcaccaact tcccctccga cggccccgta 420

atgcagaaga agaccatggg ctgggaggcc tcctccgagc ggatgtaccc cgaggacggc 480

gccctgaagg gcgagatcaa gcagaggctg aagctgaagg acggcggcca ctacgacgcc 540

gaggtcaaga ccacctacaa ggccaagaag cccgtgcagc tgcccggcgc ctacaacgtc 600

aacatcaagc tggacatcac ctcccacaac gaggactaca ccatcgtgga acagtacgag 660

cgcgccgagg gccgccactc caccggcggc atggacgagc tgtacaag 708

<210> 4

<211> 27

<212> DNA

<213> Artificial sequence

<400> 4

ggaagatctg ccactaagca agcactt 27

<210> 5

<211> 27

<212> DNA

<213> Artificial sequence

<400> 5

cgcggatcct cataggtgtg ccatcgt 27

<210> 6

<211> 2596

<212> DNA

<213> Artificial sequence

<400> 6

gccactaagc aagcactttt tatcatgctt tgttctctgt cataactcgt tttttactgt 60

agaatctgaa agtaaaaagc tatatgtttc caattatact tcttaaaaaa ctattaaaac 120

tcgtttttta acgatgcatc ttgtgggtgt atgccactgg gtttatcaac aatctgatta 180

cgtgtggttt tacataacgc aattttatga tttatctcca ttatcgagac gcacttggac 240

gtttatagcc acacattact cctactcaaa cacacaagtg gtttcagaac gcttttgcgt 300

gtcttttaac tggtttattg cattttatct gaagtctcga aatgcatttt aattgccagc 360

cgattttatg taccagtgct ttaagttgtg catttcctct atgccatggc aaatctacat 420

tacatatcac tttacaccat tattcctctc aaaaccttcc atggatatgc ttctaaattg 480

cgtcgtctca ctcatgcttc ataaaacctt ttgtgctctt tcaacattgt aaaaatagtc 540

gcggataacg gtttcctttt cttttcaagc gaggtcatat ataaacacca ttaacagtcg 600

ttacgaggct tgatttgcga ggaatggatt tgcctgaact ttatgatggc gcatgagagc 660

cccattaaga taggagagaa cgtgctgtta ttgtcatttg aagaggttcg tgcaaacaca 720

acgctatgtc ataatctgga ttacttgaat ttggcgtgaa tgtattgcat ttttaaaaat 780

attttgtaaa aaaaaaaaga agaagaagaa aacgctaagc tttttggatt cgttccaagc 840

tccagattcc atttatttta catgtaaatg tggatgactg gagagttggg tctaatgttc 900

aaaatatgtg tttcccgttt tgaagaagct aaattcaaca ttcttaagct tgaggaaaat 960

ataaaaagct ttattttgaa tatttctaaa atgaatgatt gtattttata agcatttaat 1020

actgagggaa cttttcaaac tgacattttc accctgtcct ctcgagcttg catggtgccg 1080

cacacgttgt acacacatct caaactaatt gagttttgtt gaactctcca tgttaattcg 1140

gttacaccga acagcttgtt tttatttaac cacgaggact ttagtttaat ggaccgtaga 1200

tgggtgtgct taatggcaag tgagcttagt tgaagcaaaa gacggacaac atgttaggtg 1260

ttcatagatt ttacgtaaca gtgagttaat gggaactctt aacaagataa atatcacaat 1320

tttggcgcca cgtgatgttg ggttaacgca atttgataat atactttttt tgaccaacaa 1380

aaactgattt gtgcatgtat agtgtgaaaa ttggctatat aataaagaga taatacaaat 1440

tttaaaaatc ccattttatt gccaccttcc ctttcctacg caaaatgacg caaattgtgt 1500

gcgcaccttc cgttaaattg cgtattcagt gaaatgtttt tgagacgagg tggcttcatc 1560

cagttttaaa ataaatgcat tagaatctat tactttcagc agcaatatta cagacgtggt 1620

aaatatcacc atgaaagaat gataatctaa aggagcagaa cactgaactc gaagaactgt 1680

tgctgcagtt ttatgaagtt ttaccatgac agcaatgcca aaaataccaa gttatttatt 1740

tttttttttt taaattggcg tttctcaaac tgaaagtgca acacataatt ctaaagccag 1800

taggcactaa atataacctg ttgaatgaat tggctcgtat attttctgat aagtgcaaat 1860

acgcgatcag aatgattttg ctattttttc ttttctttag ggaaaaaaca ctcaaactgg 1920

accgtgtatc ttgctatcac tggctttgtc ttaatctgct ttaccaatca aaactcaaat 1980

cggtctgaat aacgttttaa aatcctccaa atacaaaaga aaagtaatgc caaaaatgtg 2040

tttccaaaga accttcaact gatctattct taattctaat aaaatattgt ttcttagtgt 2100

aaactttttt tgttattaaa accttataat gaatcgtgtt taacgaaact gttccaatgt 2160

tagtcttctt gagctattta attcatttat tttttaggag tcatgttgag aagaagacaa 2220

tttaaaacac tcacgtaaac cttattttat ttgcttttta tatttaatat gtcgaagcac 2280

cttggttttt tcttttgttt tgccgtttaa gtggtcaata gtgacagcgt ttcccccgga 2340

gagtcaagag actattccga ttcacctgtc tttgcagtac ccaagaactg caggtgtagc 2400

acatacagtc gcaggtaaac agaacatgtt tgccaagggc attgatttga gcgtggtcag 2460

ttctgttcaa ttattgtcaa agcgcggagc agtcgaaagg aaacatgaac agaatctact 2520

gccagttgac agctttgtgt aaacagtgaa accaatggtg cggcagggtc acatggtcac 2580

gatggcacac ctatga 2596

<210> 7

<211> 236

<212> PRT

<213> Artificial sequence

<400> 7

Met Val Ser Lys Gly Glu Glu Asp Asn Met Ala Ile Ile Lys Glu Phe

1 5 10 15

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

20 25 30

Glu Ile Glu Gly Glu Gly Glu Gly Arg Pro Tyr Glu Gly Thr Gln Thr

35 40 45

Ala Lys Leu Lys Val Thr Lys Gly Gly Pro Leu Pro Phe Ala Trp Asp

50 55 60

Ile Leu Ser Pro Gln Phe Met Tyr Gly Ser Lys Ala Tyr Val Lys His

65 70 75 80

Pro Ala Asp Ile Pro Asp Tyr Leu Lys Leu Ser Phe Pro Glu Gly Phe

85 90 95

Lys Trp Glu Arg Val Met Asn Phe Glu Asp Gly Gly Val Val Thr Val

100 105 110

Thr Gln Asp Ser Ser Leu Gln Asp Gly Glu Phe Ile Tyr Lys Val Lys

115 120 125

Leu Arg Gly Thr Asn Phe Pro Ser Asp Gly Pro Val Met Gln Lys Lys

130 135 140

Thr Met Gly Trp Glu Ala Ser Ser Glu Arg Met Tyr Pro Glu Asp Gly

145 150 155 160

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

165 170 175

His Tyr Asp Ala Glu Val Lys Thr Thr Tyr Lys Ala Lys Lys Pro Val

180 185 190

Gln Leu Pro Gly Ala Tyr Asn Val Asn Ile Lys Leu Asp Ile Thr Ser

195 200 205

His Asn Glu Asp Tyr Thr Ile Val Glu Gln Tyr Glu Arg Ala Glu Gly

210 215 220

Arg His Ser Thr Gly Gly Met Asp Glu Leu Tyr Lys

225 230 235

<210> 8

<211> 217

<212> PRT

<213> Artificial sequence

<400> 8

Met Asp Ile Ile Ser Val Ala Leu Lys Arg His Ser Thr Lys Ala Phe

1 5 10 15

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

20 25 30

Leu Leu Gln Tyr Ser Pro Ser Ser Thr Asn Ser Gln Pro Trp His Phe

35 40 45

Ile Val Ala Ser Thr Glu Glu Gly Lys Ala Arg Val Ala Lys Ser Ala

50 55 60

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

65 70 75 80

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

85 90 95

Leu Val Val Asp Gln Glu Asp Ala Asp Gly Arg Phe Ala Thr Pro Glu

100 105 110

Ala Lys Ala Ala Asn Asp Lys Gly Arg Lys Phe Phe Ala Asp Met His

115 120 125

Arg Lys Asp Leu His Asp Asp Ala Glu Trp Met Ala Lys Gln Val Tyr

130 135 140

Leu Asn Val Gly Asn Phe Leu Leu Gly Val Ala Ala Leu Gly Leu Asp

145 150 155 160

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

165 170 175

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

180 185 190

His His Ser Val Glu Asp Phe Asn Ala Thr Leu Pro Lys Ser Arg Leu

195 200 205

Pro Gln Asn Ile Thr Leu Thr Glu Val

210 215

Claims

1. A construction method of a zebra fish model with type II diabetes is characterized by comprising the following steps:

(1) Constructing transgenic zebra fish;

(2) Carrying out molding on the transgenic zebra fish by adopting a medicament;

in the process of constructing the transgenic zebra fish in the step (1), firstly constructing an expression vector, wherein the nucleotide sequence of the expression vector is shown as SEQ ID NO.1, then mixing the expression vector with Tol2mRNA, and microinjecting the mixture into the zebra fish embryo;

2. The method of claim 1, wherein the expression vector comprises a gene encoding a fluorescent protein, and the gene encoding a fluorescent protein ismCherryA gene ofmCherryThe protein sequence of the gene code is shown as SEQ ID NO. 7; the above-mentionedmCherryThe nucleotide sequence of the gene is shown in SEQ ID NO. 3.

3. The construction method according to claim 1, characterized in that the drug is metronidazole, and the concentration of action of the metronidazole is 8-10mM.

4. The method for constructing the zebrafish embryos of claim 1, wherein the developmental stage of the zebrafish embryos is a 72hpf stage, and the injection amount of each zebrafish embryo is 1-2nL.

5. The use of the zebrafish model with type II diabetes mellitus obtained by the construction method according to any one of claims 1 to 4 in screening of drugs for treating type II diabetes mellitus.

6. The use according to claim 5, wherein the method for screening a medicament for treating type II diabetes comprises the steps of:

if the red fluorescence of the zebra fish model with type II diabetes is enhanced, the blood sugar concentration is reduced and the insulin concentration is increased after the medicine is applied, the medicine is judged to have the effect of treating the type II diabetes;

if the red fluorescence of the zebra fish model with type II diabetes remains unchanged after the drug is administered, and the blood glucose concentration and the insulin concentration do not change obviously, the drug is judged not to have the effect of treating type II diabetes.