CN102823522A

CN102823522A - Triploid induction of Takifugu flavidus and Takifugu rubripes

Info

Publication number: CN102823522A
Application number: CN2011101638980A
Authority: CN
Inventors: 张福崇; 刘海金; 赵振良; 赵文江; 范文涛; 赵海涛; 刘永新
Original assignee: BEIDAIHE CENTRAL EXPERIMENTAL STATION CHINESE ACADEMY OF FISHERY SCIENCES; Hebei Provincial Aquatic Products Research Institute
Current assignee: BEIDAIHE CENTRAL EXPERIMENTAL STATION CHINESE ACADEMY OF FISHERY SCIENCES; Hebei Provincial Aquatic Products Research Institute
Priority date: 2011-06-17
Filing date: 2011-06-17
Publication date: 2012-12-19

Abstract

The invention provides a method for hybridized triploid induction of Takifugu flavidus and Takifugu rubripes. The method comprises thee steps of selecting mature male and female Takifugu flavidus and Takifugu rubripes; collecting mature spawn and semen respectively; performing artificial insemination and reciprocal hybridization; 3-7 min after insemination, performing thermal shock treatment with 30-40 DEG C sea water for 5-20 min; and hatching in 15-20 DEG C sea water, to complete triploid induction by thermal shock method. The method has the advantages of simple process, low cost, strong operability, greatly improved hybridized triploid induction rate of Takifugu flavidus and Takifugu rubripes, and high economical and social benefits. According to the statistical result of experiments, the hybridized triploid artificial induction rate of Takifugu flavidus and Takifugu rubripes reaches to 100%.

Description

Huang Ju Fugu rubripes with triploid induction

Technical field

The present invention relates to a fish seed production methods, particularly relates to a daisy yellow Fugu rubripes and triploid hybrid production methods, the technology belongs to the field of marine biotechnology.

Background technology

The yellow Fugu of chrysanthemum is commonly called as ship crust, magnitude all over the sky, mainly is distributed in Yellow Sea of China, the East Sea and the Bohai Sea.And Fugu rubripes mainly is distributed in Japan, the Korea peninsula and the coastal area of china of North Western Pacific.Fugu rubripes is one of higher kind of individual maximum, economic worth.Because of its fine and tender taste, delicious flavour, protein height, nutritious, have the good reputation of " kings of fish ", receive liking of numerous gourmets deeply.

The Fugu rubripes growth is fast, but toxicity is big, and domestic the permission eats; The yellow Fugu growth of chrysanthemum is slow, and in Jiangsu, there is food custom in the area, Shanghai, commodity value is high, and market is also very big.Therefore, want to utilize the former growth vigor, through hybridization, acquisition is similar with the yellow Puffer morphological feature of chrysanthemum, but the hybridization Puffer of growing fast, to improve commodity value.

From the trend of domestic and international aquatic animal research, the triploid technology is a kind of method of potentialization.The triploid aquatic livestock is owing to have sterility, and promptly sexual gland can not be reached maturity or developmental deformity, and irreproducible offspring, does not therefore have the energy loss of sexual maturity or proliferation, has avoided the growth in breeding stage to stop or high mortality.At present, the triploid aquatic livestock has all shown good effect at aspects such as control excessive multiplication, quickening growth rate.

Therefore, according to the characteristics of Fugu flavidus to develop a method of inducing obtained with daisy yellow puffer Takifugu triploid hybrids to improve Fugu flavidus growth rate, improve their economic and social benefits that the aquaculture industry The common expectation.

Summary of the invention

The present invention aims to provide a daisy with yellow Fugu rubripes hybrid triploid induction method.

Step of the present invention is:

1) chooses Fugu rubripes milter and the raun of gonad maturity, the milter and the raun of the yellow Fugu of chrysanthemum, collecting semen and mature egg respectively; Carry out positive and negative hybridization;

2) seminal fluid is evenly mixed with the ovum grain, room temperature leaves standstill 2min, adds seawater and carries out artificial insemination;

3) triploid obtains: the fertilized egg of after fertilization 3-7min is transferred in the 30-40 ℃ of seawater bath carries out heat shock 5-20min, suppress the formation and the discharge thereof of second polar body, chromosome set is doubled.

4) relieve shock move the eggs hatch in sea water at room temperature, in accordance with the ordinary chrysanthemum yellow puffer approach to the management of seed production, cultivation of chrysanthemum with yellow Fugu rubripes hybrid triploid fish.

In step 2) in, the seawater water temperature is 15-20 ℃.

In step 3), the time of initial heat shock is after fertilization 3-7min in the seawater; The heat shock duration is 10-16min;

The heat shock temperature is 30-36 ℃.

In step 4), the temperature of seawater hatching is 15-20 ℃.

More preferably,

In the step 3), the time of initial heat shock is after fertilization 4-6min in the seawater; The heat shock duration is 14-16min,

The heat shock temperature is 34-35 ℃;

In the step 4), the temperature of seawater hatching is 17 ℃.

The employing said method is induced, and can access higher inductivity.

The present invention further program: induction get Fugu flavidus with rubripes triploid hybrids, the use of karyotype analysis, flow cytometry technique of measuring the fish multiple chromosomes, and then daisy yellow puffer with rubripes triploid hybrid breed.

Beneficial effect of the present invention is:

1 of the present invention through the use of heat shock induction methods, and by controlling the duration of induction, inhibiting the release of the second polar body, thus greatly improving the Ju Huang Fugu rubripes hybridization with triploid induction rate, according to experimental statistics, Huang Ju obtained Fugu rubripes and triploid hybrid artificial two conductivity as high as 100%.

2. technology of the present invention is simple, and cost is low, and is workable, has avoided chemical method to induce the pollution that brings.

3 using the method described in the present invention yellow chrysanthemum cultivation Fugu rubripes and triploid hybrids, with fast growth, large individual specifications, breeding high survival rate, disease-resistant ability, low toxicity, good economic and social benefits.

In a daisy with yellow puffer Takifugu hybrid triploid induction, one of the most preferred induction conditions, select the gonads mature rubripes male and female, daisy yellow males and females Puffer, semen and mature eggs were collected; conduct reciprocal crosses; 5min after fertilization of sea bathing with 35 ℃ heat shock treatment 15min, inhibiting the second polar body, and then 17 ℃ seawater incubation, the completion of triploid induction of heat shock method .

With this condition Ju Huang Fugu rubripes hybridization with triploid induction, the experimental results show that the hatching rate = 6.69%, triploid rate of 100%, the relative number of 35.42% triploid.

Description of drawings

The yellow Fugu diploid fish fry of Fig. 1 chrysanthemum cell DNA fluorescence distribution histogram.

Fig. 2 Fugu rubripes diploid fish fry cell DNA fluorescence distribution histogram.

The yellow Fugu female parent of Fig. 3 chrysanthemum * Fugu rubripes male parent triploid fry cell DNA fluorescence distribution histogram.

The yellow Fugu male parent of Fig. 4 chrysanthemum * maternal triploid fry of Fugu rubripes cell DNA fluorescence distribution histogram.

Embodiment

Following experimental example and embodiment will do further explanation to the present invention.

Experimental example 1

Huang Ju Fugu rubripes and triploid hybrids of ploidy

Adopt the German Partec Cystain DNA 1step of company dye liquor one-step method dyeing, identify ploidy through the cells were tested by flow cytometry cell DNA content.

1, preparation of histocyte suspension and mensuration

Employing is randomly drawed mode the yellow Fugu female parent of 5-8 age in days 10 tail chrysanthemums * Fugu rubripes male parent triploid and the yellow Fugu male parent of 10 tail chrysanthemums * maternal triploid of Fugu rubripes is identified, at every turn with the yellow Fugu dliploid of 10 tail chrysanthemums and 10 tail Fugu rubripes dliploids as contrast.

(1) the about 1mm of live body tail fin of each tail fish is got in histocyte extraction and DNA fluorescent staining ³, put into the 1.5mL microcentrifugal tube, add Cystain DNA 1step dye liquor 0.5ml, shred tail fin and organize to naked eyes and cannot see block, grind gently again to equal pulpous state; With the cell filtration net filtration that 300 orders Germany Partec company produces, in filtrating, add Cystain DNA 1step dye liquor 0.5ml again, further dyeing.

(2) cells were tested by flow cytometry PARTEC is that German Partec Gmbh company produces, model PA, and this machine excitation source is a negative ion laser.Before the measurement, about 1 * 106/ml, keep measuring rate during measurement at 200～300/s with sheath fluid adjustment cell concentration.Selecting the normal diploid cell suspension is control sample.

(3) detect the preceding adjustment flow cytometer of control mensuration and make pipeline unimpeded, nozzle cleaning, instrument is in optimum state, carries out FLOWCHECK and proofreaies and correct, and the coefficient of variation (CV) of each passage is stabilized in below 3%, and dna content is detected by instrument automatically.Keep cell separation purity through the control flow velocity.

2, detect principle and method for expressing

With the dliploid cellular control unit is reference calculation dliploid and triploid frequency, and cell DNA content is with DNA index (DNAindex, DI) expression.

DI=processed group cell G0/1 peak channel/cellular control unit G0/1 peak channel average

With DI=1.0 ± 0.1 is the diplontic criterion of DNA.If DI=1.5 ± 0.1 then is a triploid.

DNA to triploid processed group and dliploid control group analyzes (table 1), and the result shows the two cell

The G0/1 peak channel significant difference of DNA.

Control group that flow cytometer provides and triploid processed group cell DNA content fluorescence distribution histogram are seen Fig. 1-Fig. 4.

The DNA of table 1 triploid processed group and dliploid control group is (average) relatively

Embodiment 1

Choose Fugu rubripes milter and raun, the yellow Fugu milter of chrysanthemum and raun, the collecting semen and the mature egg respectively of gonad maturity; Carry out positive and negative hybridization; After fertilization 3min handles 10min with 35 ℃ seawater bath heat shock, in 17 ℃ seawater, hatches then, accomplishes the triploid induction of heat shock method.Experimental result shows, incubation rate=5.53%, and triploid rate reaches 90%, and the triploid number is 26.35% relatively.

Embodiment 2

Choose Fugu rubripes milter and raun, the yellow Fugu milter of chrysanthemum and raun, the collecting semen and the mature egg respectively of gonad maturity; Carry out positive and negative hybridization; After fertilization 5min handles 15min with 35 ℃ seawater heat shock, in 17 ℃ seawater, hatches then, accomplishes the triploid induction of heat shock method.Experimental result shows, incubation rate=6.69%, and triploid rate reaches 100%, and the triploid number is 35.42% relatively.

Embodiment 3

Choose Fugu rubripes milter and the raun of gonad maturity, the milter and the raun of the yellow Fugu of chrysanthemum, collecting semen and mature egg respectively; Carry out positive and negative hybridization; After fertilization 7min handles 10min with 35 ℃ seawater bath heat shock, in 17 ℃ seawater, hatches then, accomplishes the triploid induction of heat shock method.Experimental result shows, incubation rate=8.60%, and triploid rate reaches 50%, and the triploid number is 22.76% relatively.

Claims

1 A heat shock-induced Ju Huang Fugu rubripes and triploid hybrid method comprising the following steps:

1) chooses Fugu rubripes milter and the raun of gonad maturity, the milter and the raun of the yellow Fugu of chrysanthemum, collecting semen and mature egg respectively; Carry out positive and negative hybridization

2) seminal fluid is evenly mixed with the ovum grain, stir the back room temperature and leave standstill 2min, add seawater and carry out artificial insemination;

3) triploid obtains: the fertilized egg of after fertilization 3-7min is transferred to carries out heat shock in the 30-40 ℃ of seawater bath and handle 5-20min, chromosome set is doubled.

4) relieve heat shock move the eggs hatch in seawater, according to Huang Ju Puffer ordinary approach to the management of seed production, cultivation of chrysanthemum with yellow Fugu rubripes hybrid triploid fish.

(2) according to claim 1, wherein the yellow chrysanthemum Fugu rubripes hybridization with triploid induction method, comprising:

Step 2) in, the seawater water temperature is 15-20 ℃.

3 of claim 1, wherein the yellow chrysanthemum Fugu rubripes and induced triploid hybrid method, comprising:

In the step 3), the time of initial heat shock is after fertilization 3-7min in the seawater; The heat shock duration is 10-16min;

The heat shock temperature is 30-36 ℃.

4 of claim 1, wherein the yellow chrysanthemum Fugu rubripes hybridization with triploid induction method, comprising:

In the step 4), the temperature of seawater hatching is 15-20 ℃.

5 according to claim 1, wherein the yellow chrysanthemum Fugu rubripes hybridization with triploid induction method, comprising:

The time of initial heat shock is after fertilization 4-6min in the seawater; The heat shock duration is 14-16min, heat shock

Temperature is 34-35 ℃.

6 of claim 1, wherein the yellow chrysanthemum Fugu rubripes and induced triploid hybrid method, comprising:

In the step 4), the temperature of seawater hatching is 17 ℃.

7 according to claim 1, wherein the yellow chrysanthemum Fugu rubripes hybridization with triploid induction method, comprising:

Choose Fugu rubripes milter and the raun of gonad maturity, the milter and the raun of the yellow Fugu of chrysanthemum; Gather mature egg and seminal fluid respectively, positive and negative hybridization is carried out in artificial insemination; After fertilization 5min handles 15min with 35 ℃ seawater bath heat shock; Suppress second polar body and discharge, in 17 ℃ seawater, hatch then, accomplish the triploid induction of heat shock method.