CN110396518A - Special cance high-expression gene increases the method for milter reproductive capacity in a kind of gonadal tissue - Google Patents
Special cance high-expression gene increases the method for milter reproductive capacity in a kind of gonadal tissue Download PDFInfo
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
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Abstract
The invention belongs to gene expression technique fields, the method that special cance high-expression gene in a kind of gonadal tissue increases milter reproductive capacity is disclosed, extractions gonadal tissue RNA- reverse transcription is cDNA, and cDNA is template, tesk1-F/tesk1-R is primer, and high fidelity enzyme pfu carries out PCR amplification；Used carrier is the plasmid vector containing 3 ' UTR of kop promoter and nanos, kop promoter and nanos3 ' UTR can by target gene in reproduction cell specifically expressing, by NcoI and BseAI digestion, tesk1 gene is inserted between kop promoter and nanos3 ' UTR, forms transgene carrier；Microinjection obtains genetically engineered fish.The present invention special high expression tesk1 gene, discovery in milter gonadal tissue can increase the genital gland indices of milter with conspicuousness, increase it and produce essence amount, realize the purpose for improving male zebra fish fecundity.
Description
Technical field
The invention belongs to cance high-expression genes special in gene expression technique field more particularly to a kind of gonadal tissue to increase hero
The method of fish reproductive capacity.
Background technique
Currently, the immediate prior art: a kind of fish reproduction capacity of water directly determines the degree of its application.
Four large Chinese carps of large-scale commercial cultivation at present, why can national large-scale promotion cultivation, one of the main reasons is energy
It enough realizes extensive artificial propagation, obtains a large amount of seed.But famous-particular-excellent fish some for swamp eel, grouper etc., it promotes
The maximum bottleneck of cultivation is exactly that fertility is poor, cannot obtain a large amount of seed, but also these fish prices occupy height throughout the year
Under not.Therefore, the reproductive capacity for improving fish has very important significance for the further genralrlization of some fish.For hero
Property be considerably less than the fish of female, such as green crucian carp and hermaphroditic grouper etc., improve male reproductive capacity, can be certain
The problem for alleviating male rareness in degree is conducive to that it is further promoted to promote cultivation.Currently, solving some fish reproductions
The weak problem of power mostly use greatly regulation influence fish gonad development external ecology condition, as nutrition, water temperature, illumination, water flow and
Dissolved oxygen etc., these operations add somewhat to the reproductive capacity of fish, but since influence factor is too many, it is complicated for operation, increase
Aquaculture cost is added, and smaller for outdoor breeding repeatability.In addition, in recent years, " borrowing abdomen pregnant " is that fish reproduction is related
Hot spot in research, still, the technology is not promoted the use of still still in theory stage at present.In this context, by turning base
The method of cause increases milter fecundity, and by establishing family, the character that fecundity can be made to enhance is handed down from age to age, and can accomplish one
Kick and just.
Fish are that the mankind depend on for existence one of the important sources of high-quality protein, are last wild food sources.Contain in fish
There is 8 kinds of amino acid necessary to human body, unsaturated fatty acid etc., nutritive value is abundant, and there's almost no the disease that mermaid suffers from altogether
Disease, therefore by favor.The sustainable development of fishery has become the theme of countries in the world today concern.In this case, water
Producing cultivation is considered as meeting the ever-increasing unique channel to aquatic livestock albumen demand of the mankind.Supporting with merit
Breeding fish new varieties are the key that one of following aquaculture sustainable developments.Artificially breeding, introduce a fine variety with crossbreeding be obtain
The conventional method of cultured fishes new varieties with merit, but the breeding time limit of traditional breeding way overlength greatly limits
The development of fish breeding industry is made.Transgenic technology can be obtained the new varieties with merit in a short time.
Compared to mammal, fish have it is in vitro fertilization, ectogenesis, fish brood amount is big, and is easy to transgeneic procedure, embryo
Tire, which does not need to put back to after transgeneic procedure parent, which advantages, these advantage render transgenic technologies such as cultivates, can be applied to rapidly fish
Class breeding.The exploration for the first time that transgenic technology is applied to fish breeding starts from the 1980s, Zhu Zuoyan etc. utilizes mouse
The growth hormone gene that metal sulphur gene promoter is successfully driven people is expressed in crucian, cultivates first fast fast-growing in the world
Long genetically engineered fish.Later, transgenic technology is applied in fish breeding and is unfolded rapidly, by development in more than 30 years, at present
Being obtained by transgeneic procedure has that the speed of growth is fast, bait utilization is high, the genetically engineered fish of the disease-resistant degeneration-resistant merits such as strong
Nearly 40 kinds of class, and almost include important cultured fishes all in the world.
The large-scale promotion cultivation of one new fish species premise is that carry out artificial propagation, obtain a large amount of seedlings
Kind.But current some fish female-male proportion numerous imbalances, milter individual is considerably less, such as grouper, green crucian carp, these fish
Although having many merits, due to lacking male, breeding scale is severely limited, and prevents it into one
Step promotes cultivation.But at present still without for the transgenic research for increasing milter fecundity.At present about genetically engineered fish
Research main object be the biggish fish of yield, such as four large Chinese carps and carp, it is therefore an objective to which the growth for improving these fish is fast
Degree, disease-resistant, cold tolerance etc..But for these fish, fecundity is very strong, does not need to increase fertility.But mesh
The emphasis of preceding China's Fisheries Development is good in economic efficiency special aquatic products fish, and special aquatic products fish species are various, some of
Fish promote the bottleneck problem of cultivation, are exactly that reproductive capacity is poor, and therefore, it is numerous that the developing direction of fishery from now on requires research to increase fish
Grow the technology of power.
In conclusion problem of the existing technology is:
(1) some fish female-male proportion numerous imbalances, milter individual are considerably less at present；Lack male, breeding rule
Mould is severely limited, its further genralrlization is prevented to cultivate.
(2) currently without for the transgenic research for increasing milter fecundity.
Solve the difficulty of above-mentioned technical problem:
1 finds the target gene with enhancing fish reproduction power.We have discovered that tesk1 gene overexpression can enhance hero
Property fish reproduction power.
How 2 make target gene in gonadal tissue specifically expressing we select in gonadal tissue specifically expressing kop promoter and
MRNA can be positioned to the specifically expressed 3 ' UTR of nanos of gonadal tissue, so that target gene tesk1 is in the special table of gonadal tissue
It reaches.
Solve the meaning of above-mentioned technical problem:
The reproductive capacity for improving fish has the further genralrlization that some males are considerably less than the fish of female very heavy
The meaning wanted.Such as green crucian carp and hermaphroditic grouper etc. improve male reproductive capacity for these fish, it can be with one
The problem for determining to alleviate male rareness in degree, is conducive to that it is further promoted to promote cultivation.
Summary of the invention
In view of the problems of the existing technology, the present invention provides cance high-expression genes special in a kind of gonadal tissue to increase hero
The method of fish reproductive capacity.
The invention is realized in this way special cance high-expression gene increases the method for milter reproductive capacity in a kind of gonadal tissue,
In the gonadal tissue special cance high-expression gene increase milter reproductive capacity method the following steps are included:
The first step, extraction gonadal tissue RNA- reverse transcription are with tesk1-F/tesk1-R using cDNA as template for cDNA
Primer carries out PCR amplification, the clone of tesk1 gene with high fidelity enzyme pfu；
Second step, used carrier be the plasmid vector containing 3 ' UTR of kop promoter and nanos, kop promoter and
Nanos3 ' UTR can specifically expressing be inserted tesk1 gene by NcoI and BseAI digestion in reproduction cell by target gene
Enter between kop promoter and nanos3 ' UTR, forms transgene carrier；
Third step, microinjection obtain genetically engineered fish.
Further, the primer of the first step are as follows:
tesk1-F 5'-CACCCATGGGTTGCGTTAAAGTGACCCGCT-3'；
tesk1-R 5’-TTCTCCGGAGAGATGTCGCAGTGGTCGTC-3’。
Further, the transgene carrier of the second step is Tol2-kop-tesk1-nanos3 ' containing main element
UTR-Tol2。
Further, the microinjection acquisition genetically engineered fish method of the third step includes:
(1) before microinjection, the transgene carrier processing is carried out comprising go toxicity processing and anti-series connection processing；
(2) Tol2 transposase mRNA is synthesized in vitro；
(3) treated Plasmid DNA and the Tol2 transposase of the transgene carrier described in zebrafish embryo microinjection
mRNA；
(4) using the zebrafish embryo after microinjection as P0 generation, after the P0 generation cultivation sexal maturity, by with wild spot
Horse raun test cross obtains Offspring F1 generation, obtains homozygosis F2 generation using selfing after the F1 generation positive individuals are raised to sexal maturity.
Further, the external synthesis Tol2 transposase mRNA method includes:
Firstly, 396 carriers that will have transposase gene, are linearized with I restriction enzyme of Not, it is then external with SP6
Transcript reagent box is transcribed in vitro；
Secondly, carrying out removal and the reaction terminating of DNA profiling；
Finally, purifying.
In conclusion advantages of the present invention and good effect are as follows: currently, solving the problems, such as that some fish reproduction power are weak most
The external ecology condition of fish gonad development, such as nutrition, water temperature, illumination, water flow and dissolved oxygen are influenced using regulation, these operations
The reproductive capacity of fish is added somewhat to, but since influence factor is too many, it is complicated for operation, aquaculture cost is increased, and
It is smaller for outdoor breeding repeatability.The present invention is for male fish reproduction ability is increased, using zebra fish as object, in milter
Special high expression tesk1 gene, discovery can increase the genital gland indices of milter in gonadal tissue with conspicuousness, increase it and produce essence amount,
The purpose for improving male zebra fish fecundity is realized, and is expected to extend to economic fish.
Detailed description of the invention
Fig. 1 is the method stream that special cance high-expression gene increases milter reproductive capacity in gonadal tissue provided in an embodiment of the present invention
Cheng Tu.
Fig. 2 is transgene carrier schematic diagram provided in an embodiment of the present invention.
Fig. 3 is the expression schematic diagram of Tesk1 gene in zebra fish different tissues provided in an embodiment of the present invention.
Fig. 4 is that the tesk1 gene male zebra fish genital gland indices provided in an embodiment of the present invention that turn dramatically increase schematic diagram.
Fig. 5 be it is provided in an embodiment of the present invention turn tesk1 gene male zebra fish and wild zebra fish gonadal tissue slice
Observation schematic；
In figure: (a) turning tesk1 gene male zebra fish；(b) wild control.
Specific embodiment
In order to make the objectives, technical solutions, and advantages of the present invention clearer, with reference to embodiments, to the present invention
It is further elaborated.It should be appreciated that the specific embodiments described herein are merely illustrative of the present invention, it is not used to
Limit the present invention.
In view of the problems of the existing technology, the present invention provides cance high-expression genes special in a kind of gonadal tissue to increase hero
The method of fish reproductive capacity, is with reference to the accompanying drawing explained in detail the present invention.
As shown in Figure 1, special cance high-expression gene increases milter reproductive capacity in gonadal tissue provided in an embodiment of the present invention
Method the following steps are included:
S101: extraction gonadal tissue RNA- reverse transcription is cDNA, is to draw with tesk1-F/tesk1-R using cDNA as template
Object carries out PCR amplification, the clone of tesk1 gene with high fidelity enzyme pfu；
S102: used carrier be the plasmid vector containing 3 ' UTR of kop promoter and nanos, kop promoter and
Nanos3 ' UTR can specifically expressing be inserted tesk1 gene by NcoI and BseAI digestion in reproduction cell by target gene
Enter between kop promoter and nanos3 ' UTR, forms transgene carrier；
S103: microinjection obtains genetically engineered fish.
Below with reference to experiment, technical scheme of the present invention will be further described.
One, experimental procedure:
1, the clone (NCBI sequence number XM_678756.5) of tesk1 gene
Extraction gonadal tissue RNA- reverse transcription is cDNA, using cDNA as template, using tesk1-F/tesk1-R as primer, with
High fidelity enzyme pfu carries out PCR amplification, product overall length 2034bp；
SEQ ID NO:1
tesk1-F 5’-CACCCATGG(underscore is NcoI and its protection alkali to GTTGCGTTAAAGTGACCCGCT-3 '
Base)；
SEQ ID NO:2；
tesk1-R 5’-TTCTCCGGA(underscore is BseAI and its protection alkali to GAGATGTCGCAGTGGTCGTC-3 '
Base).
2, used carrier is the plasmid vector containing 3 ' UTR of kop promoter and nanos, kop promoter and nanos3 '
UTR can specifically expressing be opened tesk1 gene insertion kop by NcoI and BseAI digestion in reproduction cell by target gene
Between mover and nanos3 ' UTR, transgene carrier is formed, is Tol2-kop-tesk1-nanos3 ' containing main element
UTR-Tol2, as shown in Figure 2:
3, microinjection obtains genetically engineered fish, as follows:
(1) before microinjection, the transgene carrier processing is carried out comprising go toxicity processing and anti-series connection processing；
(2) Tol2 transposase mRNA is synthesized in vitro, and the specific method is as follows:
Firstly, 396 carriers that will have transposase gene, are linearized with I restriction enzyme of Not, it is then external with SP6
Transcript reagent box is transcribed in vitro；
Secondly, carrying out removal and the reaction terminating of DNA profiling；
Finally, purifying；
(3) treated Plasmid DNA and the Tol2 transposase of the transgene carrier described in zebrafish embryo microinjection
mRNA；
(4) using the zebrafish embryo after microinjection as P0 generation, after the P0 generation cultivation sexal maturity, by with wild spot
Horse raun test cross obtains Offspring F1 generation, obtains homozygosis F2 generation using selfing after the F1 generation positive individuals are raised to sexal maturity.
Two, result:
1, expression of the tesk1 gene in zebra fish different tissues
The expression of Tesk1 gene in zebra fish different tissues, as shown in Figure 3；
Main method: extracting zebra fish different tissues total serum IgE, and reverse transcription cDNA utilizes primer as template: RT-
Tesk1-F:5 '-GCTGCCATTGGTTGCTTATT-3 '；
RT-tesk1-R:5 '-GTGGACTTTGAAACGGCAAT-3 '；
60 degree of annealing temperature, length 154bp, agents useful for same is TAKARA (SYBR)
Using odc as the real-time fluorescence quantitative PCR of reference gene
Primer 5 software design of Primer Premier or (5 ' -3 '):
RT-odc-F:5 '-ACACTATGACGGCTTGCACCG-3 '
RT-odc-R:5 '-CCCACTGACTGCACGATCTGG-3 '
Reaction system is as follows: (template is that cDNA stoste dilutes 5 times)
Reaction condition is as follows: 95 DEG C, 3min；(94 DEG C, 15s；60 DEG C, 15s；72 DEG C, 45s；40 circulations).
2, p0 is for transgenic zebrafish genital gland indices
As shown in figure 4, genital gland indices measure: after positive zebra fish sexal maturity, solution takes its gonadal tissue, weighing, property
The ratio between gland weight and weight are genital gland indices (wild and transgenosis respectively has detected 10 zebra fish).
3, turn tesk1 gene male zebra fish and wild zebra fish gonadal tissue sections observation, as shown in Figure 5；It can from Fig. 5
To find out that turning small phyllocyst in tesk1 gene male zebra fish gonadal tissue significantly increases, there is the sperm of a large amount of maturations to be present in small
In phyllocyst, and the small phyllocyst of wild zebra fish is smaller, wherein only less sperm.
Operating method is as follows: taking the 4 DEG C of fixations in 4%PFA solution of zebra fish gonadal tissue to stay overnight, then by gonadal tissue
It is embedded in 1.5% agar, is dehydrated in following solution, permeabilization, saturating wax.Specific step is as follows: 50% 6~8h of ethyl alcohol,
75% 6~8h of ethyl alcohol, 85% 3~5h of ethyl alcohol, 95% 1~2h of ethyl alcohol, 95% 1~2h of ethyl alcohol, 100% ethyl alcohol, I 1h, 100% second
II 1h of alcohol, 100% ethyl alcohol/dimethylbenzene 40min, dimethylbenzene 40min, dimethylbenzene 30min, dimethylbenzene/paraffin 30min, paraffin (60
DEG C) 1h, paraffin (60 DEG C) 1h.
By above-mentioned processed tissue, Yao Jinhang paraffin embedding can be carried out paraffin section after paraffin, thick
Spend 6-8um.Sections stained with hematoxylin-Yihong (Hematoxylin and Eosin, HE) dyeing, staining procedure are as follows:
(1) dimethylbenzene is invaded twice, each 15min.
(2) dehydrated alcohol is invaded twice, each 5min.
(3) 80% ethyl alcohol, 5min.
(4) distilled water, 5min.
(5) PBS is washed 3 times, each 5min.
(6) hematoxylin dyes 8-10min.
(7) after flowing water washes away hematoxylin, 1% ethanol solution hydrochloride invades 5-10s.
(8) flowing water is stained with wash after, with distillation wash 5s.
(9) 0.5% eosin stains 5min.
(10) 80% ethyl alcohol, which are stained with, washes 1-2s, and 95% ethyl alcohol, which is stained with, to be washed twice, each 5s.
(11) dehydrated alcohol twice, each 5min.
(12) carbolic acid dimethylbenzene, 5min.
(13) dimethylbenzene 3 times, each 2min.
(13) dimethylbenzene is wiped away, carries out mounting processing with resin at once, after resin air-dries, can seen under microscope
It examines.
The foregoing is merely illustrative of the preferred embodiments of the present invention, is not intended to limit the invention, all in essence of the invention
Made any modifications, equivalent replacements, and improvements etc., should all be included in the protection scope of the present invention within mind and principle.
Sequence table
<110>Hunan University of Arts and Science
<120>special cance high-expression gene increases the method for milter reproductive capacity in a kind of gonadal tissue
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 30
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 1
cacccatggg ttgcgttaaa gtgacccgct 30
<210> 2
<211> 29
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 2
ttctccggag agatgtcgca gtggtcgtc 29
Claims (5)
1. special cance high-expression gene increases the method for milter reproductive capacity in a kind of gonadal tissue, which is characterized in that the sexual gland group
In knitting special cance high-expression gene increase milter reproductive capacity method the following steps are included:
The first step extracts gonadal tissue RNA, reverse transcription cDNA, using cDNA as template, using tesk1-F/tesk1-R as primer,
PCR amplification, the clone of tesk1 gene are carried out with high fidelity enzyme pfu；
Second step, used carrier are the plasmid vector containing 3 ' UTR of kop promoter and nanos, kop promoter and nanos3 '
UTR can specifically expressing be opened tesk1 gene insertion kop by NcoI and BseAI digestion in reproduction cell by target gene
Between mover and nanos3 ' UTR, transgene carrier is formed；
Third step, microinjection obtain genetically engineered fish.
2. special cance high-expression gene increases the method for milter reproductive capacity in gonadal tissue as described in claim 1, feature exists
In the primer of the first step are as follows:
tesk1-F 5'-CACCCATGGGTTGCGTTAAAGTGACCCGCT-3'；
tesk1-R 5’-TTCTCCGGAGAGATGTCGCAGTGGTCGTC-3’。
3. special cance high-expression gene increases the method for milter reproductive capacity in gonadal tissue as described in claim 1, feature exists
In the transgene carrier of the second step is Tol2-kop-tesk1-nanos3 ' UTR-Tol2 containing main element.
4. special cance high-expression gene increases the method for milter reproductive capacity in gonadal tissue as described in claim 1, feature exists
In the microinjection of the third step obtains genetically engineered fish method and includes:
(1) before microinjection, the transgene carrier processing is carried out comprising go toxicity processing and anti-series connection processing；
(2) Tol2 transposase mRNA is synthesized in vitro；
(3) treated Plasmid DNA and the Tol2 transposase mRNA of the transgene carrier described in zebrafish embryo microinjection；
(4) using the zebrafish embryo after microinjection as P0 generation, after the P0 generation cultivation sexal maturity, by female with wild zebra
Fish test cross obtains Offspring F1 generation, obtains homozygosis F2 generation using selfing after the F1 generation positive individuals are raised to sexal maturity.
5. special cance high-expression gene increases the method for milter reproductive capacity in gonadal tissue as claimed in claim 4, feature exists
In the external synthesis Tol2 transposase mRNA method includes:
Firstly, 396 carriers that will have transposase gene, are linearized with I restriction enzyme of Not, are then turned in vitro with SP6
Record kit is transcribed in vitro；
Secondly, carrying out removal and the reaction terminating of DNA profiling；
Finally, purifying.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN112011569A (en) * | 2020-09-16 | 2020-12-01 | 华南农业大学 | Method for artificially accelerating maturation of grouper spermary |
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| CN103224955A (en) * | 2013-05-09 | 2013-07-31 | 中国科学院水生生物研究所 | Vector for efficiently labeling zebra fish PGC, and preparation method and use of transgenic fish |
| CN105274141A (en) * | 2015-11-20 | 2016-01-27 | 中国科学院水生生物研究所 | Transgenic vector for target mutation of primordial germ cells, method for preparing transgenic vector and application thereof |
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2019
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| CN103224955A (en) * | 2013-05-09 | 2013-07-31 | 中国科学院水生生物研究所 | Vector for efficiently labeling zebra fish PGC, and preparation method and use of transgenic fish |
| CN105274141A (en) * | 2015-11-20 | 2016-01-27 | 中国科学院水生生物研究所 | Transgenic vector for target mutation of primordial germ cells, method for preparing transgenic vector and application thereof |
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| LIANG MENG等: "Cloning and Characterization of tesk1, a Novel Spermatogenesis-Related Gene, in the Tongue Sole(Cynoglossus semilaevis)", 《PLOS ONE》 * |
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| CN112011569A (en) * | 2020-09-16 | 2020-12-01 | 华南农业大学 | Method for artificially accelerating maturation of grouper spermary |
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