CN104745628A - Method for sex induction of hermaphrodite shrimps - Google Patents
Method for sex induction of hermaphrodite shrimps Download PDFInfo
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Abstract
The invention aims to provide a method for sex induction of hermaphrodite shrimps. According to the method, a target gene in macrobrachium is silenced by using an RNA interference technology to realize sex induction of macrobrachium rosenbergii, wherein the nucleotide sequence of the target gene is SEQ ID NO:1. By virtue of the induction technology provided by the invention, a macrobrachium individual has sex reversion and becomes hermaphrodite macrobrachium rosenbergii, and the developments of a female reproductive system and a male reproductive system are realized in the body of the hermaphrodite macrobrachium rosenbergii, so that a set of effective macrobrachium rosenbergii sex induction technology is established. Meanwhile, by using the hermaphrodite shrimps cultured by the method provided by the invention, a good scientific research material and an animal sample can be provided for sex determination, sex differentiation and researches on reproduction and fertilization of shrimps and crabs.
Description
Technical field
The invention belongs to aquatic animal sex phenotype technology, be specifically related to a kind of sex induction technology of shrimps, particularly relate to a kind of inductive technology having the telianthus Macrobrachium rosenbergii of female repro ductive system and male reproductive system concurrently.
Background technology
Telianthus refers to female proterties and male property all significantly phenomenon in an animal body, and dioecism refers to allogenic animal gamogenesis organ raw phenomenon in Different Individual respectively.The hermaphroditism of occurring in nature comes across lower animal more.Macrobrachium rosenbergii (Macrobrachium rosenbergii) is a kind of large-scale fresh water shrimp, and it has that growth is fast, feeding habits wide, meat nutrition is good and the advantage such as the culture-cycle is short.The Macrobrachium rosenbergii of occurring in nature is that dioecism is biological, and its female and male reproductive organ exists in female shrimp and male shrimp individuality respectively.There is male reproductive system in the carapace of male Macrobrachium rosenbergii, comprise spermary, vas deferens and ampulla three part, there is a pair arrenotoky hole at the 5th step base portion simultaneously.There is female repro ductive system in the carapace of female Macrobrachium rosenbergii, comprise ovary and uterine tube two portions, there is a pair female reproduction hole at the 3rd step base portion simultaneously.There is male and female two state property in the process of growth of Macrobrachium rosenbergii, if make good use of this sex character advantage, can greatly improve aquaculture benefit.But at present, shrimp crab Animal Sex determine and sexual differentiation, reproduction and fertilization molecular mechanism know little about it.
Androgenic gland is the distinctive a kind of incretory gland of crustacean, and the androgenic gland hormone of its secretion has regulation and control sexual differentiation and maintains the function of male secondary sex characteristics.The sex phenotype research of shrimps animal mainly concentrates on the application and development of androgenic gland body.On the one hand, the sex of male can be reversed by removing androgenic gland.But androgenic gland volume is small, not easily remove completely, sex reverse ratio is not high, and success ratio is lower.On the other hand, the method for transplanting androgenic gland or injection androgenic gland body extract is adopted, the physiological function of research androgenic gland in regulation and control sex phenotype.But androgenic gland sample body obtains not easily, the extract of trace not easily carries out the application of large-scale industry.
At present, from the cDNA subtractive library of Macrobrachium rosenbergii androgenic gland, screen the gene of specifically expressing in acquisition two androgenic gland bodies, inferred that it may be relevant with the sex phenotype of Macrobrachium rosenbergii.Present stage, just resolving androgenic gland body specific gene molecular structure, and utilizing molecular biotechnology and means, carrying out the study mechanism of androgenic gland specific gene in pond crayfish sex phenotype further.
Summary of the invention
The object of the invention is for the deficiencies in the prior art, a kind of sex induction technology of Macrobrachium rosenbergii is provided, for the gene of androgenic gland specifically expressing, utilize RNA perturbation technique, make goal gene in pond crayfish body reticent, realize the sex induction to Macrobrachium rosenbergii.By inductive technology of the present invention, impel pond crayfish individuality that sex occurs and reverse, form hermaphroditic Macrobrachium rosenbergii, in its body, have the growth of female repro ductive system and male reproductive system concurrently, set up a set of effective Macrobrachium rosenbergii sex induction technology.Meanwhile, the telianthus shrimp utilizing the present invention to cultivate acquisition can provide good scientific research material and animal specimen for the sex determination of shrimp crab animal and sexual differentiation, reproduction and fertilization research.
Method of the present invention, comprises following step:
1) double-stranded RNA sample preparation
First, extracting Male Reproductive System of Macrobrachium Rosenberch ampulla tissue RNA sample, reverse transcription synthesis cDNA; Take cDNA sequence as template, carry out pcr amplification, clone obtains the special goal gene fragment of androgenic gland body; Then, utilize dsrna expression vector, synthesis and preparative double-stranded RNA sample;
Wherein the Nucleotide of goal gene includes:
1) sequence is the Nucleotide of SEQ ID NO:1,
2) with 1) in nucleic acid molecule there is more than 85% homology, and have 1) nucleic acid of nucleic acid molecule effect.
Above-mentioned 2) homology in, is preferably more than 90%;
Above-mentioned steps 1) primer sequence of pcr amplification is SEQ ID NO:2 and SEQ ID NO:3;
2) RNA interference
Utilize microinjection, throw something and feed or the various ways such as immersion, object double-stranded RNA sample is sent in pond crayfish body; Choose male seedling or juvenile prawn (pond crayfish of sexual differentiation phase) carries out RNA interference, by step 1) double-stranded RNA sample prepared sends in pond crayfish body and carries out RNA interference, the nucleic acid level of goal gene is made to reduce by more than 80%, realize the efficient silence of goal gene, thus there is sex reverse in induced male Macrobrachium rosenbergii in process of growth, form the telianthus shrimp of Macrobrachium rosenbergii, in its body, have the growth of female repro ductive system and male reproductive system concurrently.
The flag check of telianthus shrimp:
Mode of appearance: described telianthus Macrobrachium rosenbergii embodies female and male outward appearance sex character on mode of appearance simultaneously, a pair arrenotoky hole is there is at the 5th step base portion of its carapace outside of belly, there is female reproduction hole at the 3rd step base portion simultaneously, namely same individuality has female reproduction hole and arrenotoky hole concurrently.
The composition of reproductive system: the reproductive system of telianthus shrimp has female and arrenotokous system composition and development characteristics concurrently simultaneously.There is male reproductive system in the cephalothorax of male Macrobrachium rosenbergii individuality, comprises spermary, vas deferens and ampulla three part.There is female repro ductive system in the cephalothorax of female Macrobrachium rosenbergii individuality, comprises ovary and uterine tube two portions.Telianthus shrimp has female and male reproductive system concurrently to be existed and grows, and the spermary in female repro ductive system in ovary tissue and male reproductive system has organized good growth.
The growth of spermary and ovary: according to different developmental phases, ovary tissue outward appearance can present oyster white, faint yellow, yellow etc., has formation and the accumulation of vitellin(Vt) (former) in ovary.Utilize paraffin section technology, in conjunction with Hematorylin and eosin stains method, clearly under the microscope can see the ovum and yolk protein that there is different developmental phases in Macrobrachium rosenbergii ovary.In addition, spermary and vas deferens tissue appearance present oyster white, structural integrity, physically well develop.Multiple seminal vesicle vesicle and a large amount of umbrella spermatid of fully-developed is there is in basis of microscopic observation to spermary.
The inventive method captures the technical barrier run in current aquaculture field shrimps sex induction and study on regulation, by the mode that RNA perturbation technique and experimentation on animals combine, carry out directed sex control, induce the individual sex of male shrimp to reverse, form telianthus shrimp.The present invention not only provides a kind of shrimps sex induction technology, also for the sex determination of shrimp crab animal and sexual differentiation, reproduction and fertilization research provide good scientific research material and animal specimen.Working method of the present invention has originality, and implementation step is clear.
Accompanying drawing explanation
Fig. 1: the double-stranded RNA sample preparation of gender-specific genes;
Fig. 2: the RNA jamming effectiveness of gender-specific genes measures;
Fig. 3: the mode of appearance of Macrobrachium rosenbergii telianthus shrimp and gonopore distribution;
Fig. 4: the reproductive system internal anatomy of Macrobrachium rosenbergii telianthus shrimp;
Fig. 5: the double-stranded RNA sample preparation figure of gender-specific genes;
Fig. 6: the RNA jamming effectiveness of gender-specific genes measures figure.
Embodiment 1
The gene that the present invention obtains two sex specifically expressings by screening from Macrobrachium rosenbergii androgenic gland cDNA subtractive library carries out RNA perturbation technique and Macrobrachium rosenbergii experimentation on animals, in pond crayfish body, realize the efficient silence of goal gene, successfully carry out telianthus shrimp sex induction.
Below in conjunction with embodiment, the present invention is described in detail.
Embodiment 1
Prepared by step (1) double-stranded RNA sample
According to the cDNA sequence of the gender-specific genes of screening, design the forward primer with restriction enzyme site and reverse primer, RNAi-PF:5 '-GC
tCTAGAgTGTGTGTTGTTCTGCTCACTCGT-3 ') (underscore place is XbaI enzyme cutting site SEQ ID NO:2) and RNAi-PR (SEQ ID NO:3:5 '-C
gAGCTCgCAGCATTCCTCCCTTGGACTTCT-3 ') (underscore place is SacI restriction enzyme site), for PCR reaction amplification, obtain goal gene sequence as described below:
Gtgtgtgttgttctgctcactcgtagcatcgcttctccctcaaccttcttcgagctatgagatcgaatgcctctccgttgactttgactgcggcgacataacgaacacccttgcctccgtctgcctgagacacaacaactacatcaacccaggacccacctacgtttccaaagagcgacgatctgctgacatctataccgttccttctacgaagtctccatcgctcgcccacccgagagctacccacttgaccatggctgacgaagaaactcagaaggtatctaaggtggaggaggagattcagcacatgacgctgagcagggaagaagcgaacaatatgctgcattcgaagcgtcgcttccggagggacagcgtgaggagaagtccaagggaggaatgctgc(SEQ ID NO:1)
Utilize dsrna expression vector, synthesis and preparative double-stranded RNA sample, the double-stranded RNA that purifying obtains is through electrophoresis detection and preserve stand-by after quantitative analysis.(Fig. 1)
Step (2) .RNA disturbs
Choose physique stalwartness, male seedling (Magna zoea larva phase) that appendage is complete or juvenile prawn (sexual differentiation period) from Macrobrachium rosenbergii breeding cultivation base and carry out RNA interference.In the present embodiment, utilize the method for microinjection, through carapace and belly intersection, the 5th step base portion peritonaeum hemostasis 5-10 μ g double-stranded RNA/only, put back in cement pit and continue to raise.Again inject same double-stranded RNA sample every other month in same group shrimp body, maintain the validity of RNAi, injection cycle is 3 months (detection of Fig. 2 RNA jamming effectiveness).In experimentation on animals process, various ways (microinjection, throw something and feed or soak) can be utilized to deliver in pond crayfish body by the RNA disturbed specimen of gender-specific genes, realize goal gene silence, Fig. 2 result shows the RNA jamming effectiveness of gender-specific genes very high, meets the requirement of interference.
Step (3). the feature of telianthus shrimp
Macrobrachium rosenbergii after gender-specific genes RNA disturbs is raised in indoor or outdoors waters, and body grows to 3-5cm, after second sex feature has been broken up, arrests and identifies hermaphroditic Macrobrachium rosenbergii.The Macrobrachium rosenbergii of occurring in nature is dioecious biology, and appearance is that female shrimp exists a pair female reproduction hole at the 3rd step base portion of its carapace, and male shrimp exists a pair arrenotoky hole at the 5th step base portion of carapace.
The mode of appearance of telianthus shrimp in the present embodiment: described telianthus Macrobrachium rosenbergii has female and male sex character concurrently on mode of appearance, a pair arrenotoky hole is there is at the 5th step base portion of its carapace outside of belly, there is female reproduction hole at the 3rd step base portion simultaneously, namely same individuality has female reproduction hole and arrenotoky hole concurrently.
The composition of reproductive system: the reproductive system of telianthus shrimp has female and arrenotokous system composition and development characteristics concurrently simultaneously.Occurring in nature, there is male reproductive system in the cephalothorax of male Macrobrachium rosenbergii individuality, comprises spermary, vas deferens and ampulla three part.There is female repro ductive system in the cephalothorax of female Macrobrachium rosenbergii individuality, comprises ovary and uterine tube two portions.Telianthus shrimp has female and male reproductive system concurrently to be existed and grows, and the spermary in female repro ductive system in ovary tissue and male reproductive system has organized good growth.According to different developmental phases, ovary tissue outward appearance can present oyster white, faint yellow, yellow etc., has formation and the accumulation of vitellin(Vt) (former) in ovary.And spermary and vas deferens tissue appearance present oyster white, structural integrity.(mode of appearance of Fig. 3 Macrobrachium rosenbergii telianthus shrimp and gonopore distribution)
The growth of spermary and ovary: utilize paraffin section technology, in conjunction with Hematorylin and eosin stains method, clearly under the microscope the ovum (ovocyte, ovogonium and ovum), sustenticular cell and the yolk protein that there is different developmental phases in Macrobrachium rosenbergii ovary can be seen.And in spermary, there is multiple seminal vesicle vesicle, there is a large amount of developmental spermatid and fully-developed umbrella sperm in seminal vesicle vesicle, show that the ovary of telianthus shrimp and spermary have good reproduction and Sperm fertility.(the reproductive system internal anatomy of Fig. 4 Macrobrachium rosenbergii telianthus shrimp)
The telianthus Macrobrachium rosenbergii related in the present embodiment, has the growth of female repro ductive system and male reproductive system concurrently in body, for the sex determination of shrimp crab animal and sexual differentiation, reproduction and fertilization research provide good scientific research material and animal specimen.Simultaneously, present embodiments provide the mode of the example evidence of capturing aquaculture field shrimps sex induction technical barrier-combined by RNA perturbation technique and experimentation on animals, induce the individual sex of male shrimp to reverse, carry out directed sex control, form the feasibility of telianthus shrimp.In addition, the present embodiment also proves that the gender-specific genes on androgenic gland plays keying action in pond crayfish sex phenotype process, can be applied to sex phenotype and the fine-variety breeding field of Macrobrachium rosenbergii.
Embodiment 2
Macrobrachium rosenbergii ampulla peptide gene (TAP) is the gene that screening obtains another androgenic gland body specifically expressing from Macrobrachium rosenbergii androgenic gland cDNA subtractive library, and its gene complete sequence can obtain (sequence number FJ595507) from gene pool.
With ampulla peptidyl because example, will elaborate by RNA perturbation technique and Macrobrachium rosenbergii experimentation on animals, in pond crayfish body, realize the efficient silence of goal gene, carry out the embodiment of telianthus shrimp sex induction in the present embodiment.
Below in conjunction with embodiment, the present invention is described in detail.
Prepared by step (1) double-stranded RNA sample
According to the cDNA sequence of ampulla peptide gene, design the forward primer with restriction enzyme site and reverse primer, RNAi-PF (SEQ ID NO:4:5 '-GC
tCTAGAcTTTTCTTGTTGATGGCACT-3 ') (underscore place is XbaI enzyme cutting site) and RNAi-PR (SEQ ID NO:5:5 '-G
gAATTCcATTCCAGGTTCATGTCGCA-3 ') (underscore place is EcoRI restriction enzyme site), for PCR reaction amplification, obtain goal gene sequence as described below:
cttttcttgttgatggcactggtctacgtgtccggtcaacagagcgtctatgacaagagtgaagaagaacaaatagagattcttggtcctctgctcagaaaattgctgatgcccagaccacgtcccgtctaccagatccaccagcttctgagggtgcacgtgcagaaatgctgcaactcgcagcctatgatggactgctgcacacctgctctctgctgcgacatgaacctggaatg(SEQ IDNO:6)
Utilize dsrna expression vector, synthesis and preparative double-stranded RNA sample, the double-stranded RNA that purifying obtains is through electrophoresis detection and preserve stand-by after quantitative analysis.(double-stranded RNA sample of Fig. 5 ampulla peptide gene)
Step (2) .RNA disturbs
Choose physique stalwartness, male seedling (Magna zoea larva phase) that appendage is complete or juvenile prawn (sexual differentiation period) from Macrobrachium rosenbergii breeding cultivation base and carry out RNA interference.In the present embodiment, utilize the method for microinjection, through carapace and belly intersection, the 5th step base portion peritonaeum hemostasis 5-10 μ g double-stranded RNA/only, put back in cement pit and continue to raise.Again inject same double-stranded RNA sample every other month to in group shrimp body, maintain the validity of RNAi, injection cycle is 3 months (the RNA jamming effectiveness of Fig. 6 ampulla peptide gene measures).In experimentation on animals process, various ways (microinjection, throw something and feed or soak) can be utilized to be delivered in pond crayfish body by the RNA disturbed specimen of ampulla peptide gene, realize goal gene silence.
Step (3). the screening of telianthus shrimp
Macrobrachium rosenbergii after ampulla peptide gene RNA disturbs is raised in indoor or outdoors waters, and body grows to 3-5cm, after second sex feature has been broken up, arrests and identifies hermaphroditic Macrobrachium rosenbergii.The Macrobrachium rosenbergii of occurring in nature is dioecious biology, and appearance is that female shrimp exists a pair female reproduction hole at the 3rd step base portion of its carapace, and male shrimp exists a pair arrenotoky hole at the 5th step base portion of carapace.
In the present embodiment, the RNA jamming effectiveness of ampulla peptide gene is fine, and nucleic acid expression level declines and reaches more than 90%, realizes the efficient silence of goal gene.But in experimentation on animals, pond crayfish is still dioecism, has female and male sex character respectively, does not screen and obtain telianthus shrimp.This experimental result illustrates, ampulla peptide gene does not affect the conversion of pond crayfish male and female sex in the disappearance that sex has just broken up period, is not the key gene of Macrobrachium rosenbergii sex phenotype.
Functional verification for shrimps sex phenotype candidate gene is provided simple and feasible technology platform by enforcement of the present invention.The physiological function of Macrobrachium rosenbergii androgenic gland body specific expression gene in sex phenotype process can be verified by technical scheme provided by the invention and operating process.In addition, utilize Macrobrachium rosenbergii sex induction technology provided by the invention, in conjunction with the information of Macrobrachium rosenbergii sex phenotype key gene, the period just can broken up in juvenile prawn sex carries out sex induction operation, make goal gene reticent and induce sex to reverse, cultivate telianthus Macrobrachium rosenbergii, as the experiment material that basic scientific research and industry are applied.
Claims (6)
1. a sex induction method for telianthus shrimp, is characterized in that, described method comprises the steps:
1) double-stranded RNA sample preparation
First, extracting Male Reproductive System of Macrobrachium Rosenberch ampulla tissue RNA sample, reverse transcription synthesis cDNA; Take cDNA sequence as template, carry out pcr amplification, clone obtains the special goal gene fragment of androgenic gland body; Then, utilize dsrna expression vector, synthesis and preparative double-stranded RNA sample;
2) RNA interference
By step 1) double-stranded RNA sample prepared sends in pond crayfish body and carries out RNA interference, the nucleic acid level of goal gene is reduced, realize the silence of goal gene, there is sex and reverse in induced male Macrobrachium rosenbergii, form the telianthus shrimp of Macrobrachium rosenbergii in process of growth.
2. the method for claim 1, is characterized in that, described goal gene includes:
1) sequence is the Nucleotide of SEQ ID NO:1,
2) with 1) in nucleic acid molecule there is more than 85% homology, and have 1) nucleic acid of nucleic acid molecule effect.
3. method as claimed in claim 2, it is characterized in that, described homology is more than 90%.
4. the method for claim 1, is characterized in that, described step 1) primer sequence of pcr amplification is SEQ ID NO:2 and SEQ ID NO:3.
5. the method for claim 1, is characterized in that, described by step 1) double-stranded RNA sample prepared sends in pond crayfish body, is by microinjection, throws something and feeds or immersion way.
6. the method for claim 1, is characterized in that, described telianthus shrimp has following feature:
Mode of appearance: described telianthus Macrobrachium rosenbergii embodies female and male outward appearance sex character on mode of appearance simultaneously, a pair arrenotoky hole is there is at the 5th step base portion of its carapace outside of belly, there is female reproduction hole at the 3rd step base portion simultaneously, namely same individuality has female reproduction hole and arrenotoky hole concurrently;
The composition of reproductive system: the reproductive system of telianthus shrimp has female and arrenotokous system composition and development characteristics concurrently simultaneously; There is male reproductive system in the cephalothorax of male Macrobrachium rosenbergii individuality, comprises spermary, vas deferens and ampulla three part; There is female repro ductive system in the cephalothorax of female Macrobrachium rosenbergii individuality, comprises ovary and uterine tube two portions; Telianthus shrimp has female and male reproductive system concurrently to be existed and grows, and the spermary in female repro ductive system in ovary tissue and male reproductive system has organized good growth;
The growth of spermary and ovary: according to different developmental phases, ovary tissue outward appearance can present oyster white, faint yellow, yellow etc., has formation and the accumulation of vitellin(Vt) in ovary; Utilize paraffin section technology, in conjunction with Hematorylin and eosin stains method, clearly under the microscope can see the ovum and yolk protein that there is different developmental phases in Macrobrachium rosenbergii ovary; In addition, spermary and vas deferens tissue appearance present oyster white, structural integrity, physically well develop; Multiple seminal vesicle vesicle and a large amount of umbrella spermatid of fully-developed is there is in basis of microscopic observation to spermary.
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| CN105647973A (en) * | 2016-01-13 | 2016-06-08 | 浙江万里学院 | Male/female sex regulation method of Macrobrachium rosenbergii |
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| CN113046372A (en) * | 2021-03-25 | 2021-06-29 | 中国水产科学研究院淡水渔业研究中心 | Freshwater shrimp PDHE1 gene and encoding protein and application thereof |
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Cited By (11)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN105647973A (en) * | 2016-01-13 | 2016-06-08 | 浙江万里学院 | Male/female sex regulation method of Macrobrachium rosenbergii |
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| CN111172189B (en) * | 2018-11-13 | 2023-06-13 | 上海海洋大学 | Establishment method of Sissy medaka model for simulating human sperm ovarian syndrome |
| CN110714009A (en) * | 2019-11-20 | 2020-01-21 | 华中农业大学 | Male macrobrachium rosenbergii sex-converted siRNA-IR sequence and application thereof |
| CN110714009B (en) * | 2019-11-20 | 2021-04-06 | 华中农业大学 | Male macrobrachium rosenbergii sex-converted siRNA-IR sequence and application thereof |
| CN111118130A (en) * | 2020-01-15 | 2020-05-08 | 深圳市泽龙生物技术有限公司 | Method for rapidly identifying sex of macrobrachium rosenbergii |
| CN111118130B (en) * | 2020-01-15 | 2023-04-07 | 深圳市泽龙生物技术有限公司 | Method for rapidly identifying sex of macrobrachium rosenbergii |
| CN113046372A (en) * | 2021-03-25 | 2021-06-29 | 中国水产科学研究院淡水渔业研究中心 | Freshwater shrimp PDHE1 gene and encoding protein and application thereof |
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