CN106478794A - A kind of artemia masculinizing factor and its application - Google Patents
A kind of artemia masculinizing factor and its application Download PDFInfo
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- CN106478794A CN106478794A CN201610946739.0A CN201610946739A CN106478794A CN 106478794 A CN106478794 A CN 106478794A CN 201610946739 A CN201610946739 A CN 201610946739A CN 106478794 A CN106478794 A CN 106478794A
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- artemia
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- masculinizing
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Abstract
The present invention provides a kind of artemia masculinizing factor and its application, particularly as being a kind of nucleic acid molecules of coding artemia masculine gene and polypeptide, and provides a kind of RNA interference method based on masculine gene silencing, realizes the production technology of artemia high female colony.The artemia masculinizing factor that the present invention provides, the aminoacid sequence of the albumen of its coding is SEQ ID NO：2.The nucleic acid based on masculine gene for the present invention and protein sequence information, the biotechnology such as integrated use RNA perturbation technique and gene expression regulation, realize the effective reticence of artemia class masculine gene, and then illustrate masculinizing factor in the function in terms of sex phenotype and its production operation practice in the induction of high female colony in animal horizontal bedding, masculine gene is provided in regulation and control animal male and female sex and to cultivate application, experimental considerations that will be splendid to Animal Sex and field of reproduction offer and skill upgrading in high female descendant.
Description
Technical field
The invention belongs to the technical field of Animal Sex and genital regulating and in particular to a kind of artemia masculinizing factor and its
Application.
Background technology
Artemia is also referred to as saline fairy shrimp, classification belongs to Arthropoda, carapace subphylum, branchiopod guiding principle, Anostraca, saline is rich
Nian Chong section, genus artemia.The artemia breeding cycle is short, and growth is rapid, as a kind of important food organisms and good laboratory animal
Material, is constantly subjected to the extensive attention of people.Artemia has special mode of reproduction, can be divided into bisexual Artemia and lonely female life
Grow artemia two big class.Meanwhile, there are two kinds of growth modes in artemia in growth course：One kind is ovoviviparity, and that is, female artemia is handed over
Join after fertilization, germ cell develops into naupliuss in female egg capsule, and discharges from parent.In normal living condition
Down or under laboratory culture conditions, artemia is all that ovoviviparity is developed with this approach.Artemia another kind growth mode be
Oviparity, that is, form the coated ovum of tunica.About 4000 cells in this ovum, are coated on one layer of polar molecule impervious
In shell, form so-called resting egg.Unique mode of reproduction and different growth modes that artemia has because of it, are increasingly becoming non-mould
Formula Animal Sex and the good experimental material of reproductive study.
Sex determination is the result that spore selects, and is also the basic feature of biological life activity.The biological property of both sexes
Individual normal development Jue Ding be not only with differentiation and existence is indispensable, be also the material base that race's procreation continues.
There is complexity and multiformity in Animal Sex decision mechanism, major endogeneous are the results that sex determining gene cascades interaction regulation and control.
In addition to there is Sex determination related gene on sex chromosome, it has also been found that many important Sex determination of animal on autosome
Exist between related gene, and portion gene and closely cascade interaction, such as sxl, tra, dsx, csd and fem gene etc..In recent years
Come, the Sex Determination Mechanism of model animal achieves certain progress, but to the research of non-mode animal also in the starting stage, but
But increasingly it is taken seriously.
Therefore, by carrying out to the masculine gene in artemia body cloning, the research such as expression characteristic analysis and gene function,
And then illustrate masculinizing factor in the function in terms of sex phenotype and its in the induction of high female colony in animal horizontal bedding
Production operation is put into practice, and provides application in regulation and control animal male and female sex and the high female descendant of cultivation for the masculine gene, will be to dynamic
Physical property is not and field of reproduction research provides splendid experimental considerations and skill upgrading.
Content of the invention
The present invention provides a kind of artemia masculinizing factor and its application, particularly as being a kind of coding artemia masculine gene
Nucleic acid molecules and polypeptide, and a kind of RNA interference method based on masculine gene silencing is provided, realize artemia high female colony
Production technology, illustrates masculine gene and in regulation and control animal male and female sex and cultivates the application in high female descendant.
The present invention provides a kind of artemia masculinizing factor, and the albumen of its coding includes：
1) aminoacid sequence is SEQ ID NO：2 albumen；
2) with SEQ ID NO:2 albumen has at least 70% amino acid sequence identity, and with 1) in protein function
Consistent albumen.
Above-mentioned masculinizing factor, its nucleotides sequence is classified as SEQ ID NO:1；
Another aspect of the present invention provides a kind of double stranded rna molecule lowering artemia masculine gene expression, described double-strand
It is SEQ ID NO that RNA molecule includes positive-sense strand and antisense strand, wherein antisense strand or positive-sense strand:The piece of the nucleotide sequence described in 1
Section, or there is the nucleotide sequence of at least 70% complementation with selected fragment.
Preferred as embodiment, the nucleotide sequence of the described positive-sense strand of double stranded rna molecule or antisense strand is SEQ ID
NO:3；
The present invention also provides a kind of double stranded rna molecule for producing the masculine gene expression that can lower artemia
DNA construct, described DNA construct is used for transcribing above-mentioned double stranded rna molecule.
The present invention also provides a kind of method reducing above-mentioned artemia masculinizing factor expression, is by above-mentioned double-stranded RNA
Molecule converts or is transfected into biological internal；
The present invention further provides a kind of method obtaining female artemia filial generation or the artemia filial generation of false female, it is by reducing
Or the artemia of the above-mentioned masculine gene expression amount of removal carrys out mate and oviposit acquisition as parent；
Described reduction or the above-mentioned masculine gene expression amount of removal, are to be built by introducing above-mentioned double-stranded RNA or DNA
Body is completing；
According to some case study on implementation, introducing described double-stranded RNA or DNA construct is by injection, immersion, percutaneous or feeding
Come to carry out.According to a certain embodiment, described double-stranded RNA or DNA construct are to be applied by injection.
The nucleic acid based on masculine gene for the present invention and protein sequence information, integrated use RNA perturbation technique and gene table
Reach the biotechnology such as regulation and control, realize the effective reticence of artemia class masculine gene, and then illustrate masculine in animal horizontal bedding
The factor, in the function in terms of sex phenotype and its production operation practice in the induction of high female colony, provides masculine gene to exist
Application in regulation and control animal male and female sex and the high female descendant of cultivation, will provide splendid experiment to Animal Sex and field of reproduction
Thinking and skill upgrading.
Brief description
Fig. 1：The amino acid sequence similarity comparison result figure of masculinizing factor zinc finger protein domain；
Fig. 2：The male and female differential expression of masculine gene
Fig. 3：The masculine allelic expression in fetal development period
Fig. 4：The masculine allelic expression of resting egg hatching process
Fig. 5：The RNA disturbed specimen preparation of masculine gene
Fig. 6：The RNA jamming effectiveness of masculine gene measures
Fig. 7：Filial generation male and female statistics after artemia (female diplotene stage) masculine gene silencing
Fig. 8：Filial generation male and female statistics after artemia (adult) masculine gene silencing
Specific embodiment
The method that the present invention provides the masculine silenced gene expression based on artemia, especially by the method for RNA interference.
Generally, RNA interference (RNAi) refers to the sequence specific post transcriptional gene silencing process being mediated by siRNA (siRNA), is
The sequence specific gene silencing phenomenon being induced by double-stranded RNA is referred on a kind of molecular biology, its mechanism is specific by hindering
The translation of gene or transcription carry out inhibition of gene expression.When importing the double-stranded RNA with endogenouss mRNA coding region homology in cell,
This mRNA occurs degraded to lead to silenced gene expression.Therefore, the present invention comprises each can lower arthropod, particularly halogen
The double-stranded RNA of worm masculinizing factor expression, Microrna, antisense oligonucleotide, children purpura nephritis (shRNA) etc..
According to the present invention, rnai agent or RNAi agent are the double-strands being engineered to transcribe in target cell double-stranded RNA
RNA or DNA construct, this double-stranded RNA comprises and SEQID NO：1 nucleotide sequence or with its homologue or fragment or part
The RNA chain of at least 70% complementation.
It should be understood that the dsRNA of the present invention can be chemically modified.Can be used for the modification of the present invention
The oligonucleotide being embodied as including the skeleton modified or non-natural nucleoside key of dsRNA compound.In addition, according to
The nucleotide of the modification of the present invention can also comprise one or more substituted sugar moieties.In addition, the oligonucleotide of the present invention can
It is chemically attached to strengthen one or more parts of activity, cell distribution or cellular uptake or the conjugate of oligonucleotide.These
Part or conjugate may include the conjugation group being covalently bonded in functional group such as primary hydroxyl or secondary hydroxyl.The conjugation of the present invention
Group includes intercalator, reporter molecules, polyamine, polyamide, Polyethylene Glycol, polyethers, the base of the pharmacodynamic profiles of enhancing oligomer
The group of the pharmacokinetic properties of group and enhancing oligomer.Typical conjugation group includes cholesterol, lipid, phospholipid, life
Thing element, azophenlyene, folate, phenanthridines, anthraquinone, acridine, fluorescein, rhodamine, Coumarinses and dyestuff.
RNAi agent can be applied to the target aquatic organism of the present invention by including but is not limited to following technology：Electroporation, note
Penetrate, microinjection, fast injection, enter, ingest (feeding), calcium phosphate-DNA coprecipitation, the transfection of DEAE- glucosan mediation, poly-
The transfection of solidifying amine mediation, liposome fusion, lipofection, protoplast fusion, retrovirus injection, biological bullet method (grain
Son bombardment, such as uses particle gun) or viral vector direct transfection cell.For arthropod, injection, microinjection, via immersion
Or ingest (feeding) and be administered orally or percutaneous procedure is preferred.RNAi agent can introduce target organism with naked form.Naked form refers to
RNAi agent does not contain and can act on to promote to immerse any delivery vehicle of target cell, such as Liposomal formulation, charged fat
Matter or precipitant.If it is desired, delivery agents can be used.RNAi agent can be delivered with compositionss, and compositionss comprise nucleic acid further and coagulate
Poly- agent such as spermidine, protamine sulfate, polylysine, shitosan or other nucleic acid condensing agent known in the art.Male
" gene delivery " or " gene transfer " of change gene refers to method or the system for exogenous nucleic acid reliably inserts target cell,
And may result in the instantaneous of genes of interest or long-term expression.
RNAi agent can be applied to arthropod in any stage of life cycle, including unfertilized ovum or germ cell or
Embryo or mezzanine level germling or larval stage or maturation period.For practical considerations, if RNAi agent will be by injection
Or microinjection to animal body deliver, animal be naked eyes visible will be preferred.In artemia, best implementation phase is in ovum
Administering RNA i agent before or during cell formation stages, embryo stage, larval stage.
In a mode of the method to target organism administering RNA i agent, using the feedstuff living organism carrying RNAi agent
Body.For example, germling and adulthood consume unicellular and many cells food source, such as plankton, similar planktonic filter
Food biology, algae and yeast.These food sources can adopt in aquaculture, available RNAi agent convert these food sources so that
Target organism finally obtains RNAi agent by food source of ingesting.Spirullina, Daphnia, Chlorella, antibacterial and small-sized primary
Animal etc. is that it can be by genetic modification to pass to artemia for feeding the little artemia of cultivation and the example of the feedstuff work-source becoming artemia
Send RNAi.Feedstuff live organism can be live body, also can be prepared as lyophilization, cold preservation or frozen form.
The compositionss of the present invention can be solid, semi-solid or liquid form.Property depending on method of application and RNAi agent
Matter, compositionss suitably can be prepared to deliver to target organism.Compositionss can further include carrier.RNAi agent can be therewith
The carrier of mixing includes conventional excipients it is possible to be aqueous solvent (such as water, saline or PBS), oil, dextrose, glycerol, profit
Humectant or emulsifying agent, solubilizing agent, stabilizer, antioxidant, preservative, coating materials, binding agent, filler, disintegrating agent, dilution
Agent, lubricant, pH buffer and the like.
For across mucosa or applied dermally, in the formulation using being suitable to the penetrating agent of the barrier being pierced.Such ooze
Agent thoroughly is generally known in the art, including such as detergent, cholate and furan western ground acid derivative.The dsRNA preparation of the present invention
Including but not limited to solution, Emulsion, foam and preparation containing liposome etc..The RNAi agent that the present invention comprises is used for suppressing in preparation
Masculinizing factor in arthropod, particularly including but not limited to artemia interior expression compositionss purposes.
The compositionss of the present invention comprise the amount at least 30% that can reduce target RNA, preferred 40%, 50%, 60%,
70%th, the amount of 80% or 90% or higher RNAi agent.Optimal dose with route of administration change, the frequency of administration of RNAi agent and
Time of application can pass through the optimal determination of simple experiment by those skilled in the art.
According to further embodiment, the present invention provides and SEQ ID NO:In 1 listed nucleotide sequence or its with
It is the Antisensedigonucleotsequence sequence of thing or fragment complementation, wherein Antisensedigonucleotsequence sequence can be with SEQ ID NO:1 or its with
It is thing or fragments specific hybridization, thus suppressing the expression of masculine albumen in arthropod.
According to some embodiments, the region of nucleotide is selected from 5 ' untranslated regions, password sub-district, termination codon sub-district and 3 '
The group of untranslated region composition.According to some embodiments, Antisensedigonucleotsequence sequence comprises at least 20 nucleotide.It should be understood that
It is that what oligonucleotide sequence was preferable includes constituting from about 290-1100 nucleotide.
The male of silence is used for the cultivation of filial generation germling with female artemia.Disclose further, the male in female male parent body
Change the expression silencing of gene, lead to part male sex in its progeny population to take a turn for the worse and become female individuals, make colony
In present the high female ratio of 71.4%-93.3% artemia distribution.By lowering artemia masculine gene expression, up to
The male artemia of 42.9%-86.7% presents feminizes.
With reference to embodiment, the present invention is described in detail.
Embodiment 1 masculine gene and the identification of polypeptide
By obtaining the sequence of complete artemia masculine gene from artemia gene bank, using ExPASx protein science clothes
Business device (http://ca.expasy.org/tools/dna.html) machine translation, and select most probable framework.By
SMART and CBS predictive server (http://www.cbs.dtu.dk/services/) evaluate further presumption aminoacid and
Peptide sequence.The masculinizing factor (Artemiafranciscana masculinization gene, AfrMasc) of artemia
Gene open reading frame 1077bp (SEQ ID NO:1), deduce peptide sequence (the SEQ ID NO generating containing 358 aminoacid:
2), there are the zinc fingerses containing CCCH of a series connection in the open reading frame of deduction
(WKEDSLCRDFLRNSCQRGERCKFVHAFG and IKELQFCHDFQNTGCPRRLCKFVHCTI).
Carrying out by the aminoacid sequence of artemia masculinizing factor zinc finger protein domain and other species of Arthropoda
Similarity compares, and result shows the zinc in the artemia of Anostraca, the Macrobrachium rosenbergii of Decapoda and three species of lepidopterous silkworm
All contain conservative C-C-C-H aminoacid sequence in finger protein domain, and contain several guarantors in two domains respectively
Aminoacid composition (Fig. 1) kept.
The spatial and temporal expression characterized of embodiment 2 masculine gene
(1) expression in male and female individuality for the masculine gene
Healthy, the ripe female of picking and male artemia, and carry out total serum IgE extracting using the method for Trizol test kit.
Subsequently pass through spectrophotography quantitative to RNA in 260nm.First chain cDNA passes through from 1ug total serum IgE in 42 DEG C of reverse transcription reaction
(Reverse Transcriptase MMLV (RNase H-)) 30 minutes, to be synthesized using Oligod (T) as primer, according to M-
The description preparation of the reverse transcriptase reaction manufacturer of MLV reverse transcriptase.The individual expression of the male and female of artemia masculine gene is passed through
Fluorescent quantitative PCR detects, by using forward primer 5 '-CAACAGTCCCAACAGTTCA-3 ' and reverse primer 5 '-
CTAAGGGTATGCCAAAGAA-3 ' specific primer proves.The Tubulin microtubule protein gene of artemia is used as positive control
(AF078670.1), from constant gene of expression 18S as internal reference, by using forward primer 5 '-
GGACATAGAGCGCCCAACTT-3 ' and reverse primer 5 '-GACAGGCGCATAGGTAACCA-3 ' specific primer prove.Will
The relative quantification of masculine gene is by using above-mentioned masculine gene primer and SRYBGreen PCR Master mixture
(ApplliedBiosystem) according to manufacturer description (SYBR Premix Ex TaqTM (TliRNaseH Plus),
Takara) carry out.By the data obtaining according to each tissue with 18S ribosomal RNA level standardization twice, then will be standardized
Data is expressed as 2-△△ct.
Shown by fluorescence real-time quantitative PCR result, the transcript of the masculine gene of artemia is in sexually matured artemia male and female
Difference distribution in individuality, in female artemia body, the content of masculine gene is apparently higher than the content (Fig. 2) in male artemia.
(2) masculine gene is in the expression characteristic of ovum and fetal development period
There are two kinds of growth modes in artemia in growth course：One kind is ovoviviparity, that is, in proper temperature, illumination, oxygen
Under the sufficient favourable conditions of existence with food, artemia discharges parent in the way of naupliuss.In the process, female artemia
Oocyte is deposited in two oviductus lateraliss, forms macroscopic " two-wire " phase；Subsequently oocyte moves to the defeated ovum in both sides
In the side pocket that pipe end is expanded, form " double ovum " phase；Treat female artemia copulation after fertilization, germ cell is transferred to the ovum that abdominal part expands
In pouch, form macroscopic " single ovum " phase；Finally, the growth that embryo experiences about 4-5 days in female egg capsule, becomes and no saves
Germling, and discharge from parent.
Ovum according to female artemia and the different times of fetal development, choose thin containing diplotene stage and double ovum phase ovum respectively
The egg capsule of born of the same parents, and the egg capsule sample containing single first day ovum phase to the 4th day, carry out the masculine gene expression in fetal development period
Feature analysiss.Fluorescence real-time quantitative PCR operation with shown on, research structure shows, masculine gene ovum formation and
Single ovum fetal development early stage assumes the expression of a large amount, and with the carrying out of fetal development, the expression of masculine gene is gradually lowered (figure
3).Thus it is speculated that male gene has important function in the Sex determination of Artemia embryos early stage and regulation process.
(3) masculine gene is in the expression characteristic of resting egg hatching process
In growth course in addition to mode ovoviviparous, another way is oviparity to artemia, that is, in living environment ratio
Under more severe situation, artemia can produce resting egg in the way of oviparity.Resting egg comprises about 4000 arrest of development and primitive guts
The cell of embryonic stage, is coated on one layer of polar molecule impervious thickness shell.Resting egg needs through certain activation condition, such as de-
After water, anoxia and freezing etc. are processed, in suitable environmental condition, just can hatch naupiar larva.Therefore, real using fluorescence
When quantitative PCR detection resting egg hatching process masculine allelic expression, little to the 0 of resting egg and hatching process respectively
When, 12 hours, 24 hours, 36 hours, the hatched blastocyst of 48 hours and 72 hours is sampled detecting.Result of study shows, male
Property gene in resting egg a large amount expression, with the carrying out of dormancy egg hatching, its expression is gradually lowered (Fig. 4).
The expression silencing of embodiment 3 masculine gene
The preparation of RNA disturbed specimen
Build and comprise GFP or the dsRNA expression vector of artemia masculine gene.Drawn by a pair of masculine gene specific
Thing enters performing PCR amplification.According to the forward primer 5 ' with restriction enzyme site for the cDNA sequence of the artemia masculine gene design-
GCTCTAGAAATCGAAACGCAGGAAA-3 ' (at underscore be XbaI enzyme cutting site) and reverse primer 5 '-
CGGGATCCCCCAGACACAACTGATGACA-3 ' (being BamHI restriction enzyme site at underscore).Artemia masculine gene will be comprised
CDNA be template, enter performing PCR amplification (94 DEG C continue 3 minutes, are followed by 94 DEG C and continue to continue within 30 seconds, 55 DEG C to hold for 30 seconds, 72 DEG C
35 circulations of continuous 30 seconds, are followed by 72 DEG C and continue 10 minutes), clone masculine genetic fragment, obtain objective gene sequence
(SEQ ID NO:3).
Pcr amplification product carries out electrophoresis on 1.2% agarose gel, is manifested in ethidium bromide using UV light, from solidifying
Glue cuts and utilizes QIAquick RCR purification kit (QIAGEN) purification.According to the description of manufacturer, using pET-T7
Plasmid construction contains the expression vector of genes of interest fragment, and the pET-T7 expression vector inserting containing genetic fragment is transformed into
In expression type bacterial strain E.coli HT115.Synthesize dsRNA, subsequent basis using dsrna expression vector pET-T7 abduction delivering
The description of manufacturer utilizes Trizol test kit to extract thalline total serum IgE, continues to make two in 20 minutes by being heated to 65 DEG C
Chain hybridizes, and then naturally cools to room temperature so as to cool down renaturation.It is subsequently added RNase A and carries out enzyme action process, remove single-stranded
RNA, phenol and chloroform purification double-stranded RNA, ethanol precipitation.Dissolving double-stranded RNA sample after purification is simultaneously quantitative (Fig. 5), is protected
There are -20 DEG C until using.
Masculine gene silencing in artemia body
The adult ' Luchung ' insect choosing physique healthy and strong carries out RNA interference.Using the method for microinjection, to the reproduction of every animal
The injection double-stranded RNA of 1 μ g masculine gene or the double-stranded RNA of GFP gene at body segment, put back to and continue in water system to raise.RNA does
After disturbing 10 days, the expression of detection masculine gene.The tissue RNA containing genes of interest for the extracting, reverse transcription synthesizes cDNA.Using
The method of quantitative fluorescent PCR, using 18S gene as internal reference, detects the expression of masculine gene, quantitative fluorescent PCR is put
Same as above.Result shows, after RNA interference, in artemia body, the nucleic acid expression level of masculine gene is significantly lowered, with GFP pair
Compare according to group, the expression of masculine gene reduces by more than 80% (Fig. 6), masculine gene in silence artemia body effectively
Expression.
The sex phenotype based on masculine gene silencing for the embodiment 4
(1) the masculine gene silencing of Adult female and male artemia
For the RNAi experiment of the long-term silence of masculine gene, choose female and the female adult artemia difference of physique healthy and strong
Carry out the RNA interference of masculine gene, the microinjection of RNA interference and operating process are as described above.Artemia after injection is normal
Culture, is placed in 28 DEG C, is cultivated in the sea water adding 1% disodiumedetate ferrum, the solution of chlorella dry powder configuration
Every day feeding once.Ovoviviparity artemia periodicity of illumination is set as illumination 16 hours, dark 8 hours, seawater salinity 4%, illumination
Intensity is 2000 luxs.
Female adult after masculine gene silencing and male worm are respectively as maternal and male parent so as to mate and oviposit produces filial generation.
Filial generation artemia continues to support in water system to adult, identifies male and female sex from mode of appearance.From observing in appearance, female artemia
Antenna secunda be in only a kick, and the antenna secunda of male artemia becomes prehensile organ, wide flat, is in obvious axe in appearance
Shape.Therefore, under the microscope or during perusal, can determine that male and female according to whether artemia has axe shape feeler, identify artemia
Male and female sex.Generally, in artemia colony the ratio of female and male close to 1:1.In masculine gene RNA interference group, filial generation
Artemia male and female sex statistical data shows, in 4 parallel groups of masculine gene silencing, female ratio accounts for colony's sum
75.0%-93.3%, assumes high female phenomenon.Data results show further, the progeny population of masculine gene silencing
Middle 50.0%-86.7% male occurs sex to reverse and becomes female individuals (Fig. 7).Above test result indicate that, parent
The sex leading to embryo and offspring individual is taken a turn for the worse by the silence of internal masculine gene, and partial male individuality is transformed into newly female
Property (false female individuality).
(2) the masculine gene silencing of female artemia diplotene stage and male artemia
Shown in the expression characteristic result in fetal development period according to masculine gene, male gene is in Artemia embryos early stage
Sex determination and regulation process there is important function.Its growth course of oocyte due to ovoviviparity artemia starts from " two-wire "
Phase, that is, the oocyte of female artemia be deposited in two oviductus lateraliss, form macroscopic " two-wire " phase.Therefore, selection is female
Property " two-wire " phase of just having germinated of artemia oocyte carry out the RNA interference of masculine gene.
Female diplotene stage choosing physique healthy and strong respectively is dry with the RNA that female adult artemia carries out masculine gene respectively
Disturb, the microinjection of RNA interference and operating process are as described above.Female adult after masculine gene silencing and male worm are respectively as mother
This and male parent are so as to mate and oviposit produces filial generation.Filial generation artemia continues to support in water system to adult, reflects in terms of mode of appearance
Determine male and female sex, authentication method is as described above.In GFP matched group artemia progeny population, the ratio of female and male is close to 1:1,
Female ratio accounts for the 45.0%-56.5% of colony's sum, is distributed in natural female-male proportion.On the other hand, masculine gene RNA
The filial generation artemia male and female sex statistical data of interference group then shows, female ratio accounts for the 71.4%-85.7% of colony's sum, presents
High female phenomenon.Data results show further, 42.9%-71.4% male in the progeny population of masculine gene silencing
Individuality occurs sex to reverse and becomes female individuals (Fig. 8).Above test result indicate that, in parent's body, masculine gene is heavy
Silent, the masculine gene silencing of especially female ovum shaping age, the sex leading to its embryo and offspring individual is occurred
Reverse, partial male individuality is transformed into newly female (false female individuality).
Above test result indicate that, in parent's body, masculine gene silencing may result in its embryo and offspring individual sex to occur
Reverse, become newly female (false female individuality).So-called false female individuality refers to its vivo carrying androgenetic information, and phenotype is (outward
See feature and reproductive system) then it is presented as female character.Therefore, by adjusting masculine gene nucleic acid in vivo and albumen table
Reach, the male and female sex differentiation of artemia class can be regulated and controled further, especially regulate and control the sex differentiation of artemia colony, induced male
Body sex reverses and forms false female individuality.Meanwhile, because the female individuality of vacation has the inherent genetic background of male and the external of female concurrently simultaneously
Phenotype, can cultivate and produce all-male progeny population, for all-male monosex cultivation with normal male stud mating, fertilization and spawning.
Animal has good male/female characteristic superiority, and these species are oriented with sex controll, carries out unisexuality
Cultivation, not only can produce huge economic benefit, and have important theory value.The inventive method is captured low at present
The technical barrier running in Animal Sex regulation and control field, by RNA perturbation technique and gene expression regulation technology, being oriented property
Do not control, induction arthropod especially artemia class Animal Sex reverses, and not only builds artemia class sex controll system and platform,
And can achieve the production of the high female based on artemia masculine gene.Operational approach of the present invention has originality, and implementation steps are clear
Clear.
SEQUENCE LISTING
<110>Wanli College, Zhejiang
<120>A kind of artemia masculinizing factor and its application
<130>
<160> 3
<170> PatentIn version 3.5
<210> 1
<211> 1077
<212> DNA
<213> 1
<400> 1
atgaacacaa atgaagaaaa agactctatg aagtggaaag aggacagttt atgtagagac 60
tttcttcgaa attcttgtca aagaggggaa agatgtaaat ttgttcatgc ttttggggat 120
ttaaaaaaag acctttctat taaggaatta caattttgcc atgattttca gaacactggt 180
tgtcctagaa gattatgcaa gtttgtccat tgtacaatcg aagaagaaca acaatacctt 240
gataaaggta tgttgccatc gcatgttata cagtctttca ttcagcgtgg cataattcct 300
gatgcaatcg taactgagcc cggtgaaaca ccaatttgta aagatttctt aaaaaatgac 360
tgttttagag gggataaatg caagtttcga catgtacaaa tcgaaacgca ggaaaaaaaa 420
ggggccagtg agttcaagcg cttggatcag gatcttgaac ctaggctaaa acgaatgaag 480
tcagataatt atatccctga tcaacaatgt gtggttgtat gtgaacccag tatctctaca 540
gaaagctcga atgaaataag acttgttcga gaagaaaaca atatcctaaa acaaaagctt 600
gaagaaatga aaaggcagct ttataattta atggcaacca atgagttcct tctggaacaa 660
aatgcccatt taagacttgg taaaatgtca tcagttgtgt ctgggattta tcacccaatt 720
aaccagctat cgtctgctgg tggcaacctt ggtgtaatta ctcaaccggc acaaagtgta 780
tcagttatta cacagaatgg acaacctgca gtatctgtct caacagtccc aacagttcaa 840
actcttgcaa caatgtccct accaactatg actgtacctg taactcttcc tcaagtggca 900
acctatccaa ataccctcaa atcctcttac aataaacact gctgcaacct ccttaggaat 960
aaccaatgtg ccatgctctc ttgcatactc ggttatgtct caaccaacta cttctttggc 1020
atacccttag ctcttttgtt atatgcaaat atattaaagt atgcttctca agcttag 1077
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Met Asn Thr Asn Glu Glu Lys Asp Ser Met Lys Trp Lys Glu Asp Ser
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Leu Cys Arg Asp Phe Leu Arg Asn Ser Cys Gln Arg Gly Glu Arg Cys
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Lys Phe Val His Ala Phe Gly Asp Leu Lys Lys Asp Leu Ser Ile Lys
35 40 45
Glu Leu Gln Phe Cys His Asp Phe Gln Asn Thr Gly Cys Pro Arg Arg
50 55 60
Leu Cys Lys Phe Val His Cys Thr Ile Glu Glu Glu Gln Gln Tyr Leu
65 70 75 80
Asp Lys Gly Met Leu Pro Ser His Val Ile Gln Ser Phe Ile Gln Arg
85 90 95
Gly Ile Ile Pro Asp Ala Ile Val Thr Glu Pro Gly Glu Thr Pro Ile
100 105 110
Cys Lys Asp Phe Leu Lys Asn Asp Cys Phe Arg Gly Asp Lys Cys Lys
115 120 125
Phe Arg His Val Gln Ile Glu Thr Gln Glu Lys Lys Gly Ala Ser Glu
130 135 140
Phe Lys Arg Leu Asp Gln Asp Leu Glu Pro Arg Leu Lys Arg Met Lys
145 150 155 160
Ser Asp Asn Tyr Ile Pro Asp Gln Gln Cys Val Val Val Cys Glu Pro
165 170 175
Ser Ile Ser Thr Glu Ser Ser Asn Glu Ile Arg Leu Val Arg Glu Glu
180 185 190
Asn Asn Ile Leu Lys Gln Lys Leu Glu Glu Met Lys Arg Gln Leu Tyr
195 200 205
Asn Leu Met Ala Thr Asn Glu Phe Leu Leu Glu Gln Asn Ala His Leu
210 215 220
Arg Leu Gly Lys Met Ser Ser Val Val Ser Gly Ile Tyr His Pro Ile
225 230 235 240
Asn Gln Leu Ser Ser Ala Gly Gly Asn Leu Gly Val Ile Thr Gln Pro
245 250 255
Ala Gln Ser Val Ser Val Ile Thr Gln Asn Gly Gln Pro Ala Val Ser
260 265 270
Val Ser Thr Val Pro Thr Val Gln Thr Leu Ala Thr Met Ser Leu Pro
275 280 285
Thr Met Thr Val Pro Val Thr Leu Pro Gln Val Ala Thr Tyr Pro Asn
290 295 300
Thr Leu Lys Ser Ser Tyr Asn Lys His Cys Cys Asn Leu Leu Arg Asn
305 310 315 320
Asn Gln Cys Ala Met Leu Ser Cys Ile Leu Gly Tyr Val Ser Thr Asn
325 330 335
Tyr Phe Phe Gly Ile Pro Leu Ala Leu Leu Leu Tyr Ala Asn Ile Leu
340 345 350
Lys Tyr Ala Ser Gln Ala
355
<210> 3
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<213> 3
<400> 3
aaaaaggggc cagtgagttc aagcgcttgg atcaggatct tgaacctagg ctaaaacgaa 60
tgaagtcaga taattatatc cctgatcaac aatgtgtggt tgtatgtgaa cccagtatct 120
ctacagaaag ctcgaatgaa ataagacttg ttcgagaaga aaacaatatc ctaaaacaaa 180
agcttgaaga aatgaaaagg cagctttata atttaatggc aaccaatgag ttccttctgg 240
aacaaaatgc ccatttaaga cttggtaaaa tgtcatcagt tgtgtctggg 290
Claims (10)
1. a kind of artemia masculinizing factor is it is characterised in that the albumen of described artemia masculinizing factor coding includes：
1) aminoacid sequence is SEQ ID NO：2 albumen；
2) with SEQ ID NO:2 albumen has at least 70% amino acid sequence identity, and with 1) in protein function phase one
The albumen causing.
2. masculinizing factor as claimed in claim 1, its nucleotides sequence is classified as SEQ ID NO:1.
3. application in adjusting artemia reproductive development for the artemia masculinizing factor described in claim 1.
4. a kind of double stranded rna molecule lowering artemia masculine gene expression is it is characterised in that described double stranded rna molecule bag
Include the whole or portion of the artemia masculinizing factor that positive-sense strand and antisense strand, wherein positive-sense strand or antisense strand are described in claim 1
Burst section；Or there is the nucleotide sequence of at least 70% complementation with selected fragment.
5. double stranded rna molecule as claimed in claim 4, the nucleotide sequence of its positive-sense strand or antisense strand is SEQ ID NO:3.
6. application in adjusting artemia reproductive development for the double stranded rna molecule described in claim 4.
7. a kind of DNA construct, described DNA construct is used for transcription and produces the double stranded rna molecule described in claim 4.
8. a kind of reduce claim 1 described in masculinizing factor expression method it is characterised in that described method be by
Double stranded rna molecule described in claim 4 converts or is transfected into biological internal.
9. a kind of method of female artemia filial generation of acquisition or the artemia filial generation of false female is it is characterised in that described method is logical
Cross and will reduce or remove the artemia of the masculinizing factor expression described in claim 1 as parent；Re-mating spawning obtains
Filial generation.
10. the method for claim 1, it is characterised in that described reduction or removal masculine gene expression amount, is logical
Cross DNA construct described in double stranded rna molecule or the claim 7 introducing described in above-mentioned claim 4 to complete.
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Cited By (1)
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| CN112481308A (en) * | 2019-09-11 | 2021-03-12 | 中国科学院分子植物科学卓越创新中心 | Novel sex determining gene HAKAI, its regulation and control action and application |
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2016
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Non-Patent Citations (4)
| Title |
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| ANONYMOUS: "ARB66312.1", 《GENBANK》 * |
| ANONYMOUS: "KY245899.1", 《GENBANK》 * |
| DONG-RUI LI ET AL: "Identification and characterization of a Masculinizer (Masc) gene involved in sex differentiation in Artemia", 《GENE》 * |
| 徐明玉: "中国两性生殖卤虫早期胚胎发育相关基因的研究", 《中国优秀硕士学位论文全文数据库（电子期刊）》 * |
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|---|---|---|---|---|
| CN112481308A (en) * | 2019-09-11 | 2021-03-12 | 中国科学院分子植物科学卓越创新中心 | Novel sex determining gene HAKAI, its regulation and control action and application |
| CN112481308B (en) * | 2019-09-11 | 2023-04-25 | 中国科学院分子植物科学卓越创新中心 | Novel sex-determining gene HAKAI, its regulation and control effect and application |
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