CN107267511A - RNAi interference fragments, interference carrier and its preparation method and application - Google Patents
RNAi interference fragments, interference carrier and its preparation method and application Download PDFInfo
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- CN107267511A CN107267511A CN201710506560.8A CN201710506560A CN107267511A CN 107267511 A CN107267511 A CN 107267511A CN 201710506560 A CN201710506560 A CN 201710506560A CN 107267511 A CN107267511 A CN 107267511A
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- C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
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Abstract
The invention provides a kind of RNAi interference fragments for Zfx genes, interference carrier and its preparation method and application.RNAi interference fragments comprise at least following sequences one or sequence two：Sequence one：F：5′‑TGTGA GGACT ACCTT ATGAT TTCAA GAGAA TCATA AGGTA GTCCT CACTT TTTTC‑3′；Sequence two：R：5 ' TCGAG AAAAA AGTGA GGACT ACCTT ATGAT TCTCT TGAAA TCATA AGGTA GTCCT CACA 3 ', described RNAi interference fragments are used to disturb the Zfx genes on animal X chromosome.The interference fragment that the present invention is provided, can effectively control the sex of animal.
Description
Technical field
The present invention relates to animal molecular genetics field, and in particular to is capable of the RNA of the related gene of silence X Sperm specific enzymes
Interference fragment and its application.
Background technology
Under field conditions (factors), the sex ratio of nearly all mammal is all close to 1: 1, it is achieved thereby that entering without interruption
Row is stably produced offspring, and the ecological balance is issued in natural condition.For husbandry sector reality, preferable sex ratio
Example tool is of great significance.The manufacturing enterprise produced using lactation, egg-laying deseription intentionally gets more females
Body, and produce meat, fibre trait showed on male dominance is more obvious.All the time, the mankind are to control animal
The sex of offspring just has great interest.Current sexual control is main from prefecundation and latter two aspect progress of being fertilized, the former
It is fertilized again using after spermatozoa isolation so that just determine the sex of offspring in the time of fertilization；The latter uses early embryo sex
Identification, the method for carrying out the sex screening of embryo controls the sex of offspring.
Mammal sex determination theory thinks that sex chromosome can just develop into female individuals for the embryonated egg of " XX ",
And sex chromosome will develop into male for " XY ".So fundamentally, the control optimal method of Livestock Sex is first
Separate X, y sperm, be then fertilized again, just can artificially control the sex of offspring, in theory this be in sexual control most
Simply, most economical approach.From after 1950s, the research report on spermatozoa isolation is a lot, and these researchs all attempt
Using the X sperms physical characteristic different with y sperm (volume, density, electric charge, motility), using centrifugal process, electrophoresis, ion
The problem of different methods such as exchange process, cell surface antigen method are to realize the separation of sperm, but have one jointly ---
Repeatability is poor.And these differences change with the difference of environmental condition, therefore, many results of study usually differ
Cause, or even opposite conclusion occur, cause some Differences of two class sperms to there is dispute.Up to the present, except X, Y essence
Outside there is sub- DNA content difference can affirm, otherwise difference shows very little, or even is difficult to determine.But with point
Sub- biology techniques are continued to develop, and research work is continued deeper into, and new achievement in research will continue to bring out.
At present according to mammal X, the principle of y sperm DNA content difference, X, y sperm are carried out using flow cytometer
Separation, preferably, purity is up to more than 90%, but its equipment is expensive, and separating rate is slower for its separating effect, and the essence of fertilization
The reason such as quantum count is few, vigor is poor, far can not meet the wilderness demand to sexing semen in production.By contrast, if energy
Enough find in two kinds of sperm developments or fertilization process, in addition during embryonic development critical X, Y chromosome specific mrna sequence
Row, and the contemporary new Protocols in Molecular Biology of combination disturbs the method for (RNA Interference, RNAi) then to have using RNA
Hope with relatively low cost, simply and quickly realize the sexual control to animal.
RNAi is the gene silencing (Post-transcriptional Gene Silencing, PTGS) after a kind of transcription
Phenomenon, it is special in the cell by double-stranded RNA (Dubble-stranded RNA, dsRNA) that is external artificial synthesized or producing in vivo
Degrade its homologous mRNA different in naturely so that corresponding gene interference, realizes the expression for preventing target gene.RNAi methods can
Quickly, corresponding gene expression that is easy, effectively, specifically lowering in cell, still one kind of research gene function does not have for it
Power instrument, and new technological means is provided for specific gene treatment, its application prospect is very wide.
All proved in the research of genome, mRNA and protein level, X, y sperm gene expression have differences really.Gene
Detection of expression research shows, in the meiotic pachytene of spermiogenesis tail, to prevent some enzymes and sex chromosome from having an effect,
The highly concentrated formative body of two sex chromosome.The formation of property body protects the table of special gene on sex chromosome, sex chromosome
Up to being closed.Therefore, there is no mRNA transcription in mammalian mature sperm, but but contain in mammalian mature sperm
MRNA, transcription of this species diversity in spermatogenesis, its translation far lags behind transcription.After these mRNA are spermioteleosis
Instruct to synthesize some necessary albumen, or after fertilization is to the supplement in egg mother cell mRNA storehouses, or as RNAi to fertility and
Embryo growth and development plays the effect of key.
The research to mouse such as Hendriksen is found, nearly all in addition to Xist gene expressions during meiosis
Sex chromosome gene all do not express, but meiosis anaphase, some sex chromosome specific genes start expression.Subtrahend point
After splitting, detecting Ubely and Sry genes on Y chromosome has higher mRNA level in-site；Ubelx bases are also detected that in X sperms
Because of mRNA high level expressions, while finding X chromosome specific gene Mhr6A expression product.Other somebody has found X, Y dyeing
The expression of other specific genes of body, such as X chromosome specific gene Akap82 (skelemin of sperm tail) and Nap-X (codings
A kind of core GAP-associated protein GAP), Y chromosome specific gene Zfy-1, Zfy-2 and Y353/B.
Wherein Zfx/Zfy genes are a pair of alleles of encoding zinc finger protein on chromosome, since meiophase
Expression, the content highest in round spermatid.Zfx genes are a member in ZFY (zinc finger-Y) gene family,
ZFY families include Zfx, Zfy and Zfa genes, they are closely similar on molecular structure.General mammal has Zfx and Zfy bases
Cause, Zfx genes are located on X sex chromosome, are the genes that female, boar X chromosome must contain, and Zfy genes are located at Y dyes
On colour solid, two genes are located at the end of sex chromosome, but are not on homology region.Zfx genes include an acid turn
The exciting region of record, a nuclear localization sequence and a DNA bonding pad, with 13 C2H2 [Cys (2) His (2)] type zinc finger knot
Structure.In mammalian cell, C2H2 zinc fingerses are one of most common protein structures.Have now been found that and plant tool more 800
There is the protein of this zinc fingers, with important physiological function.The albumen of Zfx/Zfy codings is as transcription factor, in essence
There are some functions and relevant with the generation of sperm in sub- forming process.
The content of the invention
It is a primary object of the present invention to there is provided a kind of RNAi interference fragments, interference carrier and its preparation method and application,
Main purpose is that the Zfx genes on animal (especially red deer) X chromosome are disturbed by RNAi interference carriers, with up to
To the purpose of control Animal Sex.
The object of the invention to solve the technical problems is realized using following technical scheme.
According to a kind of RNAi interference fragments proposed by the present invention, described RNA interference fragments comprise at least following sequences one
Or sequence two：Sequence one：F：5′-TGTGA GGACT ACCTT ATGAT TTCAA GAGAA TCATA AGGTA GTCCT
CACTT TTTTC-3′；Sequence two：R：5′-TCGAG AAAAA AGTGA GGACT ACCTT ATGAT TCTCT TGAAA
TCATA AGGTA GTCCT CACA-3 ', described RNAi interference fragments are used to disturb the Zfx genes on animal X chromosome.
The object of the invention to solve the technical problems can be also applied to the following technical measures to achieve further.
It is preferred that, foregoing RNAi interference fragments, wherein described RNAi interference fragments are used to disturb red deer X chromosome
Zfx genes.
The object of the invention to solve the technical problems is also realized using following technical scheme.
A kind of RNAi interference fragments application is proposed according to the present invention, described RNAi interference fragments are used to prepare control animal
The medicine or kit of sex, described RNAi interference fragments are foregoing RNAi interference fragments.
The object of the invention to solve the technical problems is also realized using following technical scheme.
A kind of RNAi interference carriers of foundation present invention proposition, the foregoing RNAi interference fragments of described RNAi interference carriers,
Described RNAi interference carriers are used to disturb the Zfx genes on animal X chromosome.
The object of the invention to solve the technical problems can be also applied to the following technical measures to achieve further.
It is preferred that, foregoing RNAi interference carriers, wherein described RNAi interference carriers are used to disturb red deer X chromosome
Zfx genes.
It is preferred that, foregoing RNAi interference carriers, wherein described RNAi interference carriers include pLentiLox3.7 carriers,
The pLentiLox3.7 carriers are connected with the RNAi interference fragments.
The object of the invention to solve the technical problems is also realized using following technical scheme.
A kind of application of RNAi interference carriers is proposed according to the present invention, described RNAi interference carriers are used in animal prefecundation
In sexual control, described RNAi interference carriers is described in foregoing any one.
It is preferred that, the application of foregoing RNAi interference carriers, wherein described RNAi interference carriers are with testis direct injection
Mode be injected in animal body, for disturbing the Zfx genes on animal X chromosome.
It is preferred that, the application of foregoing RNAi interference carriers, wherein described RNAi interference carriers are with testis direct injection
Mode be injected in red deer stags body, for disturbing the Zfx genes on red deer X chromosome.
The object of the invention to solve the technical problems is also realized using following technical scheme.
A kind of preparation method of RNAi interference carriers is proposed according to the present invention, is comprised the following steps, described in digestion
PLentiLox3.7 carriers；The RNAi interference fragments are connected with the pLentiLox3.7 carriers after digestion, connected
Practice midwifery thing, wherein, the RNAi interference fragments are for example foregoing；The connection product is converted into competent cell, and filtered out
Positive colony bacterium solution；Sequencing identification is carried out to positive colony bacterium solution, and sequencing result and the RNAi interference fragments sequence is complete
Complete consistent bacterium solution is expanded；RNAi interference carriers are extracted from the bacterium solution of the amplification.
By above-mentioned technical proposal, the present invention provides a kind of RNAi interference fragments, interference carrier and preparation method thereof and should
With at least with following advantages：
The RNAi interference fragments that the present invention is provided, are entered by the gene that played a crucial role to the red deer stags X Sperm specific enzyme stages
Row silence, influences its X sperm normal development, the fertilization chance of X sperms is improved when being mated with female red deer, is had in prefecundation
What is imitated controls the sex of red deer so that the male ratio of first filial generation red deer is greatly improved.This method will not to required spermatoblast
Cause to damage or reduce its vigor, and it is with low cost.
Brief description of the drawings
Fig. 1 red deer androgone Zfx gene mRNA expression amount statistical charts.
P：*=P ＜ 0.01；Note：P：*=P ＜ 0.01.
Interference carrier pLL3.7/a, pLL3.7/b, pLL3.7/c and the pLL3.7/d built is selected, external training is transfected into
Foster red deer testis spermatogenic cell, by RT-PCR, with the fluorescence real-time quantitative target gene and reference gene primer shown in table 3
Under sequence and PCR conditions, detection conditions in vitro, red deer androgone Zfx gene mRNA expression amounts.As shown in figure 1, with it is normal right
Compared according to group, the noiseless inhibitory action of mRNA of interference carrier pLL3.7/a, pLL3.7/b, pLL3.7/c to Zfx genes.And do
Carrier pLL3.7/d can reduce Zfx gene mRNA expressions amount in red deer androgone.Wherein interference group pLL3.7/d production of sperm
Zfx gene mRNA expressions amount reduction by 81% in cell, difference is extremely significantly (P ＜ 0.01).
Red deer first filial generation male ratio statistical chart after Fig. 2 internal injection control reagents.
By under condition of in vitro culture, effective recombinant plasmid pLL3.7/d of cell-based screening is used as property control reagent, injection
Enter red deer live body testis, natural mating counts the male and female quantity of first filial generation red deer.As shown in Fig. 2 control group is injection physiology salt
Water group, young is 47.5% for red deer male ratio；Recombinant plasmid pLL3.7/d is injected, first filial generation red deer male ratio is 76%, and right
Compared according to group, sex skew is obvious, difference is extremely significantly (P ＜ 0.01).
Fig. 3 pLL3.7 vector construction schematic diagrames.
As illustrated, the present invention application pLL3.7 slow virus skeleton plasmid carriers specific cleavage site Hpa I and
Xhol I, to optimize RNAi interference carriers, only ensure carrier infected, without virus packaging, it is ensured that the life of property control reagent
Thing security.
Embodiment
Further to illustrate the present invention to reach the technological means and effect that predetermined goal of the invention is taken, below in conjunction with
Accompanying drawing and preferred embodiment, to according to a kind of RNAi interference fragments proposed by the present invention, interference carrier and preparation method thereof and should
With its embodiment, structure, feature and its effect are described in detail as after.In the following description, a different " implementation
What example " or " embodiment " referred to is not necessarily same embodiment.In addition, special characteristic, structure or spy in one or more embodiments
Point can be combined by any suitable form.
1st, the preparation method for the RNAi interference carriers that the present invention is provided is：
According to the mRNA sequence of the family's ox Zfx genes provided in GenBank, specific primer is designed, to red deer Zfx genes
Expanded, clone obtains red deer Zfx gene orders a length of 2115bp (GenBank accession number first：KP257294), according to
To filter out 4 siRNA fragments as shown in table 1 for Blast results on RNAi design principles and NCBI.
The siRNA of table 1 Zfx cDNA templates
Hpa I and Xhol I restriction enzyme sites are added in 5 ' and 3 ' in each siRNA fragment respectively, as shown in table 2, then send
Biotech firm synthesizes siRNA fragment.Positive and negative adopted chain is combined into double-strand by annealing and is connected to plentilox3.7 (Addgene, U.S.
State) on slow virus packaging system carrier, then it is transformed into E.coli DH5 α competent cells (Tiangen, China) and is expanded
Increase, extract -20 DEG C of preservations of plasmid.
Table 2 carries the Zfx RNAi sequences of Hpa I and Xhol I restriction enzyme sites
2nd, the external interference test of Zfx genes：
Healthy red deer stags 1 are chosen, the bilateral testes of red deer are aseptically taken out by operation, using two step enzymes
Digestion method is separated to sustentacular cell of testis and androgone.These cells isolated are put into containing 10% hyclone
In DMEM/F12 nutrient solutions, interior addition HEPES (15mol/L), 100kU/L penicillin and 0.1g/L streptomysins, 1 μ g/mL tables
Skin growth factor, 100 μ L ITS (insulin, transferrins, sodium selenate), insulin (10 μ g/mL), transferrins (10 μ g/
ML), retinoic acid (3.3 × 10-7Mol/L), vitamin A (3.3 × 10-7Mol/L), 10 μ g/mL vitamin Es, 10-4Mol/L dimension lifes
Plain C, 10-3Mol/L pyruvic acid, 10-7Mol/L testosterones, 25U/L rFSH.About press 1 × 106CELL/cm2Density be inoculated in six holes
In plate, in 37 DEG C, 5%CO2, humidity be 95% under conditions of cultivate 24 hours, with the transfection reagent box of liposome 2000 as turn
The carrier built is transfected into androgone by transfection reagent, and every group in triplicate.After transfection 48 hours, androgone is extracted
Total serum IgE, with the mRNA expressions of RT-PCR detection Zfx genes, (corresponding target gene, reference gene primer, PCR conditions are shown in Table
3)。
The fluorescence real-time quantitative target gene of table 3 and reference gene primer sequence and PCR conditions
3rd, interference test in the red deer body of Zfx genes：
Choose healthy stag 10, doe totally 120, with containing dual anti-PBS that the effect through cell-based screening is notable
Interference carrier pLL3.7/d be diluted in proportion, be then expelled to respectively in 10 stag testis, injecting method：Every public affairs
Deer 3mg (1.5mg/ sides), co-injection 3 times, interval time is 10 days.After the completion of injection, 10 stags are caught up with into 10 mothers at random
Deer enclosure gives up (each colony house has 9~14 does), spontaneous estrus mating, registration stag overbit and corresponding doe overbit, breeding
Date, the young deer sex of record output, and statistical analysis is carried out to the sex ratio of first filial generation red deer.
Experimental result：
1st, RT-PCR detects the expression of Zfx gene mRNAs in androgone：
The cell transfected using empty carrier sets its mRNA expression quantity 1 as blank control group, passes through statistical side
Method shows experimental group pLL3.7/d Zfx gene mRNA expressions amount 19% to experimental group number according to analyzing, as a result, with empty carrier
Group compared to difference significantly (P ＜ 0.01), i.e., is 81% (P ＜ 0.01) as shown in Figure 1 to Zfx gene expression amounts jamming effectiveness.
2nd, interference test result in red deer body：
To inject Isodose physiological saline as a control group, the young deer sex of experimental group offspring is counted, as a result shown
Show that experimental group pLL3.7/d first filial generation red deer male ratio is up to 76%, compared with control group (physiological saline group), difference extremely shows
Write (P ＜ 0.01), such as table 4, shown in Fig. 2.
The red deer testis injection interference test first filial generation sex statistical form of table 4
Note：P：*=P ＜ 0.01；Note：P：*=P ＜ 0.01
The present invention, using red deer Zfx genes (KP257294.1) cDNA as template, designs the specific RNA of red deer Zfy genes
Interference fragment (shown in table 1), synthesis shRNA (shown in table 2) and the slow virus bone built by Hpa I and Xhol I restriction enzyme sites
Frame plasmid interference carrier pLL3.7/d (as shown in table 5), can effectively disturb the expression of red deer androgone Zfx gene mRNAs and show
Write and improve the male ratio in red deer first filial generation.The mrna expression amount of wherein Zfx genes have dropped 81%, and first filial generation red deer male ratio is
76%.
The best interference carrier oligonucleotide sequence of the inside and outside property control effect of table 5
Technology used in following examples, including gene sequencing, PCR amplification and detection, the life of vector construction equimolecular
Thing technology, is routine techniques well known by persons skilled in the art unless stated otherwise, used instrument and equipment, reagent,
Plasmid and carrier etc., only especially indicate, are that general those skilled in the art can be obtained by public approach.
1st, the RNAi interference carriers of Zfx genes are built, as shown in Figure 3：
According to the mRNA sequence of the family's ox Zfx genes provided in GenBank, design specific primer is to red deer Zfx genes
Expanded, clone obtains red deer Zfx gene orders a length of 2115bp (GenBank accession number first：KP257294), according to
To filter out 4 siRNA fragments as shown in table 1 for Blast results on RNAi design principles and NCBI.Respectively in each siRNA fragment
5 ' and 3 ' addition Hpa I and Xhol I restriction enzyme sites, as shown in table 2, then send biotech firm synthesize siRNA fragment.It is positive and negative
Adopted chain is combined into double-strand by annealing and is connected on plentilox3.7 (Addgene, the U.S.) slow virus packaging system carrier, then
It is transformed into E.coli DH5 α competent cells (Tiangen, China) to be expanded, extracts -20 DEG C of preservations of plasmid.
2nd, the external interference test of Zfx genes：
Healthy red deer stags 1 are chosen, the bilateral testes of red deer are aseptically taken out by operation, using two step enzymes
Digestion method is separated to sustentacular cell of testis and androgone.These cells isolated are put into containing 10% hyclone
In DMEM/F12 nutrient solutions, interior addition HEPES (15mol/L), 100kU/L penicillin and 0.1g/L streptomysins, 1 μ g/mL tables
Skin growth factor, 100 μ L ITS (insulin, transferrins, sodium selenate), insulin (10 μ g/mL), transferrins (10 μ g/
ML), retinoic acid (3.3 × 10-7Mol/L), vitamin A (3.3 × 10-7Mol/L), 10 μ g/mL vitamin Es, 10-4Mol/L dimension lifes
Plain C, 10-3Mol/L pyruvic acid, 10-7Mol/L testosterones, 25U/LrFSH.About press 1 × 106CELL/cm2Density be inoculated in six holes
In plate, in 37 DEG C, 5%CO2, humidity be 95% under conditions of cultivate 24 hours, with the transfection reagent box of liposome 2000 as turn
The carrier built is transfected into androgone by transfection reagent, and every group in triplicate.After transfection 48 hours, androgone is extracted
Total serum IgE, with the mRNA expressions of RT-PCR detection Zfx genes, (corresponding target gene, reference gene primer, PCR conditions are shown in Table
3)。
3rd, interference test in the animal body of Zfx genes：
Choose healthy stag 10, doe totally 120, with containing dual anti-PBS that the effect through cell-based screening is notable
Interference carrier pLL3.7/d be diluted in proportion, be then expelled to respectively in 10 stag testis, injecting method：Every public affairs
Deer 3mg (1.5mg/ sides), co-injection 3 times, interval time is 10 days.During injection, marginal not penetrates the slow pumpback puncture needle in side, has injected
Bi Hou, slowly extracts puncture needle, and injection site is carried out disinfection with the tincture of iodine.After the completion of injection, 10 stags are caught up with random
Enter 10 doe colony houses (each colony house there are 9~14 does), spontaneous estrus mating, registration stag overbit and corresponding doe ear
Number, Breeding date, the young deer sex of record output, and statistical analysis is carried out to the sex ratio of first filial generation red deer.
In the above-described embodiments, the description to each embodiment all emphasizes particularly on different fields, and does not have the portion being described in detail in some embodiment
Point, it may refer to the associated description of other embodiment.
It is understood that the correlated characteristic in said apparatus can be referred to mutually.In addition, in above-described embodiment " the
One ", " second " etc. is to be used to distinguish each embodiment, and does not represent the quality of each embodiment.
In the specification that this place is provided, numerous specific details are set forth.It is to be appreciated, however, that the implementation of the present invention
Example can be put into practice in the case of these no details.In some instances, known structure and skill is not been shown in detail
Art, so as not to obscure the understanding of this description.
Similarly, it will be appreciated that in order to simplify the disclosure and help to understand one or more of each inventive aspect, exist
Above in the description of the exemplary embodiment of the present invention, each feature of the invention is grouped together into single implementation sometimes
In example, figure or descriptions thereof.However, the device of the disclosure should be construed to reflect following intention：It is i.e. required to protect
The application claims of shield features more more than the feature being expressly recited in each claim.More precisely, such as following
Claims reflect as, inventive aspect is all features less than single embodiment disclosed above.Therefore,
Thus the claims for following embodiment are expressly incorporated in the embodiment, wherein each claim is in itself
All as the separate embodiments of the present invention.
Those skilled in the art, which are appreciated that, to be carried out adaptively to the part in the device in embodiment
Change and they are arranged in one or more devices different from the embodiment.Can be the component combination in embodiment
Into a part, and multiple subassemblies can be divided into addition.Except at least some in such feature are mutual
, can be using any combinations to the institute disclosed in this specification (including adjoint claim, summary and accompanying drawing) outside repulsion
There are feature and all parts of so disclosed any device to be combined.Unless expressly stated otherwise, this specification (including
Adjoint claim, summary and accompanying drawing) disclosed in each feature can or similar purpose identical, equivalent by offer replacement
Feature is replaced.
Although in addition, it will be appreciated by those of skill in the art that some embodiments described herein include other embodiments
In included some features rather than further feature, but the combination of the feature of be the same as Example does not mean in of the invention
Within the scope of and form different embodiments.For example, in the following claims, times of embodiment claimed
One of meaning mode can be used in any combination.The present invention all parts embodiment can be realized with hardware, or
Realized with combinations thereof.
It should be noted that the present invention will be described rather than limits the invention for above-described embodiment, and ability
Field technique personnel can design alternative embodiment without departing from the scope of the appended claims.In the claims,
Any reference symbol between bracket should not be configured to limitations on claims.Word "comprising" is not excluded the presence of not
Part or component listed in the claims.Word "a" or "an" before part or component does not exclude the presence of multiple
Such part or component.The present invention can be realized by means of including the device of some different parts.It is some listing
In the claim of part, several in these parts can be embodied by same part.Word first,
Second and third use do not indicate that any order.These words can be construed to title.
Technical characteristic in the claims in the present invention and/or specification can be combined, and its combination is not limited to power
The combination that profit is obtained in requiring by adduction relationship.It is combined by the technical characteristic in claim and/or specification
The technical scheme arrived, is also protection scope of the present invention.
The above described is only a preferred embodiment of the present invention, any formal limitation not is made to the present invention, according to
Any simple modification, equivalent variations and the modification made according to the technical spirit of the present invention to above example, still fall within this hair
In the range of bright technical scheme.
SEQUENCE LISTING
<110>Shihezi Univ
<120>RNAi interference fragments, interference carrier and its preparation method and application
<160> 1
<170> PatentIn version 3.5
<210> 1
<211> 5328
<212> mRNA
<213>Ox
<400> 1
ctgtgactga tgagaattaa aggccatgga tgaagatgga cttgaattac aaccacaaga 60
accaaactca ttttttgata caacaggagc tgatgctaca cacatggatg gtgatcaaat 120
tgttgtggaa gtacaagaaa ctgtttttgt ttcagatgtt gtggattcag acataactgt 180
gcataacttt gttcccgatg acccagactc agttgtaatc caagatgtta tcgaggacgt 240
tgttatagaa gatgttcagt gtccagatat catggaagaa gcagatgtat ctgaaacagt 300
catcattcct gagcaagtgc tggactcaga tgtaacagaa gaagtgtctt tagcacattg 360
tacagtccca gatgacgtct tagcttctga tattacttca gcctcaatgt ctatgccaga 420
acacgtcttg acgagtgagt ccatacacgt gtctgacatt ggacacgttg aacacgttgt 480
tcatgatagc gtagtagagg cagaaatcgt cactgatcct ctgacagccg atgtagtgtc 540
agaagaagta ttggtagcag attgtgcctc agaagcagtc atagatgcca acgggatccc 600
tgtggaccag caggaggatg acaaaggcaa ctgtgaggac taccttatga tttccttgga 660
tgatgctggc aaaatagaac acgatggttc ctctggaatg accatggatg cagagtcaga 720
gatcgatcct tgtaaagtag atggcacttg ccctgaagtt atcaaggtgt acatttttaa 780
agctgatcct ggagaggatg acttaggtgg gactgtagac attgtggaga gtgagcctga 840
gaatgatcat ggagttgaac tacttgatca gaataacagt attcgtgtgc caagagaaaa 900
gatggtttat atgactgtca acgactctca gcaagaagat gaagatttaa atgttgctga 960
aattgcagat gaagtttata tggaagtgat tgtaggagag gaggatgctg tgccgctgca 1020
gcagccgctg ccgcccatga agcagcagat ggatgacaac gaaatcaaaa ccttcatgcc 1080
gatagcatgg gcagcagctt acggtaataa ttatgatgga attgaaaacc ggaatggcac 1140
tgcaagtgcc ttcttgcaca tagatgagtc tgctgggctt ggcagactgg ctaaacaaaa 1200
accaaagaaa aggagaagac ctgattccag gcagtaccaa acagcaataa ttattggccc 1260
tgatggacat cccttgactg tctatccttg catgatttgt gggaaaaagt ttaagtcgag 1320
aggttttttg aaaaggcaca tgaaaaacca tcctgaacac cttaccaaga agaagtaccg 1380
ctgtactgac tgtgattaca ctaccaacaa gaagataagt ttacacaacc acctggagag 1440
ccacaagctt accagcaagg cggagaaggc cattgaatgc gatgagtgcg gaaagcattt 1500
ctctcatgct ggggctttgt ttactcataa aatggtgcat aaggaaaaag gagctaacaa 1560
aatgcacaaa tgtaaattct gtgaatacga gacagctgaa caagggttac tgaatcgcca 1620
ccttttggcg gtccatagca agaactttcc tcatatatgc gtggagtgtg gtaaaggttt 1680
tcgtcatcca tcagagctca aaaagcacat gcgaatccat actggcgaga agccgtacca 1740
gtgccagtac tgcgaatata ggtccgcaga ctcttctaac ttgaaaacgc atgtaaaaac 1800
taagcatagt aaagagatgc cattcaagtg tgacatttgt cttctgactt tctcagatac 1860
caaagaggtc cagcaacatg ctcttatcca ccaagaaagc aaaacacacc agtgtttgca 1920
ttgtgaccac aagagttcga actcaagcga tttgaaacga cacataattt cagttcacac 1980
aaaggactat ccccataagt gtgacatgtg tgataaaggc tttcataggc cttcagaact 2040
caagaaacat gtggctgccc acaagggtaa aaaaatgcac cagtgtagac attgtgactt 2100
taagattgca gatccttttg ttctaagtcg ccatattctc tcagttcaca caaaagatct 2160
tccgtttagg tgtaagagat gtagaaaggg atttaggcaa cagaatgagc ttaaaaagca 2220
tatgaagaca cacagtggca ggaaagtgta tcagtgtgag tactgtgagt atagcactac 2280
agatgcctca ggctttaaac ggcacgttat ttccattcat acaaaagact accctcaccg 2340
ttgtgagtac tgcaagaaag ggttccgaag accttcagaa aagaaccagc acataatgcg 2400
acatcataaa gaagttggcc tgccctaata atacttcggc agacttttgt agagatgttg 2460
gctttgaagc agaaaatcat tttaaaagcc agtcagtctc gttcacatac aatactgtat 2520
attgatttat gctgtgtaca aatagattta ttgcttttag ttgacttttt ttttttatat 2580
tttgttcaat agtgtgttct gaattctatt cagtttgttt aataaatggg gaaaagcagc 2640
aacaagcaag ttgcttttaa taaagtaatc cctgattcta tactgaattt ttctatctta 2700
gaagttttat atttatttaa atatttacct tgcttacctt gatggtactc ttctaagacc 2760
atttaactta aagttgaggt aattttagat tggtaactct gaaagtactc atgttgactt 2820
attttttccc ataaatttct cacaataaaa ttgtcagaga catctactaa cataaatggg 2880
agattttatg gtcaggtcta attatcctaa catggaagtc atttactctt cttgtttaat 2940
attttcagac cacgtgacaa tgaaagtttt cattcgagct tttgcgtccc tggcattgct 3000
gagtgaagag cagtggctgg gttcgggttt acctttcgtt ttgttcagca gacaaaatat 3060
cctttcaggg ggtgctttct ttgaagtatt tacacctttt gtcagcatag caaactttta 3120
gaaagcttta ctgaaaattt ttgcctgatc ctgttgtatt ttgtctgtgc tgctttgtgt 3180
tggaatgatt gtgtgctaca aatgagactt actgaggact gcatttggaa tctcctagag 3240
gtaattcgtg gctcatagga tcttttgcaa ctttatatat gtaaatgtac cctgacttat 3300
gtatatgcac atatatatga catgtatcat gtgtatcgtg tgtattgctt attttacata 3360
tttatacaca caatcccagt tagtagttgt ttaaaatcta taataatgag aagtattaaa 3420
tttacaataa catgaaagat gcagggatgc atgagagagc attttgtaaa tcatgctctt 3480
tggagagact actcaggtga agaattagaa ggaaaataag gacactagta ttttaaagag 3540
taaaggtatt ttcttttaaa tatctgtggt aactgaaaaa tagaacttaa gatgtttcta 3600
catagaatgt tttcatataa cttcagcttc atgcctttat attttcctga aaagctaatg 3660
agtatccaga tatatactcc ctcagttctc tctgagaagc ccgtgagggg actctgtgtc 3720
accaagtgag gggactaagg aagcagcggt tccatgtacc acagaatggg tgctaattac 3780
gacttcaact tggcaagttg agactgctcg ttacttctcc gttacagcta gcagcctttc 3840
aaaaacgtaa cactcacgtt ggctttgatt agaaggtaaa ggtgtattct cagtgcatga 3900
gaaaattttg aggccttatt tggcatatca ccaagtcttt cacagttgtg tttttcttta 3960
gcagttaact tttttttaac aaatagatat gattcagggt cataaataca tagaatgtac 4020
ttggttactt agaatttggt taaggtgaat tttggagaac agaaaatagg ttttggggtc 4080
tcgcttccct ggtggctcag aggttaaagc gtcttcctgc agtgcaggag acctgggtta 4140
gatccgtggg tcgggaagat cccctggaga aggaaatggc aacccactcc agtactcttg 4200
tctggagaat cccatggacg gaggagcctg gtgggctaaa gtccacaggg tcgcaaggac 4260
tcggacacga ctgaacgaat ccttagtaca ttggctgttg acacatgagt atctggcaca 4320
ttgtgggaga ttttggaatt tctttcccca cttgttagct gccttgccac ttcattatgg 4380
tgctctatcc cctttgtgct attaggtttt tagctttcat ctttgtcttt gccattgata 4440
cgtctttgca agagttatat ttttagggtt agaaatctaa tagtgtttgg caagcctctg 4500
aagtgctacg actgattgag tccatttcta aatcaagtgc tttaggctgg tacaaacacc 4560
atccttgaat gccagtgaaa gccaaggcat agaaacgcct gtgccctttg ccccccttaa 4620
cgtgcctttt ttttttaagt aacaatttcg tgattcattg ccctgcagta cagttgaaag 4680
ctctgtctgt ttttttgtga gaacctttaa aatctccctt aatttctatt tttcccacaa 4740
ataatgtaaa agaaaacaca cttaaagtag aaatttgtta attttaaaac atcctgtaaa 4800
aattaataca gaaaatataa ttggtcattt atattttttt aaactaactg aaagataaag 4860
aacacaacaa tttacacact ttatatttct cttacacggt ctggaatcat acaccattct 4920
ttccttttta aagcacaata ttgaaacctt taaaaggtat ttaagggttt ggtcaagtga 4980
atatgataaa gtgtatttgt ctgtataaag aaaaactgaa atccgtagtc actgttatgt 5040
actgacatta gttacaacct agttttaaat cttaaaatga ttttgattag caaagctaaa 5100
aggatgtttc agttaaatgt tttaaagagg tacagatttt tacaaggaca taatataagt 5160
tattgttctg tagaaatatc ctattaaata ttgtatgtcc ctccctctgt acactttgta 5220
aaaaaagtaa aatacataaa aaaaaaatca tatagggatg tgtgacatta ttgtaattgt 5280
gtacttgaga ataacgtgca aaaataaaaa tcagaatatt ttcctgtt 5328
<210> 2
<211> 55
<212> DNA
<213>Artificial sequence
<400> 2
tgtgaggact accttatgat ttcaagagaa tcataaggta gtcctcactt ttttc 55
<210> 3
<211> 59
<212> mRNA
<213>Artificial sequence
<400> 3
tcgagaaaaa agtgaggact accttatgat tctcttgaaa tcataaggta gtcctcaca 59
Claims (10)
1. a kind of RNAi interference fragments, it is characterised in that：
Described RNAi interference fragments comprise at least following sequences one or sequence two：
Sequence one：F：5′-TGTGA GGACT ACCTT ATGAT TTCAA GAGAA TCATA AGGTA GTCCT CACTT
TTTTC-3′；
Sequence two：R：5′-TCGAG AAAAA AGTGA GGACT ACCTT ATGAT TCTCT TGAAA TCATA AGGTA
GTCCT CACA-3 ',
Described RNAi interference fragments are used to disturb the Zfx genes on animal X chromosome.
2. a kind of RNAi interference fragments according to claim 1, it is characterised in that：
Described RNAi interference fragments are used to disturb the Zfx genes on red deer X chromosome.
3. a kind of application of RNAi interference fragments, it is characterised in that：
A kind of RNAi interference fragments according to claim 1 or 2, described RNAi interference fragments are used to prepare control animal
The medicine or kit of sex.
4. a kind of RNAi interference carriers, it is characterised in that：
Described RNAi interference carriers have the RNAi interference fragments described in claim 1,
Described RNAi interference carriers are used to disturb the Zfx genes on animal X chromosome.
5. a kind of RNAi interference carriers according to claim 4, it is characterised in that：
Described RNAi interference carriers are used to disturb the Zfx genes on red deer X chromosome.
6. a kind of RNAi interference carriers according to claim 4 or 5, it is characterised in that：
Described RNAi interference carriers include pLentiLox3.7 carriers, and the pLentiLox3.7 carriers are disturbed with the RNAi
Fragment is connected.
7. a kind of application of RNAi interference carriers, it is characterised in that：
Described RNAi interference carriers are used for sexual control in animal prefecundation, and described RNAi interference carriers are claim 4-
Described in 6 any one.
8. a kind of application of RNAi interference carriers according to claim 7, it is characterised in that：
The RNAi interference carriers are injected in animal body in the way of testis direct injection, for disturbing animal X chromosome
Zfx genes.
9. a kind of application of RNAi interference carriers according to claim 8, it is characterised in that：
The RNAi interference carriers are injected in the way of testis direct injection in red deer stags body, for disturbing red deer X to dye
Zfx genes on body.
10. a kind of preparation method of RNAi interference carriers, it is characterised in that：
The preparation method of described RNAi interference carriers comprises the following steps,
PLentiLox3.7 carriers described in digestion；
The RNAi interference fragments are connected with the pLentiLox3.7 carriers after digestion, connection product is obtained, wherein, institute
State RNAi interference fragments as claimed in claim 1；
The connection product is converted into competent cell, and filters out positive colony bacterium solution；
Sequencing identification is carried out to positive colony bacterium solution, and by sequencing result and the completely the same bacterium of the RNAi interference fragments sequence
Liquid is expanded；
RNAi interference carriers are extracted from the bacterium solution of the amplification.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN109735542A (en) * | 2019-01-24 | 2019-05-10 | 石河子大学 | RNAi interference fragment, interference carrier and its preparation method and application |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103131709A (en) * | 2013-02-18 | 2013-06-05 | 石河子大学 | Ribonucleic acid (RNA) interference fragment of zinc finger-x (zfx) gene and application of RNA interference fragment in mouse sex control |
| CN104232643A (en) * | 2014-08-25 | 2014-12-24 | 石河子大学 | RNAi (ribonucleic acid interference) interference segment, interference vector, and preparation method and application thereof |
| CN105255889A (en) * | 2015-09-30 | 2016-01-20 | 石河子大学 | RNAi fragment, RNAi vector, preparation method and application of RNAi vector |
-
2017
- 2017-06-27 CN CN201710506560.8A patent/CN107267511B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103131709A (en) * | 2013-02-18 | 2013-06-05 | 石河子大学 | Ribonucleic acid (RNA) interference fragment of zinc finger-x (zfx) gene and application of RNA interference fragment in mouse sex control |
| CN104232643A (en) * | 2014-08-25 | 2014-12-24 | 石河子大学 | RNAi (ribonucleic acid interference) interference segment, interference vector, and preparation method and application thereof |
| CN105255889A (en) * | 2015-09-30 | 2016-01-20 | 石河子大学 | RNAi fragment, RNAi vector, preparation method and application of RNAi vector |
Non-Patent Citations (5)
| Title |
|---|
| GENBANK: "KP257294.1", 《GENBANK》 * |
| QIANG PENG ET AL.: "Sex control by Zfy siRNA in the mouse", 《THERIOGENOLOGY》 * |
| 郭玉强 等: "猪Zfx基因RNAi片段的筛选", 《石河子大学学报(自然科学版)》 * |
| 马世俊 等: "高效抑制小鼠精子zfx基因shRNA分子的筛选", 《石河子大学学报(自然科学版)》 * |
| 魏丽敏 等: "马鹿ZFX/ZFY基因的克隆及序列分析", 《中国畜牧兽医》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109735542A (en) * | 2019-01-24 | 2019-05-10 | 石河子大学 | RNAi interference fragment, interference carrier and its preparation method and application |
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