CN109735542A - RNAi interference fragment, interference carrier and its preparation method and application - Google Patents

Abstract

The present invention is RNAi interference fragment, interference carrier and its preparation method and application.A kind of RNAi interference fragment, for interfering the Zfy gene on animal Y chromosome, the RNAi interference fragment includes following sequence: AGTTCAAACTCTAGTGATT；The albumen of Zfy negative gene responsible editor's code is related with the generation of sperm as transcription factor.RNAi interference fragment provided by the invention carries out silencing, obstruction, damage or the normal development for destroying y sperm by the zfy gene in the reproduction cell y sperm to bull.X sperm is given when mating with cow to be more fertilized chance, the gender of milk cow filial generation is effectively controlled in prefecundation to make the female sex rate of F1 generation calf increase significantly, and damage not will cause to required spermatoblast or reduce its vigor, and is low in cost.

Description

RNAi interference fragment, interference carrier and its preparation method and application

Technical field

The invention belongs to field of biotechnology, and in particular to a kind of RNAi interference fragment, RNAi interference carrier and RNAi are dry Disturb the preparation method and applications of carrier.

Background technique

Under field conditions (factors), the sex ratio of nearly all mammal is all close to 1:1, thus realize without interruption into Row steadily produces offspring, and reaches the ecological balance under the conditions of naturally.For husbandry sector reality, ideal sex ratio Example has a very important significance.It is (female to the higher husbandry sector of character requirement such as milk character (milk cow), egg-laying deseription Chicken) etc. Animal husbandries intentionally get more female individuals, and produce meat, fibre trait shows to obtain dominance on male It is more obvious.All the time, the mankind just have great interest to the gender of control animal offspring.Currently, the property of mammal It does not control and is mainly carried out from prefecundation and in terms of being fertilized latter two, the former is will to carry out again artificial insemination after spermatozoa isolation to make The time of fertilization just determines the gender of offspring；The latter uses Early-stage judgment, and the method for carrying out the gender screening of embryo is come Control the gender of offspring.

Mammal sex determination theory thinks that sex chromosome is that the fertilized eggs of " XX " can develop into female individuals, 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, can artificially control the gender of offspring, theoretically this be in sexual control most Simply, most economical approach.From after the 1950s, there are many research about spermatozoa isolation, these researchs all attempt to use X The sperm physical characteristic different with y sperm (volume, density, charge, motility)), using centrifugal process, electrophoresis, ion exchange The different methods such as method, cell surface antigen method realize the separation of sperm, but have common problem i.e. repeatable Property is poor.And these differences change with the difference of environmental condition, and therefore, many results of study are usually inconsistent, very To there is opposite conclusion, the certain Differences of two class sperms is caused to there is dispute.Up to the present, in addition to X, y sperm DNA Except there is content difference can affirm, otherwise difference shows very little, or even is difficult to determine.But with molecular biosciences The continuous development of technology and going deep into for research work, new research achievement will continue to bring out.

Currently, being separated according to the principle that mammal X, y sperm DNA content have differences using flow cytometer X, y sperm effect is best, and purity is up to 90% or more, but due to equipment valuableness, and separating rate is slow, the sperm count for fertilization The reasons such as few are measured, the wilderness demand in production to sexing semen is far from satisfying.In contrast, if it is possible to find two kinds of essences In son development or fertilization process or even when embryonic development critical X, the special mRNA of Y chromosome, in conjunction with the molecule that the present age is emerging Biology techniques are then expected to using the method for RNA interference (RNA Interference, RNAi) with lower cost, easy, fast The sexual control to animal is realized fastly.

RNAi is the gene silencing (Post-transcriptional Gene Silencing, PTGS) after a kind of transcription Phenomenon, it is special in the cell by external double-stranded RNA (Dubble-stranded RNA.dsRNA) that is artificial synthesized or generating in vivo Degrade its homologous mRNA anisotropicly, so that corresponding gene interference, realizes the expression for preventing target gene.RNAi method can Quickly, easy, effectively, specifically lower gene expression in cell, it does not study a kind of powerful of gene function still, And new technological means is provided for specific gene treatment, application prospect is very wide.

It is all proved in the research of genome, mRNA and protein level, X, y sperm gene expression have differences really.Gene Detection of expression studies have shown that spermiogenesis tail meiotic pachytene, 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 sex chromosome, the table of special gene on sex chromosome Up to being closed.Therefore, although containing mRNA in mammalian mature sperm, but the not transcription of mRNA, this species diversity are come From the transcription in spermatogenesis, however translates and far lag behind transcription.These mRNA are guidance synthesis after spermioteleosis Necessary albumen or after fertilization are to the supplement in the library egg mother cell mRNA, or as RNAi to fertility and embryo growth and development It plays a key role.

Hendriksen etc. to mouse the study found that during meiosis, it is nearly all in addition to Xist gene expression Sex chromosome gene all do not express, however meiosis anaphase, some sex chromosome specific genes start to express.Subtrahend point After splitting, Ubely and Sry gene mRNA level in-site with higher on Y chromosome is detected；Ubelx base is also detected that in X sperm Because of mRNA high level expression, while finding the expression product of X chromosome specific gene Mhr6A.In addition 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 (coding A kind of core GAP-associated protein GAP), Y chromosome specific gene Zfy-1, Zfy-2 and Y353/B.

Wherein, Zfx/Zfy gene is a pair of alleles of encoding zinc finger protein on chromosome, since meiophase Expression, the content highest in round spermatid.Zfx gene is a member in ZFY (zinc finger-Y) gene family, ZFY gene family includes Zfx, Zfy and Zfa gene, they are closely similar on molecular structure.General mammal have Zfx and Zfy gene, Zfx gene are located on X sex chromosome, are the genes that female, boar X chromosome must contain, and Zfy gene is located at On Y sex chromosome, two genes are located at the end of sex chromosome, but are not on homology region.Zfx gene includes an acidity Transcription excitement region, a nuclear localization sequence and a bonding pad DNA have 13 C2H2 [Cys (2) His (2)] type zinc fingers Structure.In mammalian cells, C2H2 zinc fingers are one of most common protein structures.Have now been found that more than 800 kinds Protein with this zinc fingers has important physiological function.The albumen of Zfx/Zfy coding as transcription factor, There is during spermiogenesis tail critical function and related with the generation of sperm.

Summary of the invention

In view of this, the embodiment of the present invention provides a kind of RNAi interference fragment, interference carrier and its preparation method and application. Main purpose is to be interfered by RNAi interference carrier the Zfy gene on animal Y chromosome, so that Zfy gene expression amount Decline, achievees the purpose that artificially to control Animal Sex with this.

In order to achieve the above objectives, present invention generally provides following technical solutions:

A kind of RNAi interference fragment, for interfering the Zfy gene on animal Y chromosome, the RNAi interference fragment includes Following sequence:

AGTTCAAACTCTAGTGATT；

The albumen of Zfy negative gene responsible editor's code is related with the generation of sperm as transcription factor.

Further, the RNAi interference fragment is used to interfere the Zfy gene on bull Y chromosome.

Further, a kind of RNAi interference fragment according to claim 1 or 2, the RNAi interference fragment are used In the drug or kit of preparation control Animal Sex.

On the other hand, a kind of RNAi interference carrier, the RNAi interference carrier have above-mentioned RNAi interference fragment；

The RNAi interference carrier is used to interfere the Zfy gene on animal Y chromosome.

Further, the RNAi interference carrier is used to interfere the Zfy gene on bull Y chromosome.

A kind of above-mentioned RNAi interference carrier, it is characterised in that:

The RNAi interference carrier includes pLentiLox3.7 carrier, the pLentiLox3.7 carrier with it is described The connection of RNAi interference fragment.

On the other hand, a kind of application of RNAi interference carrier,

The RNAi interference carrier is used for sexual control in animal prefecundation, and the RNAi interference carrier is above-mentioned RNAi interference carrier.

Further, the RNAi interference carrier is injected in animal body in a manner of testis direct injection, for interfering Zfy gene on animal Y chromosome.

Further, the RNAi interference carrier is injected in bull body in a manner of testis direct injection, for interfering Zfy gene on animal Y chromosome.

A kind of preparation method of RNAi interference carrier, includes the following steps:

(1) digestion pLentiLox3.7 carrier；

(2) the RNAi interference fragment is connect with the pLentiLox3.7 carrier after digestion, obtains connection product；

Wherein, the RNAi interference fragment sequence is as described in claim 1；

(3) 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 sequencing result and the RNAi interference fragment sequence is completely the same Bacterium solution expanded；

(4) RNAi interference carrier is extracted from the bacterium solution of the amplification.

Compared with prior art, the present invention at least has the advantage that

RNAi interference fragment provided by the invention is sunk by the zfy gene of y sperm in the reproduction cell to bull It is silent, obstruction, damage or the normal development for destroying y sperm.X sperm is given when mating with cow to be more fertilized chance, To make the mother calf ratio of F1 generation greatly improve.This method, which not will cause required spermatoblast, significantly to be damaged or reduces it Vigor, and it is low in cost.

Detailed description of the invention

Fig. 1 is ZfymRNA in RNAi interference fragment of the present invention, interference carrier and its preparation method and application in cell Expression；

Fig. 2 is the vector construction schematic diagram in RNAi interference fragment of the present invention, interference carrier and its preparation method and application.

Specific embodiment

For the present invention is further explained RNAi interference fragment, interference carrier and its preparation method and application, reach expected Goal of the invention, in conjunction with the preferred embodiment, to a kind of reinforced grape wine proposed according to the present invention and preparation method thereof, Specific embodiment, structure, feature and its effect, detailed description is as follows.In the following description, different " embodiment " or What " embodiment " referred to is not necessarily the same embodiment.In addition, special characteristic, structure or feature in one or more embodiments can be by Any suitable form combination.

A kind of reinforced grape wine of the present invention and preparation method thereof is cooked below in conjunction with specific embodiment further detailed It is thin to introduce:

Embodiment 1.

Design and synthesize the RNAi interference fragment for interfering the Zfy gene in animal testis androgone.This reality below Example is applied by taking the RNAi interference fragment of the Zfy gene in bull testis spermatogenic cell as an example, to technical solution provided by the invention into Row explanation.It is specific as follows:

3 siRNA fragment templates are designed according to the mRNA sequence of ox Zfy gene and filter out 1 siRNA fragment, wherein Table 1 is the ZfycDNA template for siRNA；

Table 1 is used for the ZfycDNA template of siRNA

Then according to the requirement of the pLentiLox3.7 carrier and institute's tape starting, the oligomerization core of 1 couple of shRNAi is synthesized Nucleotide sequence.The shRNAi interference fragment is shRNAA, as shown in table 2.

Wherein, used Zfy gene order is from NCBI gene pool, according to RNAi design principle and on NCBI It compares analysis result and filters out 3 siRNA fragments, as shown in table 1, the zfy cDNA template for siRNA.Respectively each Then the 5 ' of siRNA fragment and 3 ' addition Xhol I and I restriction enzyme site of Hpa synthesize siRNA fragment and filter out 1 couple of shRNAi Interference fragment, as shown in table 2.

The shRNA sequence of 2 pLL3.7-A interference carrier of table

Embodiment 2.

The screening technique of shRNAi interference fragment described in embodiment 1 is specific as follows:

Used zfy gene order is designed from NCBI gene pool (accession number: NM_177491.1) according to RNAi Principle and the comparison analysis result on NCBI filter out 3 siRNA fragments, as shown in table 1, the zfy cDNA for siRNA Template.Xhol I is added in the 5' and 3' of each siRNA fragment respectively and then I restriction enzyme site of Hpa send life as shown in table 3 Object company synthesizes shRNA segment.Positive and negative justice chain is combined into double-strand by annealing and is connected to plentilox3.7 (Addgene, the U.S.) (Tiangen, China) on slow virus packaging system carrier, is then transformed into E.coli DH5 α competent cell to be expanded, Extract -20 DEG C of plasmid preservations.

Table 3. has the milk cow Zfy gene shRNA sequence of HpaI and XhoI double enzyme site

2, the external interference test of zfy gene:

External interference test setting blank control group (with no treatment), empty carrier group (transfection empty carrier) and test group (transfecting pLL3.7/a, pLL3.7/b, pLL3.7/c respectively).

Acquiring 18 1 more than the monthly age, healthy adult bull testis, 75% alcohol drenches disinfection, dual anti-PBS soaking flushing, In cell culture room, sustentacular cell of testis and androgone are isolated using two step enzyme digestions.The cell that these are isolated Be put into the HamF12/DMEM culture solution containing 10% fetal calf serum, interior addition HEPES (15mol/L), 100kU/L penicillin with And 0.1g/L streptomysin, 1ug/ml epidermal growth factor, 100ulITS (insulin, transferrins, sodium selenate) { insulin (10 μ G/ml), transferrins (10 μ g/ml) }, 3.3 × 10^-7Mol/L retinoic acid, vitamin A (3.3 × 10^-7Mo/L), 10 μ g/ml are tieed up Raw element E, 10^-4Mol/L vitamin C, 10^-3Mol/L pyruvic acid, 10^-7Mol/L testosterone, 25U/LrFSH.About press 1 × 10⁶CELL/ cm²Density be inoculated in six orifice plates for being placed with coverslip, in 37 DEG C, 5%CO₂, under conditions of humidity is 95% culture it is 24 small When, use liposome 2000 that the carrier built is transfected into androgone as transfection reagent, every group is in triplicate.Transfection 48 The total serum IgE that androgone is extracted after hour, with the mRNA expression of RT-PCR detection zfy gene.It is as shown in table 4 purpose base Because of primer sequence and PCR condition.

4 Zfy gene primer sequence of table and PCR condition

3, interference test in the animal body of zfy gene:

It picks out the best pLL3.7/a of interference effect and carries out interference experiment in animal body.

The pLL3.7/a plasmid of extraction is diluted with dual anti-PBS solution is added, then presses the amount pair of 2.5mg/ pipe Plasmid is dispensed.Test ox uses 14 close at epididymis one third along the testis longitudinal axis according to the dosage of every side testis 2.5mg The injection of number puncture needle.When injection, marginal not penetrates the slow pumpback puncture needle in side, after injection, slowly extracts puncture needle, and to note Perforation strict sterilization measure.It is primary to be spaced 7d injection, co-injection 3 times.5d starts to acquire bull semen, system after third time is injected Make straw frozen semen, Liquid nitrogen storage.The wonderful mode manually inseminated of jelly of production is bred, registration breeding cow overbit, breeding It is date, for statistical analysis to the sex ratio quantity of offspring.

As a result as follows:

1, RT-PCR detects the expression of zfy gene mRNA in androgone:

As shown in Figure 1, as the result is shown: the table of zfy gene mRNA in test group pLL3.7/a, pLL3.7/b, pLL3.7/c Blank control group and empty carrier group are below up to level.Wherein pLL3.7/b, pLL3.7/c are compared with control group and empty carrier group Significant difference (P < 0.05)；PLL3.7/a and control group and empty carrier group comparing difference are extremely significant (P < 0.01).So choosing PLL3.7/a carries out interference test in animal body.

2, it participates in the experiment the sex ratio of bull offspring:

As shown in table 5, the female calf Rate for injecting the bull F1 generation calf of pLL3.7/a is 74.8%, F1 generation calf before injecting Female calf Rate is 49.2%, significant difference (P < 0.05).

5 F1 generation calf male and female sex ratio statistical form of table

Note: compared with the control group, the expression difference for being above designated as A is not significant；On be designated as B expression significant difference (P < 0.05)。

Embodiment 3.

A kind of RNAi interference carrier, including sequence are RNAi interference fragment, i.e. siRNA A shown in SEQ ID NO.1: AGTTCAAACTCTAGTGATT, preparation method include the following steps:

(1) digestion pLentiLox3.7 carrier；

(2) the RNAi interference fragment is connect with the pLentiLox3.7 carrier after digestion, obtains connection product；

Wherein, the RNAi interference fragment sequence is SEQ ID NO.1, i.e. siRNA A:AGTTCAAACTCTAGTGATT；

(3) connection product is converted into competent cell, with containing pLentiLox3.7 carrier, restriction enzyme site and Hairpin structure screens and obtained bacterium solution is carried out sequencing identification, and sequencing result and the RNAi interference fragment sequence is complete Complete consistent bacterium solution is expanded；

The restriction enzyme site is HpaI and XholI；The hairpin structure sequence is SEQ ID NO.2；

(4) RNAi interference carrier is extracted from the bacterium solution of the amplification.

Wherein vector construction schematic diagram is as shown in Figure 2.

The above is only the preferred embodiment of the embodiment of the present invention, not makees any shape to the embodiment of the present invention Limitation in formula, any simple modification to the above embodiments of technical spirit according to an embodiment of the present invention, equivalent variations With modification, in the range of still falling within technical solution of the embodiment of the present invention.

SEQUENCE LISTING

<110>Shihezi Univ

<120>RNAi interference fragment, interference carrier and its preparation method and application

<160> 3

<210> 1

<211> 19

<212> DNA

<213>artificial sequence

<400> 1

agttcaaact ctagtgatt 19

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<211> 55

<212> DNA

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tagttcaaac tctagtgatt ttcaagagaa tcactagagt ttgaactctt ttttc 55

<210> 3

<211> 2406

<212> DNA

<213> mRNA

<400> 3

atggatgaag atgaatttga attacagcca caggagccaa actcatgttt tgatggaata 60

ggaactgatg ctacacacat ggatggtgat caaattgttg tggaagtaca agaaactgtt 120

tttgtttcag atgttgtgga ttcagatata actgtgcata attttgttcc tgatgatcca 180

gactcagttg taatccaaga tgttattgag aatgttgtta ttgaggatgt tcagtgctca 240

gatatcttag aagaagcaga tgtgtctgaa aatgtcatca ttcctgagca aatgctttcc 300

tcagatgtaa cagaagaagt gtctttagca cattgcacag taccagatga cgtcttagct 360

tctgatatta cttcagcttc aatgtctatg ccagaacatg tcttgaccag tgagtctgta 420

catgtgtctg atgttggaca tgttgaacac attgttcatg gtagtgtagt agaggcagaa 480

atcgtcactg atcctctgac agccgacgta gtgtcagaag aagtattggt agcagattgt 540

gcctcagaag cagtcataga tgccaacgga atccctgtgg accagcagga tgatgacaaa 600

ggcaactgtg aggactacct tatgatttcc ttggatgatg atggcaaaat ggaacacgac 660

tgttcctctg gtatgaccat ggacgcagag tcggaaatcg atccttgtaa ggtggatggc 720

acttgccctg aagtcatcaa ggtgtatatt tttaaagctg accctggaga ggatgactta 780

ggtgggactg tcgacattgt ggagagtgag cctgagaatg atcatggagt tgaactcctt 840

gatcagaata acagtattcg tatgccaagg gaaaagatgg tttatatgac tgtcaatgac 900

tctcaacaag aagatgaaga tttaaatgtt gctgaaattg cagatgaagt ttatatggaa 960

gtgattgtag gagaggaaga tgctgctgtt gctgcagcag ctgctaccac tgttcatgaa 1020

caggaaatgg atgacagtga aatcaaaacc ttcatgccaa tagcatgggc agcagcttac 1080

ggtaataatt ctgatggaat tgaaaatcgg agtggcactg caagtgctct cttgcacata 1140

gatgagtctg ctggacttgg cagactgact aaacataaac caaagaaaag gagaagacct 1200

gattccaggc agtaccaaac agcaataatt attggtcctg atggacatcc cttgactgtc 1260

tatccttgta tgatttgtgg gaaaaaattt aagtcgagag gttttttgaa aaggcacatg 1320

aaaaaccatc ctgaacacct taccaaaaag aagtaccgct gtactgattg tgattacact 1380

accaataaga agataagttt acacaatcac ctggagagcc acaagcttac cagcaagtca 1440

gagaaggcca tcgaatgtga tgactgtggg aagcatttct cccatgctgg ggctttgttc 1500

actcacaaaa tggtgcataa ggaaaaagga gccagcaaaa tgcataaatg taaattctgt 1560

gagtatgaga cagctgaaca agggttatta aatcgccacc ttttggcagt ccacagcaag 1620

aactttcccc atatatgtgt agagtgtggt aaaggttttc gtcacccatc agagctcaaa 1680

aagcacatgc gaatccatac tggagagaaa ccgtaccaat gccagtactg cgaatatagg 1740

tctgcagact cttctaattt gaagacgcat gtgaaaacta agcatagtaa agaaatgtct 1800

ttcaagtgtg acatttgtct tctgactttc tccgatacca aagaggttca gcaacatgct 1860

cttatccacc aagaaagcaa aacacaccag tgcgtgcatt gtgaccacaa gagttcaaac 1920

tctagtgatt tgaaacgtca cataatttca gttcacacaa aggactaccc ccataagtgt 1980

gacatgtgtg ataaaggctt tcacaggcca tcagaactca agaagcatgt ggctgcccac 2040

aagggtaaaa aaatgcacca gtgtagacat tgtgacttta agattgcaga tccatttgtt 2100

ctaagtcgcc atattctctc agttcacacg aaagatcttc cgtttaggtg taagagatgt 2160

aaaaagggat ttaggcaaca gaatgagctt aaaaagcata tgaagacaca cagtggcagg 2220

aaagtgtatc agtgtgagta ctgtgaatat agcactacag atgcctcagg ctttaaacgg 2280

catgttattt ccattcatac aaaagactat cctcaccgtt gtgagtactg caagaaaggg 2340

ttccgaagac cttcagaaaa gaaccagcat ataacacgac accataaaga agttggtctg 2400

ccctaa 2406

Claims (10)

1. a kind of RNAi interference fragment, for interfering the Zfy gene on animal Y chromosome, which is characterized in that the RNAi interference Segment includes following sequence:

AGTTCAAACTCTAGTGATT；

The albumen of Zfy negative gene responsible editor's code is related with the generation of sperm as transcription factor.

2. RNAi interference fragment described in claim 1, it is characterised in that:

The RNAi interference fragment is used to interfere the Zfy gene on bull Y chromosome.

3. a kind of application of RNAi interference fragment, it is characterised in that:

A kind of RNAi interference fragment according to claim 1 or 2, the RNAi interference fragment are used to prepare control animal The drug or kit of gender.

4. a kind of RNAi interference carrier, which is characterized in that

The RNAi interference carrier has RNAi interference fragment described in claim 1；

The RNAi interference carrier is used to interfere the Zfy gene on animal Y chromosome.

5. RNAi interference carrier described in claim 1, it is characterised in that:

The RNAi interference carrier is used to interfere the Zfy gene on bull Y chromosome.

6. a kind of RNAi interference carrier according to claim 4 or 5, it is characterised in that:

The RNAi interference carrier includes pLentiLox3.7 carrier, and the pLentiLox3.7 carrier and the RNAi are dry Disturb segment connection.

7. a kind of application of RNAi interference carrier, it is characterised in that:

The RNAi interference carrier is used for sexual control in animal prefecundation, and the RNAi interference carrier is claim 4- Described in 6 any one.

8. a kind of application of RNAi interference carrier according to claim 7, which is characterized in that

The RNAi interference carrier is injected in animal body in a manner of testis direct injection, for interfering animal Y chromosome Zfy gene.

9. a kind of application of RNAi interference carrier according to claim 8, which is characterized in that

The RNAi interference carrier is injected in bull body in a manner of testis direct injection, for interfering animal Y chromosome Zfy gene.

10. a kind of preparation method of RNAi interference carrier, which comprises the steps of:

(1) digestion pLentiLox3.7 carrier；

(2) the RNAi interference fragment is connect with the pLentiLox3.7 carrier after digestion, obtains connection product；

Wherein, the RNAi interference fragment sequence is as described in claim 1；

(3) connection product is converted into competent cell, and filters out positive colony bacterium solution；

Sequencing identification carried out to positive colony bacterium solution, and by sequencing result and the completely the same bacterium of the RNAi interference fragment sequence Liquid is expanded；

(4) RNAi interference carrier is extracted from the bacterium solution of the amplification.