CN102206676A - Vector for controlling gender of animals and application thereof - Google Patents

Abstract

The invention discloses a vector for controlling gender of animals and a preparation method thereof. The sequences of the vector provided by the invention contain one or two selected from the following segments of: SEQ 1:5'-AGCTTCAAATTCAAGAGATTTGAAGCTCACACTGTTGTTTTTTGGAAA-3' SEQ 2:5'-CAACAGTGTGAGCTTCAAATCTCTTGAATTTGAAGCTCACACTGTTGG-3'.

Description

A kind of other carrier of control animality and application thereof

Technical field

The present invention relates to control other carrier of animality, be specifically related to the siRNA recombinant expression vector and the application thereof of Zfy gene.

Background technology

For many years, people have carried out big quantity research to the difference that exists between X, the y sperm, and attempt to utilize these differences to come separated sperm, are fertilized with the sperm that obtains again, and the time of fertilization has just determined embryo's sex like this.Except the dna content of X, y sperm has difference can affirm, otherwise difference performance is very little or be difficult to determine but up to the present.The sperm that utilizes the fluidic cell partition method to obtain, though accuracy is higher, conception rate is lower, this instrument costs an arm and a leg in addition, and the popularization of this technology is restricted.Yet, if can find the specific mrna of X, y sperm key,, be expected to lower cost in conjunction with emerging RNAi technology in the present age, easy, realize sex control apace to animal.

The Zfy gene is that (testis-determining facter's testicular decisive factor in early days, candidate gene TDF), the zinc finger protein of its coding and nucleic acid play an important role in the embryo gender decision in conjunction with relevant.The Zfy gene is positioned on the Y chromosome galianconism (Yp11.3), and 11 exons and 1 13 " zinc-refer to " structures of repeat region are at random arranged ^[8], 801 amino acid are arranged approximately, 30 amino-acid residues are made of 2 halfcystines and 1 zine ion of 2 Histidine coordinations.It has two nuclear localization signals and DNA binding site, can specificly combine with target gene, and the guiding target gene passes nuclear membrane, be positioned in the spermatid nuclear, its proteins encoded may have certain function as transcription factor in the sperm forming process, relevant with the generation of sperm.

Summary of the invention

The objective of the invention is to make up with animal Zfy gene is the siRNA interference vector of target spot, reaches the purpose of man's activity offspring sex.

In order to realize purpose, the present invention takes following technical scheme:

One aspect of the present invention relates to a kind of other carrier of control animality, and its sequence comprises one or two in the following fragment:

SEQ?1：

5′-AGCTTCAAATTCAAGAGATTTGAAGCTCACACTGTTGTTTTTTGGAAA-3′

SEQ?2：

5′-CAACAGTGTGAGCTTCAAATCTCTTGAATTTGAAGCTCACACTGTTGG-3′

In a preferred embodiment of the present invention, above-mentioned SEQ 1 and/or SEQ 2 screenings are from the RNAi target sequence of mouse Zfy gene.

In a preferred embodiment of the present invention, described screening is that length is no more than 100 oligonucleotide chains from the RNAi target sequence fragment of mouse Zfy gene.

In a preferred embodiment of the present invention, above-mentioned carrier comprises the plasmid fragment.

The invention still further relates to the application of above-mentioned carrier in other medicine of preparation control animality, test kit, preferred animal is mouse or people.

The invention still further relates to the preparation method of above-mentioned carrier, comprising following steps:

From the RNAi target sequence of mouse Zfy gene, screen sequence;

In sequence, introduce restriction enzyme digestion sites;

Synthetic hair clip shape RNA and complementary strand thereof are connected in the known carrier of digestion with restriction enzyme closing chain.

In a preferred embodiment of the present invention, described preparation method also comprises the process of extracting plasmid.

In a preferred embodiment of the present invention, above-mentioned carrier is the pSilencer5.1-H1Retro carrier.

Description of drawings

Zfy gene mRNA expression situation behind Fig. 1 RNAi.

Embodiment

Below in conjunction with specific embodiment, the present invention is described in detail.

1.Zfy gene RNAi construction of recombinant vector

The RNAi target sequence of design Zfy gene has filtered out two siRNA (seeing Table 1).Respectively 5 ' and 3 ' end introduce the restriction enzyme site of restriction enzyme BamHI and HindIII, synthetic hair clip shape RNA (Short Hairpin RNA, shRNA) oligonucleotide chain and complementary strand thereof (seeing Table 2).

Respectively Zfy205 (control group) is closed chain with the upstream and downstream of Zfy2012 (experimental group), be connected into pSilencer5.1-H1Retro carrier (the Ambion Shanghai safe biotechnology of Ji company limited) again through BamHI and HindIII (TaKaRa company) double digestion, be built into siRNA expression vector pSilencer5.1/Zfy 205 (being called for short p205), pSilencer5.1/Zfy2012 (being called for short p2012), and be transformed in the E.coli Top10 competent cell (the biochemical company limited of sky, Beijing root), extract test kit (the biochemical company limited of sky, Beijing root) by specification operation with plasmid DNA and extract plasmid, behind EcoR I and Hind III double digestion evaluation recombinant plasmid, send Bioisystech Co., Ltd's order-checking.

Checking on the cell in vitro level shows, the RNAi technology of siRNA expression vector mediation can specificity suppress Zfy expression of gene in the mouse androgone.

Table 1. is used for the Zfy cDNA template of siRNA

Table 2. has the Zfy RNAi sequence of BamHI and HindIII restriction enzyme site

1.2.2Zfy RNAi test in the genosome

1.2.2.1Zfy the segmental importing of gene RNAi

Grope RNA interferential top condition in the body by pre-stage test, determine to adopt the testis local injection to import in the mouse body for g/ time through plasmid 40 μ of little fat parcel.64 male mouse are divided into 4 groups (16 every group) at random, and test I group, II group are injected recombinant plasmid p205 and p2012 respectively, and negative control group injection empty carrier pSilencer5.1-H1Retro organizes in contrast.The physiological saline of blank group injection equal volume.Injection in 10 days is once injected 4 times continuously at interval.Begin 32 female mouse abdominal injection PMSG 5U during last injection 15d, injection 5U HCG (the Ningbo second hormone factory) behind the 42-48h, then with every group of 8 male mouse by mating (this moment is apart from injecting plasmid 17d for the last time) at 1: 2.Morning next day, check the cloudy bolt of female mouse, see that the single cage of female mouse of cloudy bolt is raised the sex ratio of statistics first filial generation.Mate and simultaneously each is organized remaining 8 male mouse execution, get testis tissue rapidly and put into the liquid nitrogen flash freezer preservation.

1.2.2.2qRT-PCR measure testis Zfy gene mRNA abundance

Extract testis RNA with Trizol (American I nvitrogen) method, carry out reverse transcription.The reverse transcription total reaction volume is 20 μ L, system is: the total RNA of 2 μ g, 0.5mmol/L dNTP and 5 μ mol random primers, at 75 ℃ of following sex change 5 min, immediately put cooled on ice, add 20 U RNA enzyme inhibitorss (RNase inhibitor), 10U ThermoScript II (AMV RTase), 4 μ L, 5 * RT Buffer again, 42 ℃ of reaction 60 min are at last at 95 ℃ of following sex change 5 min.(primer information sees Table 3 for stratagene, MX3000P) the mRNA expression level of PCR method detection Zfy gene to utilize fluorescence real-time quantitative.

The cumulative volume of PCR reaction system is 25 μ L, wherein contains 12.5 μ L SYBR@Premix Ex TaqTM (2 *) (Dalian is precious biological), each 0.5 μ L of upstream and downstream primer (10 μ mol/L), and 2 μ L cDNA templates add ddH20 to final volume.Response procedures is: 95 ℃ of pre-sex change 30s, and 95 ℃ of sex change 5s, annealing 15s (annealing temperature sees Table 3), 72 ℃ are extended 15s, 40 circulations.Test is carried out 3 replications to all samples, and establishes negative control when each test.There is the pulsating plasmid of purpose to carry out behind the gradient dilution as standard substance the production standard curve clone.

Table 3 goal gene primer sequence and PCR condition

1.3 data statistics

Two calibration curve methods that the RT-PCR detected result adopts relative quantification to resolve are that internal standard gene carries out stdn with Gapdh, utilize the one-factor analysis of variance (one-way ANOVA, the significance of difference of experimental result between LSD) check group among the SPSS 13.0.Utilize the χ2Jian Yan method to carry out the significance of offspring mouse sex ratio difference between the statistical study group.

2 results

2.1Zfy the expression abundance of gene mRNA

As seen from Figure 1, utilize qRT-PCR to detect the expression level of the control group Zfy mRNA of test group and injecting normal saline and empty carrier behind RNAi.The result shows, injection recombinant vectors p205, and the Zfy mrna expression significantly is lower than control group (P＜0.05); Injection recombinant vectors p2012, the poor heteropole of Zfy mrna expression and control group is (P＜0.01) significantly; Significant difference (P＜0.05) between recombinant vectors p2012 group and the p205 group

2.2 offspring's sex ratio

As shown in Table 4, the sex of birth mouse is found by statistics, in the control group, 147 of products of injecting normal saline, 75 of female mouse (51.02%); 144 of products of injection empty carrier, 70 of female mouse (48.61%).146 of products of injection p205 in the test group, 81 of female mouse (55.47%); 145 of products of injection p2012,114 of female mouse (78.62%).The ratio of female of test group and control group compares, and the difference of injection p205 is not remarkable, and the difference of injection p2012 is (P＜0.01) extremely significantly, and two test group differences are (P＜0.05) significantly.

Table 4 is respectively organized offspring's male and female mouse quantity

Annotate: the right oblique angle of the ratio of female between two groups has the identical person of arbitrary letter not remarkable for difference, and capitalization represents that difference is extremely remarkable, and the lowercase alphabet differential is different significantly.

Should be understood that, for those of ordinary skills, can be improved according to the above description or conversion, and all these improvement and conversion all should belong to the protection domain of claims of the present invention.

Claims (8)

1. control other carrier of animality for one kind, it is characterized in that its sequence comprises one or two in the following fragment:

SEQ?1：

5′-AGCTTCAAATTCAAGAGATTTGAAGCTCACACTGTTGTTTTTTGGAAA-3′

SEQ?2：

5′-CAACAGTGTGAGCTTCAAATCTCTTGAATTTGAAGCTCACACTGTTGG-3′

2. carrier according to claim 1 is characterized in that the RNAi target sequence of described SEQ 1 and/or SEQ2 screening from mouse Zfy gene.

3. carrier according to claim 2 is characterized in that the RNAi target sequence fragment of screening from mouse Zfy gene is that length is no more than 100 oligonucleotide chains.

4. according to any described carrier of claim 1-3, it is characterized in that carrier comprises the plasmid fragment.

5. the application of any described carrier of claim 1-4 in other medicine of preparation control animality, test kit.

6. the preparation method of any described carrier of claim 1-4 is characterized in that comprising the steps:

From the RNAi target sequence of mouse Zfy gene, screen sequence;

In sequence, introduce restriction enzyme digestion sites;

Synthetic hair clip shape RNA and complementary strand thereof are connected in the known carrier of digestion with restriction enzyme closing chain.

7. preparation method according to claim 6 describedly is characterized in that also comprising the process of extracting plasmid.

8. preparation method according to claim 6 is characterized in that described known carrier is a pSilencer5.1-H1 Retro carrier.

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