CN112063657A

CN112063657A - Method for evaluating sheep oocyte in-vitro culture system by using MPP1 gene

Info

Publication number: CN112063657A
Application number: CN202010979195.4A
Authority: CN
Inventors: 曹鸿国; 陶金萍; 周铭生; 陈宏权; 周建军
Original assignee: Anhui Province Tianchang City Zhoushi Sheep Co ltd
Current assignee: Anhui Province Tianchang City Zhoushi Sheep Co ltd
Priority date: 2020-09-17
Filing date: 2020-09-17
Publication date: 2020-12-11

Abstract

The invention discloses a method for evaluating a sheep oocyte in-vitro culture system by utilizing an MPP1 gene, which is characterized in that a gene amplification primer is designed in a promoter region of an MPP1 gene of an X chromosome of a sheep, and the evaluation method of the sheep oocyte in-vitro culture system is established by a molecular marker gene MPP 1.

Description

Method for evaluating sheep oocyte in-vitro culture system by using MPP1 gene

Technical Field

The invention relates to the technical field of bioengineering and gene molecular marking, in particular to a method for evaluating a sheep oocyte in-vitro culture system by utilizing an MPP1 gene.

Background

The sheep has the characteristics of moderate physique, high growth speed and the like, the whole body of the sheep is precious, the mutton is tender, contains high protein and rich vitamins, and the sheep can tonify qi, supplement deficiency, promote blood circulation and enhance body resistance after being eaten frequently. Compared with cow milk, goat milk is rich in more fat and protein, is an ideal food for patients with kidney diseases, and is a natural supplement for weak people. Wool is an important raw material in textile industry, and has the advantages of good elasticity, strong hygroscopicity, good heat retention property and the like. In addition, the sheep manure is a good organic fertilizer after fermentation, and the sheep manure can be used as a fertilizer to improve soil quality, prevent soil hardening and has good economic value. With the continuous improvement of the living standard of people, people need sheep varieties with different characteristics to meet the increasing material demand of people. Therefore, the cultivation of sheep breeds meeting different requirements of people increasingly becomes the era requirement of the development of modern animal husbandry. The improvement and the cultivation of sheep varieties and the related research of the basic reproductive development without leaving sheep oocytes, at present, ovary tissues abandoned after sheep slaughter, ovary tissues in aborted fetuses, and the like are high-quality sheep oocyte resources. Therefore, an ovary tissue culture system is established by utilizing the ovary tissue abandoned after slaughtering or the ovary tissue in the aborted foetal sheep, and a large amount of important experimental materials can be provided for the relevant research on the development of the sheep oocyte.

With the progress of scientific technology, modern cell engineering and molecular engineering technologies can greatly accelerate the variety improvement and the breeding speed of sheep, so a molecular marker gene is searched, a method for evaluating the in vitro culture quality of sheep oocytes by using the molecular marker gene is explored, the in vitro culture system of the sheep oocytes can be effectively established, the optimization of the in vitro culture system of the sheep oocytes is realized, a high-efficiency culture system of the sheep oocytes is established, the high-quality breeding of sheep is promoted, and the method plays an important role in developing and utilizing the sheep oocytes and rapidly improving the breeding quality of the sheep.

Disclosure of Invention

The invention aims to: provides a method for evaluating a sheep oocyte in-vitro culture system by using an MPP1 gene, so as to solve the defects.

In order to achieve the above purpose, the invention provides the following technical scheme:

the method for evaluating the sheep oocyte in-vitro culture system by utilizing the MPP1 gene is characterized in that the MPP1 gene is a molecular marker gene MPP1 of sheep X chromosome, and the nucleotide sequence of the MPP1 gene is shown in SEQ ID NO. 1.

Preferably, the MPP1 gene is sheep Chromosome X-NC-040278.1 MPP1 gene.

Preferably, the promoter region of the MPP1 gene of sheep X chromosome is selected to design gene amplification primers.

Preferably, the method comprises the following steps:

s1, extraction of DNA genome of sheep ovarian tissue: taking sheep ovarian tissues, smashing the sheep ovarian tissues to form cell suspension, and preparing a sheep ovarian tissue DNA genome by using a DNA extraction kit;

amplification of S2 and sheep MPP1 gene promoters: designing sheep MPP1 gene promoter amplification primers by using Vector NTI advanced 11.5.1 software, synthesizing the primers after adding restriction sites and protecting bases, amplifying by adopting PCR (polymerase chain reaction), recovering an MPP1 gene promoter sequence by adopting agarose gel electrophoresis to prepare a sheep MPP1 gene promoter sequence, and selecting an MPP1 gene promoter to amplify a correct sequence after sequencing the promoter sequence;

s3 and construction of an MPP1 gene promoter pLV-mCherry reconstruction vector: respectively using restriction enzymes Cla I and BamH I to carry out enzyme digestion on a sheep MPP1 gene promoter sequence and a pLV-mCherry vector, respectively recovering the MPP1 gene promoter sequence and the pLV-mCherry vector by adopting agarose gel electrophoresis, connecting the MPP1 gene promoter sequence and the pLV-mCherry vector by using T4 ligase, and constructing a pLV-mCherry reconstructed vector of which the MPP1 gene starts mCherry expression; carrying out PCR identification on thalli containing the reconstruction vector by using a sheep MPP1 gene promoter identification primer so as to detect whether the construction of the MPP1 gene promoter pLV-mCherry reconstruction vector is correct or not;

s4, mediated MPP1 gene promoter-mCherry transfection virus package: packaging and transfecting a virus of pLV-MPP1 gene promoter-mCherry plasmid by using a cell culture system, and collecting a packaging virus culture solution;

s5, in vitro culture of sheep ovarian tissue: collecting sheep ovary tissue, removing ovary primary follicle, culturing ovary primary follicle in culture dish microdroplet, and standing at 37 deg.CContaining 5% CO₂Culturing in a saturated humidity incubator, and after the ovary primary follicle adheres to the wall, adopting a packaging virus mediated MPP1 gene promoter-mCherry expression transgenic technology;

expression of S6, MPP1 gene promoter-mCherry: centrifuging, filtering and resuspending the packaged virus culture solution collected in S4, adding an infection enhancer Polybrene, and uniformly mixing to obtain virus infection solution containing MPP1 gene promoter-mCherry; absorbing the culture microdroplet of the ovary primary follicle adherent prepared by S5, discarding a follicle culture solution, adding the prepared virus infection solution containing the MPP1 gene promoter-mCherry for infection culture, absorbing the virus infection solution containing the MPP1 gene promoter-mChery, adding a follicle culture solution for culture, and selecting the ovary primary follicle strongly expressing red fluorescent protein according to the expression condition of the ovary primary follicle mChery red fluorescent protein for oocyte microdroplet maturation culture; according to the expression level of the mCherry red fluorescent protein of the oocyte, the in-vitro culture system of the sheep oocyte can be evaluated by combining the growth and development conditions of the primary follicle of the sheep ovary and the oocyte, and the whole process of evaluating the in-vitro culture system of the sheep oocyte by utilizing the molecular marker gene MPP1 is completed.

Preferably, a sheep MPP1 gene promoter amplification primer before the enzyme cutting site is added, and the nucleotide sequences of an upstream amplification primer and a downstream amplification primer are respectively as follows:

an upstream primer F: 5'-ACTTTGTTATAGGTTGCGCG-3', respectively;

a downstream primer R: 5'-TCATCTTATGGGAGAGCCGG-3', respectively;

the gene sequence length of the MPP1 gene promoter is 2008 bp.

Preferably, the restriction enzyme site of the upstream amplification primer is Cla I, and the nucleotide sequence of the upstream amplification primer of the sheep MPP1 gene promoter after the restriction enzyme site of Cla I is added is as follows:

F：5’-ATCGATACTTTGTTATAGGTTGCGCG-3’；

the enzyme cutting site of the downstream amplification primer is BamH I, and the nucleotide sequence of the downstream amplification primer of the MPP1 gene promoter after the BamH I enzyme cutting site is added is as follows:

R：5’-GGATCCTCATCTTATGGGAGAGCCGG-3’。

preferably, the protection base of the upstream amplification primer is AGC, after the Cla I enzyme cutting site is added to the upstream amplification primer, the protection base AGC is continuously added to the 5' end of the primer, and the nucleotide sequence after the protection base is added is as follows:

F：5’-AGCATCGATACTTTGTTATAGGTTGCGCG-3’；

the protective base of the downstream amplification primer is CGT, after the downstream primer adds a BamH I restriction enzyme site, the protective base CGT is continuously added at the 5' end of the primer, and the nucleotide sequence after the protective base is added is as follows:

R：5’-CGTGGATCCTCATCTTATGGGAGAGCCGG-3’。

preferably, in the step S3, primers for identifying the promoter of the sheep MPP1 gene are designed by using primer design software premier 6.0;

the nucleotide sequences of an upstream identification primer and a downstream identification primer of the MPP1 gene promoter identification primer are respectively as follows:

an upstream identifying primer F: 5'-AGCCATCCATCAATCACA-3' the flow of the air in the air conditioner,

downstream identifying primer R: 5'-CTCGCAGTATTACAAGCAG-3' the flow of the air in the air conditioner,

the identified fragment length is 159 bp.

The invention has the beneficial effects that:

the method for evaluating the in vitro culture quality of the sheep oocyte utilizes the MPP1 gene, selects the promoter region of the MPP1 gene of the sheep X chromosome to design a gene amplification primer, establishes the evaluation method of the in vitro culture system of the sheep oocyte through the molecular marker gene MPP1, has high evaluation efficiency, is simple and convenient, lays a foundation for the optimization of the in vitro culture system of the sheep oocyte and the establishment of the oocyte culture system, effectively promotes the high-quality breeding of sheep, and has great popularization value.

Detailed Description

To facilitate understanding of those skilled in the art, the present invention will be further described with reference to specific examples.

A method for evaluating a sheep oocyte in vitro culture system by utilizing an MPP1 gene comprises the following steps:

s1, extraction of DNA genome of sheep ovarian tissue:

taking sheep ovarian tissues, smashing the sheep ovarian tissues to form cell suspension, and preparing a sheep ovarian tissue DNA genome by using a DNA extraction kit (a product of Tiangen reagent company) according to a genome DNA extraction operation instruction, wherein the method comprises the following specific steps:

(1) taking sheep ovary tissues to break up to form cell suspension, then centrifuging at 12000rpm for 1min, pouring out supernatant, adding 200 mu l of buffer solution GA, oscillating until complete suspension, adding 20 mu l of protease K, uniformly mixing, standing at 56 ℃ until the tissues are dissolved, and centrifuging briefly to remove water drops on the inner wall of a tube cover.

(2) Adding 200 μ l buffer solution GB, fully reversing and mixing, standing at 70 deg.C for 10min, cleaning the solution, and centrifuging briefly to remove water droplets on the inner wall of the tube cover; add 200. mu.l of absolute ethanol, mix well for 15s with shaking, at which time a flocculent precipitate may appear, and centrifuge briefly to remove water droplets on the inner wall of the tube cap.

(3) Adding the solution and flocculent precipitate obtained in the previous step into an adsorption column CB3 (the adsorption column is placed into a collecting pipe), centrifuging at 12000rpm for 30s, pouring off waste liquid, and placing adsorption column CB3 back into the collecting pipe. Add 500. mu.l buffer GD (check whether absolute ethanol was added before use) to adsorption column CB3, centrifuge at 12000rpm for 30s, discard, place adsorption column CB3 in the collection tube.

(4) Adding 600 μ l of rinsing solution PW (checking whether anhydrous ethanol is added before use) into adsorption column CB3, centrifuging at 12000rpm for 30s, pouring off waste liquid, and placing adsorption column CB3 into a collection tube; repeating the previous step; putting the adsorption column CB3 back into the collecting pipe, centrifuging at 12000rpm for 2min, and pouring out waste liquid; placing the adsorption column CB3 at room temperature for a plurality of minutes to thoroughly dry the residual rinsing liquid in the adsorption material; transferring the adsorption column CB3 into a clean centrifugal tube, suspending and dripping 50-200 mu l of elution buffer TE into the middle part of the adsorption film, standing at room temperature for 2-5 min, centrifuging at 12000rpm for 2min, and collecting the solution into the centrifugal tube, thereby preparing the sheep ovarian tissue DNA genome.

Amplification of S2 and sheep MPP1 gene promoters:

designing sheep MPP1 gene promoter amplification primers by using Vector NTI advanced 11.5.1 software, synthesizing the primers after adding restriction sites and protecting bases, amplifying by adopting PCR (polymerase chain reaction), and recovering an MPP1 gene promoter sequence by adopting agarose gel electrophoresis to prepare the sheep MPP1 gene promoter sequence, wherein the specific steps are as follows:

(1) sheep MPP1 gene promoter amplification primer design

The sheep MPP1 gene is a molecular marker gene of sheep X Chromosome, in particular to an MPP1 gene of sheep Chromosome X-NC-040278.1, and the nucleotide sequence of the MPP1 gene is shown in SEQ ID NO. 1.

The promoter region of MPP1 gene of sheep X chromosome was selected to design gene amplification primers using Vector NTI Advance11.5.1 software.

Firstly, adding sheep MPP1 gene promoter amplification primers before enzyme cutting sites, wherein the nucleotide sequence of the upstream amplification primer is shown as SEQ ID NO.2, and the specific nucleotide sequence is as follows: an upstream primer F: 5'-ACTTTGTTATAGGTTGCGCG-3', respectively;

the nucleotide sequence of the downstream amplification primer is shown as SEQ ID NO.3, and the specific nucleotide sequence is as follows: a downstream primer R: 5'-TCATCTTATGGGAGAGCCGG-3', respectively;

the gene sequence length of the MPP1 gene promoter is 2008 bp.

Then, the restriction enzyme site of the upstream amplification primer is Cla I, the nucleotide sequence of the upstream amplification primer of the sheep MPP1 gene promoter after the restriction enzyme site of Cla I is added is shown as SEQ ID NO.4, and the specific nucleotide sequence is as follows:

F：5’-ATCGATACTTTGTTATAGGTTGCGCG-3’；

the enzyme cutting site of the downstream amplification primer is BamH I, the nucleotide sequence of the downstream amplification primer of the MPP1 gene promoter after the BamH I enzyme cutting site is added is shown in SEQ ID NO.5, and the specific nucleotide sequence is as follows:

R：5’-GGATCCTCATCTTATGGGAGAGCCGG-3’。

and finally, the protective base of the upstream amplification primer is AGC, after the Cla I enzyme cutting site is added to the upstream amplification primer, the protective base AGC is continuously added to the 5' end of the primer, the nucleotide sequence after the protective base is added is shown as SEQ ID NO.6, and the specific nucleotide sequence is as follows: f: 5'-AGCATCGATACTTTGTTATAGGTTGCGCG-3', respectively;

the protective base of the downstream amplification primer is CGT, after the downstream primer adds a BamH I restriction enzyme site, the protective base CGT is continuously added at the 5' end of the primer, the nucleotide sequence after adding the protective base is shown as SEQ ID NO.7, and the specific nucleotide sequence is as follows:

R：5’-CGTGGATCCTCATCTTATGGGAGAGCCGG-3’。

(2) amplification of sheep MPP1 gene promoter:

taking sheep MPP1 gene promoter upstream primer

F: 5'-AGCATCGATACTTTGTTATAGGTTGCGCG-3', respectively; a downstream primer R: the 5'-CGTGGATCCTCATCTTATGGGAGAGCCGG-3', RBP1 gene promoter sequence fragment is 2008 bp. Primers were prepared into 100. mu.M stock solution and 10. mu.M working solution by adding sterile double distilled water according to the primer synthesis instructions.

The MPP1 gene promoter was PCR-amplified, and the following reaction products were added to a sterilized PCR amplification tube to prepare a 50. mu.l reaction system. Easy Taq DNA polymerase is a product of Beijing Quanjin Biotechnology Limited.

The PCR amplification reaction conditions were as follows, for a total of 35 cycles.

Preheating at 94 deg.C for 5min, denaturation at 94 deg.C for 30s, annealing at 55 deg.C for 30s, extension at 72 deg.C for 2min and 30s, performing 35 cycles, extension at 72 deg.C for 10min, and storing at 4 deg.C.

(3) Recovery of MPP1 gene promoter sequence by agarose gel electrophoresis

The preparation method of the 1% agarose gel plate is as follows: a200 ml Erlenmeyer flask is taken, 0.7g of agarose is weighed, 70ml of 1 XTAE buffer solution is added, and the mixture is placed into a microwave oven to be heated (high fire for 3min) until the agarose is melted and transparent. Cooling to about 50 ℃, adding EB (ethidium bromide) to the final concentration of 0.5 mu g/ml, pouring into a gel tank, selecting proper comb teeth, taking out the comb and the partition plate after cooling and forming, putting into a horizontal electrophoresis tank, pouring 1 XTAE solution into the electrophoresis tank, and submerging the gel by buffer solution for 1-2 mm.

The Marker 20006 μ l was added to the first well of the gel plate and the sample was added to the subsequent well by mixing a small amount of 6 XDNA loading buffer in the amplification tube and adding 50 μ l of the corresponding amplified gene to the subsequent well. And (3) carrying out electrophoresis by adopting the conditions of U160V, I200 mA and T30 min, and turning off the power supply when the bromophenol blue moves to the distance of 1/2-2/3 of the gel. And (4) taking out the gel block after the electrophoresis is finished, and detecting and analyzing by using an ultraviolet analyzer and a gel imager.

And comparing the size of the MPP1 gene promoter with that of the DNA Marker 2000 fragment, and recovering the MPP1 gene promoter sequence, wherein the MPP1 gene promoter sequence fragment is 2008 bp. The agar blocks containing the corresponding gene fragments were cut with a surgical blade in an ultraviolet transreflective analyzer and placed in a 1.5ml centrifuge tube. The DNA fragment of the corresponding gene was recovered according to the protocol of DNA gel recovery kit (Hangzhou thinking Biotechnology Co., Ltd.). The method comprises the following specific steps:

firstly, cutting an MPP1 gene promoter sequence gel strip under the condition of purple fluorescence, and putting the MPP1 gene promoter sequence gel strip into a 1.5ml centrifuge tube.

Secondly, adding 1ml of the binding buffer solution into a centrifugal tube containing the MPP1 gene promoter sequence gel belt, carrying out water bath at 75 ℃ for 10min to melt the gel, and uniformly mixing the gel once every 2min when heating to melt the gel.

Thirdly, transferring the melted glue solution to a UNIQ-10 adsorption column sleeved in a 2ml collection tube, standing for 2min at room temperature, and centrifuging for 1min at 8000rpm at room temperature.

And fourthly, taking down the UNIQ-10 adsorption column, pouring off waste liquid in the collecting pipe, putting the UNIQ-10 adsorption column into the same collecting pipe, adding 750ml of eluent, and centrifuging at room temperature of 8000rmp for 1 min.

Fifthly, repeating the step (iv).

Sixthly, taking down the UNIQ-10 adsorption column, pouring the waste liquid in the collecting pipe, putting the UNIQ-10 adsorption column into the same collecting pipe, and centrifuging at the room temperature of 12000rmp for 1 min.

Seventhly, putting the UNIQ-10 adsorption column into a new centrifuge tube, adding 30 mu l of eluent into the center of the column membrane, and standing at room temperature for 2 min.

And (3) centrifuging at 12000rpm for 1min at room temperature, wherein the liquid in the centrifuge tube is the recovered DNA fragment, and selecting a gene promoter to amplify a correct sequence after sequencing the recovered DNA fragment.

S3 and construction of an MPP1 gene promoter pLV-mCherry reconstruction vector:

the MPP1 gene promoter sequence and pLV-mCherry vector are cut by enzyme digestion, the MPP1 gene promoter sequence and pLV-mCherry vector are cut by restriction enzyme Cla I (Thermo product) and BamH I (Thermo product), 50 mul enzyme digestion system is used, the system composition is as follows:

after mixing, the mixture is digested for 3h at 37 ℃.

And after enzyme digestion, respectively recovering the MPP1 gene promoter sequence and the pLV-mCherry vector by adopting 1% agarose gel electrophoresis, and connecting the MPP1 gene promoter sequence and the pLV-mCherry vector by using T4 ligase to construct a reconstructed vector. The specific operation steps are as follows:

mixing the MPP1 gene promoter sequence and pLV-mCherry vector components after enzyme digestion in a microcentrifuge tube (200 mul PCR amplification tube) uniformly, wherein the reaction system is as follows:

a total volume of 5. mu.l of ligation reaction was constructed and ligated overnight at 16 ℃.

The ligation product was transformed into Stbl3 competent cells by the following specific procedures:

first, 50. mu.l of the cells were frozen in competent cells at-70 ℃ and thawed at room temperature, and immediately placed on ice after thawing.

② taking 1 mul of the ligation product, shaking up gently, and placing on ice for 30 min.

Thirdly, thermally shocking the mixture for 90s in a water bath at 42 ℃, and rapidly placing the mixture on ice for cooling for 2-3 min.

Fourthly, adding 500 mul of LB liquid culture medium into the tube in a super clean bench, evenly mixing, and then shaking for 45 min-1 h on a shaking table at 200rpm and 37 ℃.

Fifthly, spreading the bacterial liquid on a LB culture medium plate with 90mm containing ampicillin, and lightly beating the plate to make the distribution uniform.

Sixthly, culturing for 12-16 h in a constant-temperature incubator at 37 ℃.

Taking a plate connected with the transformed colony, and carrying out the following operations in a super clean bench:

firstly, 90ml of prepared TB culture medium, 10ml of dipotassium phosphate buffer solution and potassium dihydrogen phosphate buffer solution and 100 mu l of 50mg/ml Amp solution are mixed in a reagent bottle.

② marking 10 sterilized 10ml centrifuge tubes.

③ taking 2ml of the mixed TB culture medium, putting the mixed TB culture medium into a centrifuge tube with the number of 1, and repeating the process until the number is 10.

And fourthly, clamping toothpicks (sterilized) by using forceps of the ophthalmology department, picking bacterial colonies from the flat plate, putting the bacterial colonies and the toothpicks into the centrifuge tube, picking round bacterial colonies, putting each bacterial colony into one centrifuge tube, and putting 10 bacterial colonies into 10 centrifuge tubes in total.

And fifthly, putting 10 centrifuge tubes into a water bath constant temperature oscillator, and oscillating for 6-7 hours at 200rpm and 37 ℃.

A sheep MPP1 gene promoter identification primer is designed by adopting primer design software premier 6.0, the designed MPP1 gene promoter identification primer has the nucleotide sequence shown as SEQ ID NO.8, and the specific nucleotide sequence is as follows:

an upstream identifying primer F: 5'-AGCCATCCATCAATCACA-3', respectively;

the nucleotide sequence of the downstream identification primer is shown as SEQ ID NO.9, and the specific nucleotide sequence is as follows: downstream identifying primer R: 5'-CTCGCAGTATTACAAGCAG-3', which identifies a fragment of 159bp in length.

Primers were prepared into 100. mu.M stock solution and 10. mu.M working solution by adding sterile double distilled water according to the primer synthesis instructions.

PCR identification of thallus containing a reconstruction vector comprises the following specific operation steps: the following reaction mixture was added to a sterilized PCR amplification tube to prepare a 25. mu.l reaction system.

The PCR amplification reaction conditions are as follows, preheating at 94 ℃ for 5min, denaturation at 94 ℃ for 30s, annealing at 55 ℃ for 30s, extension at 72 ℃ for 2min for 30s, 35 cycles, extension at 72 ℃ for 10min, and storage at 4 ℃. For a total of 25 cycles. And (3) carrying out 1% agarose gel electrophoresis on the thallus PCR product, and identifying whether the MPP1 gene promoter-mCherry reconstructed vector bacteria are positive or not in an ultraviolet analyzer according to the existence and the approximate length of the amplified fragment. In a clean bench, 0.8ml of the positive bacteria liquid is taken out and put into a centrifugal tube of 1.5ml, 0.2ml of 80% glycerol is added, and the mixture is mixed evenly. The strain was stored at-80 ℃. Extracting the MPP1 gene promoter-mCherry reconstructed vector from the positive bacterial liquid, and sequencing 10 mul of the MPP1 gene promoter-mCherry reconstructed vector to detect whether the MPP1 gene promoter sequence in the reconstructed vector is correct.

S4, mediated MPP1 gene promoter-mCherry transfection virus package:

the pLV-MPP1 gene promoter-mCherry virus is packaged in a 60mm culture dish, and the specific steps are as follows:

the cell is transferred to 293T cells 24h before virus infection, and the density is about 1X 10⁶And a 60mm dish, observing cells the next day, wherein the cells are clear and smooth in boundary, free of clumping, full and provided with three to four protrusions, and can be packaged when the cells are approximately 50-60% converged. And replacing fresh culture solution for cells 2h before packaging.

Two 1.5ml centrifuge tubes were first removed and designated as tube A and tube B, 250. mu.l of 2 XHBS was added to tube B, embryo culture water was added to tube A, and 25. mu.l of 2.5M CaCl was added₂Mixing, adding carrier plasmid and auxiliary plasmid in turn, mixing to make the final volume of tube A be 250 μ l, and adjusting the total volume of water according to the total volume of plasmid. After mixing, the solution in tube A is slowly added into tube B, and mixing is carried out while adding.

pLV-MPP1 gene promoter-mCherry: 5 μ g, pMDLg/pRRE: 2 μ g, pRSV REV: 2. mu.g, pVSV-G: 2 μ g. Standing at room temperature for 5-10 min.

The mixture was added dropwise to 293T cells and gently shaken well. And replacing the fresh 293T cell culture solution after 10-16 h. And collecting the virus 48h after the liquid is changed.

S5, in vitro culture of ovarian follicles of sheep ovary tissue:

collecting sheep ovary tissue, separating to remove primary ovarian follicle, culturing primary ovarian follicle in culture dish droplet, and placing at 37 deg.C with 5% CO₂After the ovary primary follicles are attached to the wall, the packaging virus mediated MPP1 gene promoter-mCherry expression transgenic technology is adopted, and the specific steps are as follows:

(1) preparing an ovarian follicle culture solution:

the ovarian follicle culture solution contains 10% FBS DMEM/F12 and alpha-MEM (1:1), and the culture solution contains DMEM/F12 and alpha-MEM (1:1), FBS, pyruvic acid, glutamine, non-essential amino acids, FSH, LH and double antibody,

(2) droplet preparation for follicular culture:

preparing a primary follicle culture solution small drop in a 3.5cm culture dish (Thermo) by using the prepared follicle culture solution, wherein each primary follicle culture solution small drop is 15 mul/drop/15 pieces/dish, and then covering 2.5-3 ml of paraffin oil; in this step, the prepared primary follicle culture micro-drop dish is placed at 37 ℃ and contains 5% CO₂The saturated humidity incubator is preheated.

(3) Acquisition of primary follicles in sheep:

the collected ovaries were placed in fresh medical normal saline containing 10% FBS preheated at 37 ℃, primary follicles were dissected under a stereomicroscope, and the primary follicles were washed with fresh medical normal saline containing 10% FBS preheated at 37 ℃.

(4) Microdroplet culture of sheep primary follicles:

transferring the stripped and cleaned primary follicles into the preheated primary follicle culture dish microdroplets, and continuously placing the culture dish at 37 ℃ and containing 5% CO₂The culture is carried out in a saturated humidity incubator, and after the follicles adhere to the wall, a packaging virus mediated MPP1 gene promoter-mCherry expression transgenic technology is adopted.

Expression of S6, MPP1 gene promoter-mCherry:

(1) centrifuging the virus liquid collected in the step at 4 ℃ (3500rpm/min) for 30min, discarding supernatant, adding 250 mu l of the follicle culture solution prepared in the step into the virus sediment containing the MPP1 gene promoter-mCherry, sucking all the liquid into the virus sediment containing the MPP1 gene promoter-mCherry after heavy suspension, continuously re-suspending, adding an infection enhancer Polybrone (the addition amount is 1/1000 volume ratio) into the re-suspending mixed liquid, and uniformly mixing for later use.

(2) Absorbing and discarding culture solution in the culture microdroplets after the primary follicles are attached to the walls, and simultaneously supplementing the prepared virus infection solution containing the MPP1 gene promoter-mCherry with the same volume (30 mu l/drop); the follicle culture dish was marked and placed at 37 ℃ with 5% CO₂The saturated humidity incubator is used for infection culture for 12 hours.

(3) After the follicle is infected and cultured for 12h, the virus infection liquid containing MPP1 gene promoter-mCherry in microdroplet is absorbed and discarded in the same way, and the equal volume (30 mul/drop) of the follicle culture liquid prepared in the method is added; the follicle culture dish was placed at 37 ℃ in 5% CO₂The culture is continued in the saturated humidity incubator.

(4) The primary follicles of the sheep cultured in the above way are changed into new culture solution every 3 days, and the development condition of the primary follicles is observed by photographing with a fluorescence microscope every 48h until the primary follicles develop to the mature follicles.

(5) And (4) selecting the mature follicles to perform microdroplet maturation culture on the sheep oocytes according to the expression condition of the mature follicle red fluorescent protein.

Wherein the sheep oocyte basic culture solution is TCM 199/alpha-MEM (1:1) of 10% FBS, and the specific components of the culture solution are TCM 199/alpha-MEM (1:1), FBS, pyruvic acid, glutamine, non-essential amino acid, beta-E2, FSH, LH, EGF and double antibody;

manufacturing an oocyte culture dish: making small drops of oocyte culture fluid in a culture dish (Thermo) of 3.5cm, wherein each small drop is 30 mu l/3 per dish, and then covering with 3-4 ml of paraffin oil; in the step, the prepared oocyte culture micro-drop dish is placed at 37 ℃ and contains 5 percent CO₂The saturated humidity incubator is preheated.

After the oocyte micro-droplets are matured and cultured for 24 hours, the expression level and maturation condition of the sheep oocyte red fluorescent protein are detected under a fluorescent microscope.

According to the level of the red fluorescent protein expression of the sheep oocyte under a fluorescent microscope, the sheep follicle and the growth and development conditions of the oocyte are combined, and the in-vitro culture system of the sheep oocyte is adjusted, optimized and perfected, so that the quality of in-vitro culture of the sheep oocyte can be evaluated, and the whole process of evaluating the in-vitro culture system of the sheep oocyte by utilizing the molecular marker gene MPP1 is completed.

The MPP1 gene is a stage specific molecular marker gene of oocyte growth and development in a sheep follicle development process, and an MPP1 gene promoter is selected to construct a red fluorescent protein expression driving vector, so that the sheep follicle and oocyte in-vitro culture system can be accurately detected and reflected, and an important scientific basis is provided for optimizing the sheep follicle and oocyte in-vitro culture system.

The foregoing is an illustrative description of the invention, and it is clear that the specific implementation of the invention is not restricted to the above-described manner, but it is within the scope of the invention to apply the inventive concept and solution to other applications without substantial or direct modification.

Sequence listing

<110> Zhou sheep industry Co., Ltd, Tianchang, Anhui province

<120> method for evaluating sheep oocyte in vitro culture system by using MPP1 gene

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ccgcgtccca ggcggcccgg ctctcccata agatgacgct gaaggcgagc gagggcgagg 180

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acaaccggcc cgaggtgagc ggctcccggg gtccccccaa cggcatgcgg tgagggccgg 300

tccccgactc tgggcctcgc cgtccgccct cctgccctcg gggaggggtc ctgacccccg 360

gggcttgtgc gggacttctg ggcgccttgg ctcgggactc cgcggcgcct ctaggcggga 420

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cgtggacgga gtcggggtca cggagaagac tttcaggaac agagcccaga ggaggtctgc 540

ttaagtgctt gtgtcagcag aaagggttgg tggtaagagc caacgctggc ctcaaagtgc 600

tcagtcacca tgccgtggca tttctgcggg ggcctctggc gctggggggc acccgggtgg 660

cctgtgtctg tgggcaggca tcatctcagg agtaattctg cgccccccca ccaagattcc 720

tgcgatggcc cataaatagc aactcacctc tatcctcaaa gatggcccat acgggagagt 780

tgattctctt gttatcttgg ggcttccctg gtggctcaga gggtaaagcg tctggtttgg 840

ttcctgggtc gggaagatct cctggagaag gaaatggcca cccactccag tacccttgcc 900

tggaaaatac cctggacgga ggatcctggt aggctacagt ccatggatct caaagagtct 960

gatatgactg agcgacttca aggtaaggct gcatgccagg cccactcact cacctgaaga 1020

ctgaagctgg gtctcagtgg tcagccatca gccagcaccc actggcagcc cttccatgtt 1080

ccctggcctc ctcacagcct gctggccagg ttccaagaga ggatgtccac agagacaccc 1140

agagacagag ggctcatcag acactagctg gcctgggaac tcacgagtgt gaatcttttt 1200

ctagacctcc aagatcttcc agggcagggc tgtttgcaca ctcacttttt ccaggtgaaa 1260

tgcctgatcc gtggggcctt ggaaatggtt cccaaattac taagtgcctg aagaggtgga 1320

cgtgttatgc agcgcaccca tccacttggg catatgatga aactgcagat cccccaggtc 1380

ccagcagact ggtgtgcagg ctgcggactg tggactggga ggcatcagag ggaggctgca 1440

ttctgtatct gcattgagct tgctgtttcc ctcagggtgc catattcaca cttttccgtc 1500

tttgcaaatc aggattgcgg tagtctaggc ttccaggagc agtgggatga tcatgtgggg 1560

aaggcaaagt tgcaagttag gcagcggcaa gtgcgctagg aggggatcaa gctctgaaac 1620

tccttgattc cccataagct tggaaatgtg attttttttg cttttcttcc catcagtcaa 1680

ctcacttggc tgagccttaa ctccgtcccc accccctgcc aaactcgctt tattgaattt 1740

tcttttggca actttattga gatttacttt gcattaatag aattcactga tttgtgcagt 1800

tcattgacat tgggcatgta catcccaccc tgtagctgtg accacagtac ttgagggttg 1860

ccccccaacc ccctgccatc cctagaagca tggaacaccc ccacctccac ccccccccac 1920

cgccagcagc atttggaaaa cactatcact ttgccttttt gaggctgtcc tacaaatgga 1980

ctccagctta cataggcctt tctgtgtgcc tgctttcact ttgcacaacc tgagcgccac 2040

ctgtaactca gtgtcccgac cccttggaga tctttctgac cctggtggaa aggctgggga 2100

gattcattgt gtgcagtttt gaacagaagc tctgtcacta ctctctgagt aagcagcagt 2160

ttttgtggag ccatgagttt atcctgcaat accagataca gtctcctgcc tatgtagttt 2220

gtttgcatga aaggatttga ggatgggata agaaaaattg gtaaagcaca gaatgaaaag 2280

cttccagaac tgcccaactg tggacctcgg agggagaacc tccacaccat gaggggtggc 2340

ctgaccccac aaactggctg atttcttatt tcctgaggtt caccttccct ccgaggaagc 2400

tgttggcttt aaagcaattc agaaattcct gttgccaaac atttcattga ggaggattgc 2460

ttttggtaaa agttctaaag actcaaaaac caaagtttcc cttctgattg tagtctgtat 2520

ttgataactg aaaaagtctt atcagaacac aatgttgcaa tacatttgaa gtggaggctg 2580

cctcctctat ttaactatag gtgtccttcc ttgaagaaac agtttatgag ctccctgtga 2640

ccttgttctt tatggcagta acttagtgca cataaaatgt gttgtaaaat tcctacttgt 2700

ctctgctggc ctgtttgaag ttaccccttt cccatgagct gaagaaatag gttgcaagtc 2760

cctgaaagtt cacttgggcc taatggccca cgaatgaagt ctggctccag ctgagtatcc 2820

actcagcaga gctcctggcc agcagattcg gcctcatctg ctgttgcaca ggtttgaggg 2880

tggagatatc cagggtgcca tcggccaaca gtgctccagg gaggggacat tcagggctca 2940

cagaggtctg gagatttcgc cagaggtccc ctggggtggg ggagatgaaa tcaggtgcat 3000

ttcatggatg gcagggattt tttttttttc ccttaagcac cagttgctca agtttttaac 3060

tgttgctcaa aggagaactg actccacagc tgggaaatgg gaagacatgc tccaaattag 3120

ggaaccagag tacagcttgg tgtaacagaa ttttctgagg cttcactttg cctttttcct 3180

tcacgtggag gcatagagtt gagccgtatc catacgcatt cattcctccg tccctatggt 3240

tcatccacga ctataagcct ccagccacca gcagaggcca ccgaccctgg actgaattgt 3300

gtggtgctgc ggggttgact gtgcaagaaa tgctcatgcc agcggacctt tggcagctca 3360

caaccatcgt gtcccagggc cgattgtaca cgtttgtgct ctgaagtcac tagcctgcca 3420

gtcctgctgc aggtgctttg tttgcctgca tcccaggtac ctggttcatg aggactgaag 3480

ccaggacttg cgtaaaatcc tgcctctgag gctctcctcc cagccctttt gtgcagctct 3540

catatttgtg gggaaacagt gtcccctttt ttctggtacc ttgagccctc ttgggttttc 3600

tctttttttt tttctctcct tgtgttcttt cccaatgtgg agcctgccct ggcccaggag 3660

gactgagcag ctgatgtctg gtgtgggcca tcacttgcct acttggcctg ggccttcttc 3720

tgagtctgtt caagtcgctc agtcatgtcc aactctgggc gaccccactg acagtacagt 3780

ctgtggaatt ctccaggaca ggatcttgga gtgggtagca gcctttccct tctccagggg 3840

atcttcccaa cccagggact gagcccaggt ctcccgtact acaggtagat tctttcccag 3900

ctgagccccc agggaagccc aagaatgctg gagtgggtag cttagccctt ctccaaggga 3960

acttcctggc ccaggaatca aacctgggtc tcctgcattg caggcagctt ctttcccaac 4020

cgagctctca gggaagcccc acggtacctg gaaacagtgg gcagagggct tatccatcag 4080

cctaacagat gttgattctt tccctcccgt cactggcacc agctcttaat gtgacttggt 4140

ggcccctgct caagtcatcc acctctgcca cctgccgcta agtttgtttt ttcatctctc 4200

tctctttttt aatcccagct tcctctgagc tgctgcctct gccctttgaa cccactgcag 4260

atgtttcctc tgaagagggt ttcagaggcc aggtcttgcc accccagcag tttgtgtgat 4320

tatgagcccc tgagaggctg cagagcaaag ggcagccctg caccctcctc atgcgggaca 4380

gccatccccc atcttgcctg ggtgtggctg ggggcctccc tggccagagg gggtactggt 4440

atcacagaaa gcaatcgaac acctttgccc gatgttgttt gctcagcatt gcttgtatca 4500

ctttgtaatc acctggcacg ggtgctcatg gcactgggaa ggactcaaca ttgtggtcca 4560

gtgtggggcc agtaagtccc agaacaaacc atggaaagtc tctgccaccc tttggaaggc 4620

acgtgtctta ccagctgtgg aaacacgcca agttccagca gttgaggggc aggacaggag 4680

agacggggaa ccaattgcag cctgcagccc ctgctccgca cacatctgtc tgtctgtctt 4740

gcccttcctc gcaattgtct gcgggctggg ggtgccaggg gctggcatgc caggtgccat 4800

caaagagcgg tcagagcagg ggtatcaggt ggtgcttggg gtgcgcccca tgggtgcgag 4860

ttggttcttc gggtgccagg tcagagcatt ccgggtatct ctctcatcac tggcagcttc 4920

tgagggagtg taatagtggc tctgagtcat taggaagggg gactgtcttg acggataagc 4980

acattgaaaa gcccagcgtt tggcatagca agtgttgatc ttttctgggt ttgatgtgtt 5040

ctacctctgg caggctttgt gagtccttaa tttaaaggct gacgtgtcct ggagctgaat 5100

gaagagatga ggcccccaat cacgcaggca tccaccccct tgccaagttc actcggtctt 5160

ctgtggactg acttttgact ctgtccttgt tgagaatgtt atgtagggct ccaccatgtc 5220

tgcaaaccca gatgaatcat aagaaatgct ttcttgatgg tactttacag cacatgataa 5280

gagaatctga tctgtttcca aactttatgg ccacctttac ccgctagggt tactgatgaa 5340

tccctggtta tatggatcga acccaaatct cctgcattgc aggcagattc tttaccagca 5400

ttgctgggtt gtaagacatg taagtggtac atgtttggag aaggcaatgg caacccactc 5460

cagtattctt gcctggcaaa tcccatggat ggaggagcct ggtaggctgc agtccatggg 5520

gttgctagga gtcagacaca actgagcgac ttcactttca cttttcactt tcatgcattg 5580

gagaaggaaa tggcaaccca ttccagtgtt cttgcctgga gaatcccaag gatgggggag 5640

cctggtgggc tgccgtctct ggggtcgcag agttggacac gactgaagtg acttagcagc 5700

agcagcagca gcagcaagtg gtagttgctg gcagatccat ggaatgaatc tgcgggtttg 5760

attttgagct ttctcacccc acgattacta gtgaacaaag atttgtaagt cagaatcagg 5820

gcaagtcagg catgcagtca gtgatccttg aactcctcag tctatccgtt caaatctctt 5880

aatacctgca acatggtgaa aacaaataag gtgcttaatt taacagaaat aattcttcca 5940

acttgatgtc acatgcttcc acgtcgctgt gggtaatgct ttgagaagct ctggggtcac 6000

catgtgctgt agcctgttca ttccaacccc agattcctgc aactttctcc acggtttgtt 6060

gttgcctctc cccattggtt actgtgctct cttagttaca gctctttggg tgcaaacaag 6120

tgcaacctgc cagctgtttt gcaacttttt tgcaataact tcaactctgt ctcctacgag 6180

atcaataata gtacattgtc aaaaccatgg acttgacatt gctgtgacat gtaagtgtag 6240

ctctgggcca ttttgtcacg tgtgtgaaga tacgtaacca ccaccactag tctgttttcc 6300

agctctgtaa ttttgtcatc ccaataacgt tatatacctg gaatcaaatc attggcaata 6360

tttctattgc tgggaatcat cccaaagtct ggatggatca tagtctgttg agctgctcac 6420

ttgtgataga tactcctggt tgtttccagg tttgagttac tgtgagtaaa tctgtatgaa 6480

ccttcagttc agttcagtca ctcagtcgta tccaactctt tgcaacccca tgaactgcag 6540

cacaccaggc ctccctgtcc aataccaact cctggagtct acccaaactc atctccattg 6600

agccagtggt gccatccaac catcttatcc tctgtcgtcc ccttctcttc ctgccttcaa 6660

tctttcccag catcagggtc agtcaggtcc cagtcagctc tttgcatcag gtggccaaag 6720

tattggagtt tcagcttcaa catcagacct tccaatgaac acccaggact gatctccttt 6780

aggatgaact atgtggatct ccttgcaatt caagggactc tcaagtgtct tctccaacac 6840

cacagttcaa aagcatcaat tcttcggtgc tcagctttct ttatagtcca actctcatat 6900

ccatacatga ccactggaaa aaccatagcc ttgactagac ggacctttgt cgacaaagta 6960

atgtctctgc tttttaatat gctgtctagg ttggtgataa ctttccttcc aaggaataaa 7020

gtgaaagtga agtcactcag taatgtctga ctctttgtga ccccatggcc tgtagcctac 7080

caggcttctc tgtccatggg attttccagg caagaatact ggagtgggtt accatttcct 7140

tctccaggac atcttcccga cccagggatt gaacccaggt ctcctgcatt ataggcagac 7200

actttgccgt ctgagccacc agggaagcag taagtgtttt ttaatttcat ggctgaagtc 7260

accatctgca gtgattttgg agtgcccccc caaataaaat ctctcactgt ttccactgtt 7320

tccccgtcta tttgccttga agtgatggga ccagatgcca tgatcttagt tttctgaatg 7380

ttgtgcttta agccaacttt ttcactctcc tctttcactt tcatcaagag gctttttaat 7440

tcctcttcac tttttgccat aagggtagta tcatctgcat atctaaggtt attgatgttt 7500

ctcccggcaa tcttgattac agcttgtgct tcatccagcc caggtacgaa ccttcatgta 7560

gcaggtttta catttacatt tgtatttttc cattacagtg aatacagtga aagtctctca 7620

gtcgcgtcct attcttcacg accctatgga ctatgcagtc cgtggaattc tccaggccag 7680

aatactggag tgggtagcct ttcccttctc caggggatct tcccaaccaa gggatctaat 7740

ccaggtctcc ctgtgattct ttaccagcat tgctcacttg taaagtactc attttgtttt 7800

ctaagtaatc acccaattct tttcctgaat ggctgtccca ttttacactg ccaccagcca 7860

tggatgagag attcagtttc tttccatcct tatgactagt cttgtcactg gttttgatca 7920

tagctgctct gatggctgca tggtgatatt tccttgtggt tttctttctt ttttttaata 7980

gtactggttt gtttttctgt ttttgtctct gctgggtctt catggctgag ctcaggcatt 8040

ctctagttgc agcaagaagg gactcctctt ggttccacaa ggacaagcct tctgactgtc 8100

actaaatgca acccaagcgt gtggccaaaa tccagcagaa ctaaaaattc tccgttcatt 8160

cctttgacag ctttccttct cgctggtctc ccaggattgg aagacccttc ccttcattct 8220

tttctgtcgt gtgtgtgagg accttgatgt tgtaagggtt ctgacattta cagtctgttc 8280

tttacctaca attgttttgc aacttgctgt aggtcgatgc taaaaaggga acatcggtag 8340

aggcttttgt attattctga ctggtggcca acccaacagc tttattctgt ttctcactga 8400

tccagcttct ccctgatctt gttagggatg accagatgcc gagctcagtt cttttttcag 8460

ccccttgtgc agcctgggac agcctgatct ccagttctgg ttgatgagac tccagcttcc 8520

tccttcctgc aatcctcccc agctagtttc ttttgggtgc acattcttgg ctctgccgtc 8580

tgctggaaca tttttgaaat gtctcatcct catgacttcc ttttgtgtgt gtgtaggttt 8640

ctatttacct tccttccttg ggatctcctt tcagtagggc ccagggactt ttccaaggct 8700

cagacttttc catgggttca atccctgctc tgagaactag atcctgccga ctgcaactaa 8760

gacccagcac agtcaaataa atgagtatct aaaaaaaaaa aatagggtcc acactacaca 8820

caagtcctta agctcccaac ttgaatctaa atgacactca cttaggaaaa ttctatcttg 8880

ccacaagaga aggctttcat gtgcctgttt agagattaat ctgaaatgaa tccagttttc 8940

ttctgagccc cttctcttgt ctcccagttc ctccatgttg ttcacatgtc cttccagaga 9000

gagatgacat gtttacaagc agatatgaag attttcttct gtttcacagc atacctttcc 9060

ttcccccacc tttttttttt cacttaacaa catttctttg aggtcaaccc ctgaacctcc 9120

ttttcatggc tccatggtat ctgtgtgtgc taggacactt tgcacagagg tagaggtaac 9180

tcatctactg tagatggtct tgaagtgccc tgtagctctg gtgtcatgag ttacatcagt 9240

tacatgagtt acatgagtta catctcatca gttactttgc taaatacaat cctttgttag 9300

acacttctgc tttcatagcg ctgtggaaag ccatgtcacg tgagtttgga gaagtgctag 9360

ctgctgccca gggcagtttc tcttttttgg gactttcctg attgcaatag atctggagag 9420

acagatcata gatggagcta gctgttcctc cctggggaat cttcccattt ctcctttgcc 9480

accaactggc tgatgaggtg tcctcctggt cccacccatt gggaatatct tccctcccca 9540

tggcctctga ggggcaccgc cacccctgct gtggctggat cacagccgcc atccatgtgc 9600

agcagaatcc ttccctattg tcctgattga atatttgacc cggagtgggt gagagcattt 9660

tcagaagtat ctctcatatt ctacccagcc taccccttcc catcccatca gaggcaacca 9720

ccatcttcaa tgttgttatt gtcatttagg catgttctaa aacctcacga atgggctcat 9780

ctaatgtgta ctcttttgcc taaagcttct ttcactcaac atcacacttt tcagactgat 9840

gcatgttgac ccctttgtca acattttggt ccttttcttg tttctgcaac ctcctgtggg 9900

tgaataaagc tgctgtgaac atccatgtgc aaccctgtgt cttttcttct gggtgaacac 9960

ttcgaagtgt ggttgacagg gcctagggtc agtgtgtgct aaacttgtct ttttcaccat 10020

cagctcccta gtacatctta tgaaaaagtg caaaagaact ttttggccca cccagtattt 10080

ccctgattac agtgcttgta gggtcttgta gtgcatgtgg ccttctgaaa tgaagcgtcc 10140

gttcaaacat tctgtctttt cctcgcttgt catcctgtta ttgagtcata agagttcttc 10200

acatgttctg ggtataaata agtctctgtc agacgtatgt attgcagctt ttacagtcag 10260

gctatggcat gccttcatac ctcttgatcg tcttttggtg agaaggtgtt agttttgatg 10320

ggacaactgt ctgcatctat gattatcgct ctctgtgcct actaaacctt tgcctgtctc 10380

aggggagtta cttcatttcg agctatttat attttttagt tgtcgtttgg ctcttttgtg 10440

tcagatagaa ggcacttctg cttcctcccc ggttttgaca atgcgctgtg ctgcttttct 10500

caaagcttgg ctgtctcacc ttttctgttt ggcgttgcca actgtctggt tttgtctaaa 10560

gtcaggtggg gatgaacatt cctgctgccc atgtggggtt gacctggcac catggattac 10620

caagaccagc cttctgtctg tcctcctgcc caggccagag gccttaaatt aacttcagat 10680

gtatcatgca gagtctttcc agagtgtaat tcatccggct gtgctgtctg catggttggg 10740

gtcaatgaat cacagttcac gtggcacctc aggccaatgg gaggggtggc ccattggata 10800

aataacagtt cagttcagtt cagttcagtc actcagtcat gtccgacttt ttgcgacccc 10860

atgaatcgca gcacgccagg cctccctgtc catcaccaac tcctggagtt cactcaaact 10920

cacatccatc cagtcggtga tgccatctag ccatctcatc ctacgtcgtc cccttctcct 10980

cctgccccca atccctctca gcatcagagt cttttccaat gagtcaactc ttcacatgag 11040

ctggccaaag tactggagta tcagctttag cgttattcct tccaaaggaa cccagggcag 11100

atgtccttta gaatggactg gttggatctc cttgcactcc gagggactct taagagtctt 11160

ctccaacacc acacttcaaa agcatcaatt ctttgccact cagcattatt cagggtccaa 11220

ctctcacatc catacatgac cactggaaaa aacaaagcct tgactagaca gacctttgtt 11280

ggcaaagtaa tgtctctgct tttcaatatg ctatctaggt tggtcatagc tttctttcca 11340

aggagtaagc gtcttttaat ttcatggctg cagtcaccat ctgcagtgat tttggagccc 11400

ccaaaaatta agtctgacac tgtttccact gtttccccat ctatttgcca taaagtgatg 11460

ggaccagatg ccatgatctt cgttttttga atgttgagct ttaagccaac tttttcactc 11520

tcctctttca ctttcatcaa gaggcttttt agttcctctt cactttctgc cataagggtg 11580

gtgtcatttg catatctcag gttattgata tttctcccag caatcttgat tccagcttgt 11640

gtttcttcca gtccagcgtt tctcatgagg tactctgcat ataaattaaa taaacagggt 11700

gacaatatac agccttgacg tactcctttt cctgtttgga acccgtctgt tggtccatgt 11760

ccagttctaa cagttgcttc ctgacctgca tataggtttc tcaagaggca ggtcaggtgg 11820

tctgatattc ccatctcttt cagaattttc cacagtttat tgtgatccac acagtcaaag 11880

gctttggcat agtcaataaa gcagaaatag atgtttttct ggaactctct tgctttttcc 11940

atgatccagc ggatgttggc agtttggttt ctggttcctc tgccttttct aaaaccagct 12000

tgaacatcag gaagttcacg gttcacgtgt tgctgaagcc tggcttggag aattttgagc 12060

attactttac tagcgtgtga gatgagtgca attgtgcagt agtttgagca tgctttggta 12120

ttgcctttct ttgggactgg aatgaaaact gaccttttcc agtcctgtgg ccactgctga 12180

gttttccaaa tttgctggca tattgagtgc agcactttca cagtatcatc tttcaggatt 12240

tgaagtagct catctggaat tccatcacct ccactagctt tgttcgtagt gatgctttct 12300

aaggcccact tgacttcaca ttccaggatg tctggctcta ggtgagtgat cacaccatca 12360

tgattatctg agtcgtggag atcttttttg tacctttctt ctgtgtattc ttgccacctc 12420

ttcttaatat cttctgcttc tgttaggtcc agaccatttc tgtcctttat cgagcccatt 12480

tttgcatgaa atgttccctt ggtatctcta atttccttga agagatttct agtctttctc 12540

attctgttgt tttcctctat ttctttgcat tgatcactga agaaggcttt cttatctctt 12600

cttgctattc tttggaattt tgcattcaga tgcttatata tttctttttt tttttttttt 12660

ttttaaattt ttatttattt atttggctgc tctgggtctt agttgtagca tgtgggatct 12720

agctccctga ccagggatcg aacctgggcc ccctgcactg ggagctcaga gtcttagcca 12780

ctggaccacc agggaattcc ccagatgctt atatatttcc ttttctcctt tgcttttcac 12840

ttctcttctt ttcacagcta tttctaaggg ctccccagac agccattttg cttttttgta 12900

tatcttttcc acggggatgc tcttgactcc tgtctcctgt acagtgtcac aaacctctgt 12960

ccatagttca tcaggcactc tgtctatcag atctagtccc ttaaatctat ttctcacttc 13020

cactgtataa tcatcaggga tttgttttag gtcacacctg aatggtctag tggttttccc 13080

cactttcttc aatttaagtc tgaatttggc aataaggagc tcatgatctg aaccacagtc 13140

agctcccagt ctagtttttg ttgactatag agcttctcca tctttggctg caaagaatat 13200

aatcaatctg attttggtgt tgaccatctg gtgatgtcca tgtgtagagt cttctcttgt 13260

gttgttggaa gagggtgttt gctatgacca gtgcgttctc ttggcaaaac tctattagcc 13320

tttgccctgc ttcattccat attccaaggc caaatttgcc tgttactcca ggtgttcctt 13380

gacttcctac ttttgcattc cagtccccta taatgaaaag gacatctttt ttgggtgtta 13440

gttctaaaag gtcttgtagg tcttcataga accgttcaac ttcagcttct tcagcattac 13500

cggttgaggc atagacttgg attactgtga tattgaatgg tttgccttgg aaatgaacag 13560

aaatcattct gtcttttttg agatcgcatc caagtactgc attttggact cttttgttga 13620

ccatgatggc tactccattt cttctaaggg atccctgccc acagtagtag atataatggt 13680

catccaagtt aaattcaccc attccagccc attttagttc gttgattctt agaatgttga 13740

ctttcactct tgtcatctcc tgtttgacca cttccaattt gccttgattc atggacctaa 13800

tattccaggt tcctacgcca tattgctctt tataagcatc agaccttgct tctgtcacca 13860

gtcacatcca caactgggta ttgtttttgc tttgactcca tcccttcatt ctttctggag 13920

ttatttctcc actgatctcc agtagcatat aaggcaccta ctgacctggg gcatttctct 13980

ttcagtatcc agtatgaaaa ggcaaaatga taggataaat aacatggccc atcaaattgg 14040

atgcagtcag ctaccagcct aaggggataa gcattggttt atgtagcttg ggattccctg 14100

gtggctcaga tggtaaagcg cctgcccgca atgcaggaga cctgggttcg atccctatgt 14160

caggaagatc ccctggagaa ggaaatggcc acccactcca gtactcttgc ttagaaaatt 14220

ccctggatgc aggagccttg tgggctacag tccaaggggt cgcaaagagt cagacacgac 14280

tgagcgactt cactttcact ttctgtagct ttgggggttg taaacctgca gtgatatcct 14340

aggggtcgcc tcagccactt gcttcatcca atgctctgct cttttgcccc accagcctgt 14400

gtcaccccag ctgagtgctg tgatggagga catgtacacc aatgggcccg ctgccctggg 14460

gagcccggcc cagactcagg gccaggaggc ccggaaggtg cggctcatcc agttcgagaa 14520

ggtcaccgag gagcccatgg taacccttgt tttttgccac catcaccccc caacccccac 14580

cccatgcctc cgcctcagag cgggcttcct ggtcccttcc agtggttgac gccccaggtg 14640

atggagacag aaagcctttc tcccaagaaa tcactttcag atgtcttaaa gatcagctca 14700

gcctggagac ggaaacagca accaactcca gtattcctgc ctggaaatgt ccatggacaa 14760

agaggagcct ggcgggctac agtccgttgg gttacaggac tgagcatgca cacatgaggg 14820

tggagggttg gtagcaataa actgatagaa ctaaaacaaa caaaacaaga tcagctcaga 14880

aatagctcac agtcaagcat gctatggctt tgtctgtctt ccagggaatc acgctgaagc 14940

tgaacgagaa gcagtcatgt acggtggcca gaattctcca tgggggcatg gtccacagac 15000

aaggtatagg tccagccttc ttctatactt tgtgacacga ctgtatttcc tgttaggtaa 15060

catttgagca attacaatat gcaacccctt tcattcgcct ctgaatacat agcagtgaag 15120

caaagagacc atctctgcct tctggggcta ccagttaaac tgagaaaata gggaattccc 15180

tggcacatca gtatttagga ctcagtattc tcacggccgt gggccctagt tcaatccctg 15240

gtcagggaac taagattccg ttagctaaac agcatggcca gaaaaaaaaa aaaaagaaaa 15300

aaccaactaa gcaaatagac attaatacgt atttcagtaa ctgctgtatc tccttcatgg 15360

gaagcagcca tcaatttgca gatgcatcct ttagtaaaaa cacttttctt tgtatctagg 15420

ggagtgcagg taagataacc actcttcaca gagaatcaca tagattctaa acagtgatta 15480

acttgaccac ttggtagtgt ttggaggttt tagcttaaca gcagaaaaac atcaagcttg 15540

ctggtgactt cagagtgcca agttcttcac tttctggttt ctttgagtgt gtatagctgt 15600

tatcagaagc cccagagatc aggggcactc tagttccagg acattttaag gatcaggtaa 15660

cttttattct tcactcggag ttctgaagtc aaggcagaag acatagtcag cttcccggtg 15720

ctggtcctca taggacccag agggccatca atagaatatt ccagtctctt gtggtcgaat 15780

acttccaacc ctgtgtgttt ttttttacac aatcttttgt atcacgtgct acaaatcaca 15840

gtttctttcc tgcagtgttt tgactcctat tgacagctct tgtctgaacc tgtgttcttt 15900

tgatggtagg ctcacttcac gttggggacg agatcctaga aatcaacggc acaaacgtga 15960

ctaatcactc ggtagatcag ctgcagaagg cgatggtggg tatccactct gcccttgtct 16020

tggtgaactg tggatcggaa ttgctgtcca cgtaccgcat ttttctgtgg aagtgtttat 16080

acctcattga agggcctctt atcactcaag aaatgacaac tctctgtttt catttagtaa 16140

acttctaaaa caaggcagat gacccagtgt gtctagaaag agctctgtac tgagtgccag 16200

aagtcctcag tctttaacgg cagccctgaa tcccattctt ctttactcca gccctgtgat 16260

ttggagcaaa attcacttct ctaagtcatc tgtaacacga agatgctagt aatcactatt 16320

cttctgttat agatttctta cagagagcaa ttggactaat agatgaaacg tcaaaaagac 16380

agaatgatct ctgttcattt ccaagacaaa ccagtcaata tcacagtaat ccaagtctat 16440

gacccaacca ctaaagctta agaagctgaa gttgaatggt tctctgaaaa cctaaaagac 16500

attctagaac taaaaccccc aaagatgtcc ttttcattat aggggactgg aatgcaaaag 16560

taggaagtca ggagacacct agagtaacag gcaaatttgg ccttggagta cagaatgaag 16620

cagggcaaag gctaacagaa ctttgccgag agaatgcact ggtcatagca aacaccctcc 16680

aacacaggag aagactctac atatggacat caccagatgg tcaataccaa aatcagattg 16740

attatattct ttgtagccag agatggagaa gctctataca gtcagcaaaa gctagactgg 16800

gagctgactg tggctcaaat catgaactcc ttattgccaa attcaaactt aaattggaga 16860

aagtagggga aaacactaga ccatttaggt atgacctaaa tgaaatccct tacaattata 16920

tagtggaagt gacaaataga ttcaagggat tagatctgat agacagagtg cctgatgaac 16980

tatggacaga ggttcatgac attgtacagg aggcagtgat caagaccatc cccaagaaaa 17040

agaaatgcaa aaaagcaaga tgttgtctga ggaggcctta caaatagctg agaaaagaag 17100

agaggcaaaa ggcaaaggtg aaaaggaaag atataccacc taaatgcaga gttccaaaga 17160

atagaaagaa gagataagaa agccatcctc agtgatcaat gcaaagaaat agaggaaaat 17220

aatagaatgg gaaagactag agatctgttc aagaaactta gagataccaa gggaacattt 17280

catgcaaaga tgggcacaat aaaggacagg aatggtgtgg acctaacaga agcagaagat 17340

gttaagaaga gctggcaaga atacacagaa gagctatacc aaaaagatct tctgacacag 17400

ataatcatga tgatgtgatc actcacctaa agccagccat cctggaatgc aaagccaaga 17460

gggccttagg aagcatcact acgaacaaag ctactggagg tgatggaatt ccagttgagc 17520

tatttaaaat cctgaaagat gatgctgtga aagtgctgca cccaatatgc cagcaaattt 17580

ggcaaactca acagtggcca caggactgga aaaggtcagt tttcattcca atcccaaagg 17640

gcaatgccaa agaatgctca aactactgcg caattgcact catctcacac actagcaaag 17700

taatgctcaa aattctccaa gccaggcttc agcagtacct gatccgtgaa cttccaaatg 17760

ttcaagctgg ttttagaaga ggcagaggag ccagagatca cattgccaac atctgttgga 17820

tcatagaaaa agcaagagag ttctagaaaa acatctactg ctgtcaaagc ctttgactgt 17880

gtagatcacc acaaactgga aaattcttta agagatggga atatcagacc acctgaccac 17940

gcctcctgag aaatctgaat gcaggtcaag aagcaacagt tagaactgga catggaacaa 18000

cagagtggtt ccaaattggg gaagaatttc gtcaagctgt atattgtcac cctgcgtatt 18060

taatttacat gcatagtaca tcatgagaaa tgccaggctg gataaacaca agctggaatc 18120

aagattgcca ggagaactat caataacttc agacatgcag atgccaccac ccttacggca 18180

gaaagtgaag aggaagtata gagcctcttg atgaaagtga aacaggagag tgaaaaacct 18240

ggcttaaaac tcaacgttca aaaaactaag atcaaggcat ccggtcccat aacttcatgc 18300

caaatacatg gggaaacaat ggaaacagtg acagactttg ttttgggggg ctccaaaatc 18360

actgcagatg gtgactgcag ccatgaaatt caaagacatt tgctccttgg aagaaaagct 18420

atcaccaacc tagacagcat attaaaaaca gagacatcac tttgccatca aaggtccatc 18480

tagtcaaggc tatggttttt ccagtagtca tgtatggatg tgagagttgg accataaaga 18540

aagctgagca ccaaagaatt tatgcttttg aactgtggtg ttggagaaga ctcttgagag 18600

tcccttgaac tgcaaggaga tccagccagt ccatcctata cgaaatcagt tctgaatatt 18660

cattgaaagg actgatgctg aaactccaat acttgggcca tctgatgtga agaactgact 18720

catttgaaaa accctgattc tgggaaagat tgaaggcagg aggagaacgg gactacagag 18780

gaagggatgg ttagatagca tcatcgactt gatggacatg agtttgagca tgctctggga 18840

gttggcgatg gacagagaag cctgtcctgc tgaactccat ggggtcacaa agattcggac 18900

atgactgagc gactgaactg aactgagatg agacattgaa tgtgccctct agattcagtt 18960

gtgctgggtg tttgcttctc cagattcttc accatggttc ttcagccttg cacacaatgg 19020

agactattcc tgtaagcaaa gcaggaaaat cacatggagt agttgggtgt cattggccat 19080

ggctagtagg aagcacccct caggcactta gcatgggaat gaagcagaga atagaaactt 19140

gaaaacaagt gtggtttgaa ccagaaatga aaaaaactga acattgccag aattacatgt 19200

taacattcct tttaaatttc tgccaatctg acaggtgcaa agtacttcat ggttgcttta 19260

ttcttctaat tttattccca gagccaaaag ttgagcactt tttaagagct cattggctat 19320

atgcttttcc ttcttgtatc tttttgccct gttgtctatc gggttggtta tctatttata 19380

acagagtttc agtgagcctg attgttagtt gctcagtcat gtccaactct ttgtaatccc 19440

atgcaatgta acccaccagg ctcctccgtc catggacttc tccaggcaag aacactggag 19500

tgggttgcca tttccttctc caggggatct tcctaaccca aggaccgaac ccgggtctcc 19560

agcattgcag gcagactctt taccgtctga gccaccaggg aagcccatat aataaagttt 19620

agaagcactt gaaatattgg ttattaactg tctgcctact atatgcacaa taatatgttt 19680

ccaggaagta agtcagtgtt agtcactcag tcgtgcctga ttctttgcga ccccatggac 19740

tgcagcccac caggctcctc tgtccgtgag attttccagg caaggatact ggaatgggtt 19800

gccatgtcct tctccagggg atcttcccaa cccagggatg gagcccaggt ctcctgcact 19860

gcaggcagat tctttatcga ctgagctgcg agggaagccc attatgtttc caagtgtatt 19920

gtataaggta tcttttgcca catgacacaa attaactttt acatagttga ttatatttat 19980

acaaatcaaa aagaagtata aattttgaat attcatatat ttgaaacagt tgttccaaga 20040

ctaagggata cagccttgct gtgggctcag aaaaaaacct gtgcatcaca ttctgttgag 20100

aagaaatgat taaggccaag tgcagaggat cgtgttctgt gcagggtaca ctgaggatat 20160

gagtcagcta ctcgtgctgg tgagcatgaa acactccagc cagggtcctg gcaggagata 20220

ggtgacccac tccaaaaggt ggataagatg ctgaatgaag gggttgttta cagagggtaa 20280

ggcctaaaga tagaggggaa caaaaatccc tggtggctgg gaaagattga aggcatgggg 20340

agaaggggac aacagaggat gagatgattg gatggcatcc ccgactcaat ggacctgagt 20400

ttgagtaaac tcaggacagg gttatggaca gggaagcctg gcgtggtgca gtccatgggg 20460

tcacaaagag tcacacacga ctgggcaact caataacaac aacaattact actgcatggg 20520

tctggggcac atgctttggg tatttgcatc aggatccagg tcacgtccac acactgcagt 20580

tgacggatac atcttttaag actctttgaa tccttctgtt ttctgatctg atctttgtgg 20640

aagaaacttg gttgttggtc ctatagagtt tctcctagtg agagtggaat gtctatagaa 20700

aagtaagttg aggatagctc tgaaagtgtt cttaccattt gattccacat ttctctcatg 20760

gggttctagt ctgtagaaac caaatgcaaa tatataagga catatgttga agggggtctc 20820

tggcagcagg ttgtaagaaa gaaagaaagt gaagttgctc agttgtgtcc gactctttgc 20880

gagcccctgg actgtagccc acctggctcc tccatccatg gaattttcta ggcaagagta 20940

ctggaatggg tggccattgc cttctccagg ggatcttcct gacccaggga tcgaacctgg 21000

gtctcccaca ttgcgggcag gcgctttact gtctgagcca ccagggaagc gcaggtcgta 21060

gaagaaaaaa caaaaactgg tgacaatgga cacgcctact aatggggatg gtggctgagg 21120

ccaccatcac acaagggatg taactgagcc atcagagaga gtgaataaag tcttcatata 21180

ttgacctgga agcaagttgt tgagtcatgc ttatagtata atcccatttt gttaaaaaca 21240

acaaaaatgc ataaatatga atacgtgtat aaatatctgt acatgtttgt attgtataga 21300

aaagtgtgga catatgtgtt cattgtaggg gttcatttta agtacttctg gggatctttt 21360

ggggtcacca gtgaagagga gggacaatca ttagcctttg gttcatgttg ttatgctggt 21420

ttatgggacg aagaaagtgt tctgctacat ttcaaagtgc aatgatggct attgggggga 21480

aaaaaagaaa cctcatgggc ctttgggagg ggcccatgac ttcatgagac ccccagtcag 21540

ccaagttcat tttacttaac agttatttca tgtttcttgt tttaatagaa agaaaccaaa 21600

ggaatgatct cattgaaagt aattcccaac cagcaaaatc ggcttccagc actacaagta 21660

ggtggcagca ttttctctgt cgcgatgact gcaggattca gacccacggt gttctcagaa 21720

tttgattgaa tgatgtgatt ctaatcagtg gagttgccta gtactctctg ggccaaattg 21780

acacaaattg actggcaggc ctttgctgcg tatcctgcat gtgagagcaa aggcccatct 21840

cctgattctg caacccccct tcacacacag cgcatcatgg cttaggaggg aaatcgcttg 21900

ggcagggccc tggcagatga actttgacaa aaatcactga agtttctgtt ccaggttact 21960

taatgtgtga tggtataatt ttcttgtcaa gtaaactttt taagattgaa gttttggtat 22020

atatgagcac atagacaaca aagtacacaa atcctaagta taaaaccaga tgaattttca 22080

caaacttaac acacccatgt gaccagcacc ctaagagtcc ccatccccat ttccagtccg 22140

ccaaccctga cttctgactc cagagatgag ttttgcccct tttgggaatt tgtgtaaatt 22200

gactcacaca gtacgaattc ttcagtgtct cgttttgttc actgggtgtg aagtctgtgc 22260

tatagcaact gatgattccg tctctttttt tgtctgaatt atttttattt tttaaaaatt 22320

ttaattgaag gctaattact ttacaatatt gtggcgattt ttgccataca ttcacatgca 22380

tcagccatgg gcgtgcatgt gagtccttct tattcacttc tcatgaacat gctcttgagg 22440

gccttctgca attccagctt tttggttctt ccaaacaaaa accaaaaaac agtgctgcca 22500

tgaacttcat tgtatctgag tttcagtgca tttctgacga gttgtggagc acgttcaact 22560

gtagtgatgt tgcccaactg ttttccaagg tagctgtacc attttctgtt cccagcttcc 22620

agttccagct ccaccgcatc ctcgctgaga attctctggc attttccccc ttccttttgg 22680

ccattgtgtt ggtggtggta aaatttgctc tctgcttgga agtggcgtct tctcttgttt 22740

tttagtcggc ctgaatttca taaatcttca gagaacagtg tcgcctctct tgatcctttc 22800

aactctattt ttctgtttca ttgctttctg ctccttcctg ctttctttat ttctttgggt 22860

tggatttaca ccccgatctt cctatccgag atagctggta ttaagttctg acctaatagc 22920

ttcttccagt ctttttttgc ttccatgtat aaagaaatac taaatttcaa gcatcgtagg 22980

tgtttttatt tgataatttg ataatcaaac acattttatt tgcttgattt gttttgattt 23040

gctttgattt gaaatgatca tctcatttta tgtgatatca gcagcaagac ttggggcatg 23100

ttcataaagt agctgctttg tactccagga cggacagctg agtcccagta accacacacc 23160

atctttgcct ccctgctgtc tacgcagtgc agaccctgct tatttacagc caaactttcc 23220

actaatgcag catccctcgc ctgctttttc agatgttcat gcgcgcgcag ttcgactatg 23280

atcccagaaa ggacaaccta atcccttgca aggaggcggg actgaaattc atcacggggg 23340

acatcatcca gatcatcaac aaggatgaca gcaactggtg gcagggccgg gtggaaggct 23400

catcccagga gtcagcgggc ctgatcccct cccctgagct gcaggaatgg tgagccactg 23460

atggacgagg acccatgccc accacagaga actgcccagc ttccacaatc tgtcatgccg 23520

tgtatccttt ctgttgctca atggagccaa cctcccagtt ttcctagtac aatgggcgga 23580

gggcaggggc aatgatgccc ttatgagctt gactggctgt tttatggcta aaggagcctg 23640

attagttctc cctgcatcca cggatctctg tggccacaga tgctttccct cagcttacaa 23700

agcaggccgt ggcactggga ctcaatcccg cctcccccag cgcccttctg agccatgatc 23760

ttctgagtcc tgcagaaaaa atgaaagtga agtcactcag tcgtgtctga ctctttgtga 23820

ccccatggac tgtagcccac caggttcctc tgtccagggg attctccagg caagaggact 23880

ggagtgggtg tccattgtct tctccagagg atctttctga cccagggatc gaacctgggt 23940

ctcccacatt gtaggcagac tctgagcact gctgagaaca gtcaaatttt aaggatgtgt 24000

ccattatgca catttgtggt acataggaaa ggttatccct gtgcccaagt tggattgcca 24060

tccctatcaa aacacccgta acatttttca ccaatgtaga acaaccctga aacttatttg 24120

gaaccacaca cacacacaca caaaaacaaa ccctgagttt ccaatgcagt cttaagaaaa 24180

aagaacaaag ctggagttag cacatttctt gacttcagaa tatactacaa agctacagtt 24240

gtcaaaagag catagtccta catcactata tttaaaatgg gtaaccaaca agggcctact 24300

gtatagcaca ttgaactctg ctcgatgtca tgtggcagcc tggatgggag gggagtttgg 24360

gggagaatgg atacacgtat atggatggct gagtcccttt gctgttcacc tgaaactatc 24420

acaatattgt taattggcta taccccgata caaaataaaa agtttaaaaa atagcatagt 24480

agtggcacaa gaacagacac acagaccaat ggaatggaac agagagccca gaaataaaca 24540

catgtactta acatcaatta acttatgaca gaggaggtaa gaatatgcaa aggaggaaag 24600

acagtatctt caataagtgg tgctaggaaa actggacaac tacatttact ttacaaaatt 24660

aaattagaat attttttcac accatttaca aaaagaaaag ggattaaaga ctgaagtgtt 24720

agactagaaa gtataaaacc ccaagaaaaa ctgactgcag catgtggctt gcagcatctt 24780

agttgctcaa ccatgcattg aacccacact cttgacagtg aaaatgcaga cctctaagca 24840

ctgcactgcc aggggattcg ctatattcca tttaaagtta taaaataacg gccgtatgtc 24900

cctatactat ataatacatc cttgtagctt atttatttta tgcatagcag tttttgtatt 24960

gatacctctt aatcctctac gcctatattg cccctccctc ttctttttcc tcagtggtaa 25020

ccacaagttt ttcctccata tctgtgagtc tgtttgtttt gtcttattca ttcattcgtt 25080

ttatatttta gatttcacgt acaagtgata acttaaagtt ttgtttttcc ctgagttttc 25140

actaagccta atactctcta ggcccatcca tgttattgta attggcagag tttcattctt 25200

tttattaatg gctgagtaat attcctctgt gtgtgtatgc tcatatgtgc atatgcgcat 25260

gttacatctt catccgttca tctgttggtg gacatatagg ttgtttccac gtcttggcta 25320

ttgtaaataa tgccttgaga gaaagggccg gaggcagatg gttctagggg tttcatgtag 25380

agggaccgca caggcaagtg acccagggct tttgcaccaa gaagccaccg atgtcctctg 25440

ttgtatcttc cctccaggcg agtggcgagt ggggcccact ctgctccaag tgaggcccct 25500

agctgcagcc cctttgggaa aaagaagaag tacaaagaca agtacctggc gaagcacagc 25560

gcaagtacgc tccccgactc gccgctgctc ctggtctcct atgaccacat ccctctgcct 25620

ggctccacac agtcagggca tgtgggagga aggctgctca tcacactttg cccctaggga 25680

tccaccctga ccgccttcca gacctttctc aatacatcag ccgacatcag tatgacccag 25740

tgcagggttc gagccacctc agacagcggc ctgttttcct gaactaactc tgactgtgtt 25800

ctttacctgc tgtgggcaca agcagattgt agataagaaa ggaagaatca tccatgtctg 25860

ctactgtctg ttaaatgcct acttcctggg gggagcatgt tcgagaaata caggagcaaa 25920

tcgcaggacc tgaaagcacc taccaagtca ggagtctcca tggagccaca gagggcagtt 25980

gtgtgacatg ggaggcagca ggatcagagt caggatgaca ctcatgtccc ctgagacaaa 26040

ggccagtaca ggcggtactg gggagatgac ggtggtaggg cagatagctg gggccaaaag 26100

cttacagaaa atctgagtgc aaagaactga ccaacagctg tgttgagttt cagtggcagg 26160

gggttgggaa aaccaagaca tagaccccaa ggaggggcac ccaccatcaa gggtcagtct 26220

cacccgcagt tgtccccacc agtgttcgag cctgtctggc tgatctcagt ctgccctgtt 26280

tgcccagctc tgggatgacc ttggtttgct tttctttgtc cctagttttt gaccagttgg 26340

atgttgtttc ctacgaggaa gtggttcggc tgcccgcgtt caagaggaag accctggttc 26400

tcattggtat cttcttgttg ctttgctact ggacacgaca tttctgtttg tgttcccctg 26460

cctgcaggat gtaccctact tccattgcct ccctgcctgg agcatgtaac ctacctccat 26520

gccttctccc tactctcttc ctccaaaaaa agtagggggc agtgaggaag aaaaggaagc 26580

aagaagttgg gtgtcttggg cctccatatg gagcagattt aagagtttac tgtgtcctta 26640

ccaggactca ccttggttca tgttgctaga gagggtaaac ctgatacagt tttcagcaac 26700

taattctttt aggtgattta tctgtctggc cagcagtagg gatgctctag gcctatgctt 26760

gcattctcca gaaaaggatt cagggctctt tgaagtggag caagttctcc ctgtctctag 26820

aaggaaaagg ttagttggtt agaggatctg tttaaacttg caggaaaagt ctgttccatt 26880

catttggggg gagtctagga aggcaaatag ccttcagaaa gaaaccgaaa atgccctgtg 26940

gtagccagct cctatataca aatgacagct gtgttctgaa ttgtcatcag gacaccaggc 27000

cctgaaaccc ttgatcctcc tgaccccagc ccttcagaag cccatgcaca ttgctttgac 27060

ctgtcactga tcgtgggctc tgagagttga aaacccacgt gttagtcctc tttctagccc 27120

tgtctttgtc tctggggaaa gaaatggaag cattgatctg catgcctgtc tctgtccatg 27180

caggagccag tggggtgggc cgcagccaca tcaagagtgc cctgctcagc cagaacccgg 27240

acaagtttgc ataccctgcc ccgtgtgagt agctcagggg cctgagggcc atggttccag 27300

agtggtctgt cattttgtgt tttttttttt tttccttctt agccatgcca cgtagctggg 27360

gatatcccaa tctccccggc cagggactga acccatgctt tggcagcaaa gacatagagc 27420

cctaaccact agacggccag ggaagtccct tctttgtgat ttttttaccc caaacatttg 27480

atgatcaaaa gaaaagatca tttggaaagg tacaaatttg cccctagagt tgctgcaact 27540

aacaaagtag tcatgctgtt ggtctgcata agggacatag tggcacttag ggtcattcaa 27600

taacttaaaa tgcaaactga gttcacaaga gtgggtgaaa gtaagaagaa gttgggtcaa 27660

agaaagactt tcagtgcgct tcccagaaat ccagactata tatagcctgc agatcggttt 27720

tattttttaa attttttcac aacactgcat ggaacgtggg atcttagttc cctgactagg 27780

gttcaagctt gtgccccagc ctgcattgga gggtggagtc ttaaccactg gactgcctgt 27840

gaagtctctg cagatctgct ctacaaagat ttccagcttc tgtgtccctg gggttggtgt 27900

tggagagaga ggcaacttta tgactgcagg agggtgggcg ctcactctgg agcatttaaa 27960

cgtgttcctg gcccacttca ttttcttgag tcaggcccta aggatcctaa taagaaagtg 28020

aagtcactca gtcatatctg acttggcgac ccccatggac tgtagcccac ccggcttctc 28080

cagccatgga attttctcag caagagtact ggggtgggtg gcccttgcct tctccagggg 28140

atcttctaga cccagggatc aaacccaggt ctcccatttt gcgggcagat gctgtaccgt 28200

ctgagacacc agggaagccc ttagaagatt ccctgaatca gaagcccctg cactaaaggc 28260

agctggggtg gcagtggcga caccagggtc tgtgcccccg ggagccacgg aggggggccc 28320

ccgagtggct cggattgatc ggggttctcg gggtgcagat acgacacggc cagccaggaa 28380

gagcgaagaa gatggcaagg aatatcactt catctccacg gaggagatga cgcggagcat 28440

ctccgccaat gagttcttgg agtttggcag ctaccagggc aacatgtttg gcaccaagtt 28500

cgagaccgtg caccagattc acaagcaggg caaggttgcc atcctggaca tcgagcctca 28560

ggtgggtcag tgggggaggc cggagcagat aagagcatgg tgctgaagag cacagctcag 28620

ggggacctgc aggatgtgca ccccggcttc acgggctgtc actggggtca ccaggaggaa 28680

gagctggacc agccatgcca gagacagtcc tggccttggc aaggccctaa gatgctgatt 28740

gccaggggtc tgctgtgacc gctgactctt tttcccctct cttctccctc cctccactcc 28800

gttttttttc cccagaccct gaagatcgtt cggacagcag aactttcacc tttcattgtg 28860

ttcattgcac ccactgacca aggcattcag gtgggaatgt agggtctgtg caggggggtg 28920

ggagacccga catatggttg gcagggtgct ggcagagctt aacgtcttag ctgctgttgt 28980

cttcctgccc ttgaggccaa gaagacagag cacttgcgtc cagtctgttc agttcagttg 29040

ctcagtcgtg tctgaccctt tgcgacccca tggaccacag cacgccaagc ctccctgtcc 29100

atcaccaact cccagagtct acccaaactc ctgtccatca agttggtgat gccatctaac 29160

catctcatcc tctgttgtcc ccttctcctc ccaccttcag tctttcccag catcagagtc 29220

ttttctagtg agtcagctct tcacatcctg tggcgaaagt attggagttt ctgttcaatg 29280

gggagttgtt aacccactag ccaggccgtt gttccagaag cacagagatg gatggtgggc 29340

ttaagagggg caaatgtggg ggacagggac cctggacata tgtggcccta ggcccacagg 29400

ggtcatgctg tggggagggg ggaccatgta gggaagcctc ctgggtccct agagccaacg 29460

agctccaaac cccgctgcag attgcttttc actgaacgca tctgtggaga ccttcctggg 29520

cctttctcag gagctgtggt ctcccatcat gtggatttag ctgaattatt tagttcccta 29580

ggtattccct agcggttcag atggtaaagc gtctgcccac aataagggag acccgggttc 29640

gattgctggg tcaggaagat cccctggtga aggcaatggc cacccactcc agtactcttg 29700

cctagaaact tccatggatg gaggagcctg gtgggctaca gtccacgggg tcacaaagag 29760

tcggatatga gtgagtgact tcactgtagt tccctcactg ccttcttgac ggctgtttag 29820

gtttaaaact tttaatagca tcaatatgac aatccatgat tctgtgcgtg tgcctcccat 29880

gtacaaatct gtctgtggaa gattgcaggc agcgtcatgt tgggaatttt ggatgtggcg 29940

ggggagttgc ccccctgttt caggagcatg tcctgcactt tcatctttgg agtctgctga 30000

atggggaagt ggaatctcat tgaacttggg tgtttgactg gaagtggtaa agcatcttgt 30060

ctagactccc aagggccagg tctgtctgtt tttctctgaa ctacctggtc agctcctgag 30120

cttatttttg ttcagtcatt ggccttatta atttctggga actctgtgtg aggatcatta 30180

gcccttaata gtatcagttg cacccccccc ccaatttggc cttttagtgt ttattgggcc 30240

tccctgttgg ctcagctggt aaagaatctg cccacaatgc aggaaacctg ggttcgatcc 30300

ctgggttggg aagatcccct ggagaaggga aaggctaccc actccagtat tctggcctgg 30360

agaattccat ggactgtata gtccatgggg tcaccaagag tccgacatga ctgagcaact 30420

tgcactttgc ctttcacttt catgtttatt gctactgaga cgtcttcagg gagcacttct 30480

gcatacccca gcccattaac attctcaggc aagggcttca gggttttagg tttttttttt 30540

ttttcagtga atgaaagtca ctcagtcgtg tccagctctt tgcaatcccg tggactgtag 30600

ctcaccaggc tcctctgtcc atgggatttc ccaggcaaga atactggagt gggttgtcat 30660

ttccttctcc agggaatctt cctgatccaa ggattgaacc agggtctcct gcattgcagg 30720

cagattcttt accatctagc caccagggaa gcccttaggc ttttcagcac gtgattcaaa 30780

gttgggtggc gggcacggcg atcaaggcct ccacttgaac caccctgaac ctcccaggca 30840

gggcacacgc agcctgctcc atccatgggc ctctcccttc tctcctccac agacggacgc 30900

cctgcagcag ctgcagaagg actcggaggc catccgcatc cattatgcgc actactttga 30960

cctctcgctg gtcaataaca gcgttgagga aaccctgaag acattgcaag aagcatttga 31020

ccaggcgtgc cgttccccac agtgggtgcc tgtctcctgg gtttactaag ctttgcacca 31080

tagtggggag tgactggggg cccctgttca gcctttggaa gtccaccccg ccttgctgtt 31140

ttaagaccac aggctcgcta gctttgtgtc agcttctagc tctctacaac tctcctcctc 31200

atgaggctgg tgggtgtctt gtcttcctca catttctgaa gggctgggct gatttcctga 31260

cagagcagga ttttcaaagt atccctgcac actgagggcc agaagcactc ggccaaggca 31320

gctgctgatc acagctagtg cccacaggga agaaaagcaa tggaccccct ggcccccctg 31380

aagcctgcac cccatttacc tttggtcaca ccaggcattt catgtgtctt taaagacagc 31440

atttgtcact gcgactttct accctgccaa atcaaacagc tgtgcaatag gatgggtctg 31500

ctctagggaa accctcaaaa gcaataaaag tattgctgtg ttaaaatgcc a 31551

<210> 2

<211> 20

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 2

actttgttat aggttgcgcg 20

<210> 3

<211> 20

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 3

tcatcttatg ggagagccgg 20

<210> 4

<211> 26

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 4

atcgatactt tgttataggt tgcgcg 26

<210> 5

<211> 26

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 5

ggatcctcat cttatgggag agccgg 26

<210> 6

<211> 29

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 6

agcatcgata ctttgttata ggttgcgcg 29

<210> 7

<211> 29

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 7

cgtggatcct catcttatgg gagagccgg 29

<210> 8

<211> 18

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 8

agccatccat caatcaca 18

<210> 9

<211> 19

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 9

ctcgcagtat tacaagcag 19

Claims

1. The method for evaluating the sheep oocyte in-vitro culture system by utilizing the MPP1 gene is characterized in that the MPP1 gene is a molecular marker gene MPP1 of sheep X chromosome, and the nucleotide sequence of the MPP1 gene is shown as SEQ ID No. 1.

2. The method for evaluating an in vitro culture system of ovine oocytes using the MPP1 gene according to claim 1, wherein the MPP1 gene is MPP1 gene of sheep Chromosome X-NC-040278.1.

3. The method for evaluating an in vitro culture system of ovine oocytes using the MPP1 gene of claim 1, wherein the promoter region of the MPP1 gene of the X chromosome of ovine is selected to design gene amplification primers.

4. The method for evaluating an in vitro culture system of ovine oocytes using the MPP1 gene according to claim 1, comprising the steps of:

s3 and construction of an MPP1 gene promoter pLV-mCherry reconstruction vector: respectively using restriction enzymes Cla I and BamH I to carry out enzyme digestion on a sheep MPP1 gene promoter sequence and a pLV-mCherry vector, respectively recovering the MPP1 gene promoter sequence and the pLV-mCherry vector by adopting agarose gel electrophoresis, and then using T4 ligase to connect the MPP1 gene promoter sequence and the pLV-mCherry vector to construct a pLV-mCherry reconstruction vector of which the MPP1 gene starts mCherry expression; carrying out PCR identification on thalli containing the reconstruction vector by using a sheep MPP1 gene promoter identification primer so as to detect whether the construction of the MPP1 gene promoter pLV-mCherry reconstruction vector is correct or not;

s5, in vitro culture of sheep ovarian tissue: collecting sheep ovary tissue, separating to remove ovary primary follicle, culturing ovary primary follicle in culture dish microdrop, and placing at 37 deg.C with 5% CO₂Culturing in a saturated humidity incubator, and adopting packaging virus after the ovary primary follicle adheres to the wallA mediated MPP1 gene promoter-mCherry expression transgenic technology;

5. The method for evaluating the sheep oocyte in vitro culture system by utilizing the MPP1 gene according to claim 4, wherein the sheep MPP1 gene promoter amplification primers before the enzyme cutting site are added, and the nucleotide sequences of the upstream amplification primer and the downstream amplification primer are respectively as follows:

an upstream primer F: 5'-ACTTTGTTATAGGTTGCGCG-3', respectively;

a downstream primer R: 5'-TCATCTTATGGGAGAGCCGG-3', respectively;

the gene sequence length of the MPP1 gene promoter is 2008 bp.

6. The method for evaluating an in vitro culture system of ovine oocytes using the MPP1 gene according to claim 5,

the restriction enzyme site of the upstream amplification primer is Cla I, and the nucleotide sequence of the upstream amplification primer of the sheep MPP1 gene promoter after the restriction enzyme site of ClaI is added is as follows:

F：5’-ATCGATACTTTGTTATAGGTTGCGCG-3’；

R：5’-GGATCCTCATCTTATGGGAGAGCCGG-3’。

7. the method for evaluating an in vitro culture system of ovine oocytes using the MPP1 gene according to claim 6,

the protection base of the upstream amplification primer is AGC, after the Cla I enzyme cutting site is added to the upstream amplification primer, the protection base AGC is continuously added to the 5' end of the primer, and the nucleotide sequence after the protection base is added is as follows:

F：5’-AGCATCGATACTTTGTTATAGGTTGCGCG-3’；

R：5’-CGTGGATCCTCATCTTATGGGAGAGCCGG-3’。

8. the method for evaluating the in vitro culture system of the sheep oocyte with the MPP1 gene according to claim 4, wherein in the step S3, the sheep MPP1 gene promoter identification primer is designed by using primer design software premier 6.0;

the identified fragment length is 159 bp.