Application of hormone-sensitive lipase HSL gene in improving the developmental ability of porcine embryos in vitro

Abstract

The application discloses application of a hormone-sensitive lipase HSL gene in improving in-vitro embryo development capacity of pigs and belongs to the technical field of embryo development. The application aims to improve in-vitro embryo development capacity of pigs. The application provides application of the hormone-sensitive lipase HSL in improving in-vitro embryo development capacity of pigs, wherein the amino acid sequence of the hormone-sensitive lipase HSL protein is shown as SEQ ID NO. 3. The application proves that overexpression of the hormone-sensitive lipase HSL gene can provide technical support for improving in-vitro early embryo development capacity of pigs and improving pig reproductive capacity, and provides a brand-new way for good-breed pig breeding work, and has good application value.

Description

Technical Field

[0001] The invention belongs to the technical field of embryonic development, and particularly relates to the application of hormone-sensitive lipase (HSL) gene in improving the developmental ability of pig embryos in vitro. Background Art

[0002] In vitro embryo production (IVP) primarily includes oocyte maturation (IVM), in vitro fertilization (IVF), and in vitro embryo culture (IVC). IVP technology can fully tap the genetic potential of my country's elite pig breeds, expand superior breeds, promote genetic improvement, and support the development of disease model pigs and organ donor pigs. However, the developmental capacity of mammalian embryos in vitro is far lower than that in vivo.

[0003] It is currently believed that the main reasons why the developmental capacity of in vitro embryos is far lower than that of in vivo embryos include oxidative stress and abnormal epigenetic modification. Although the developmental capacity of in vitro embryos can be improved by effectively clearing excess ROS in cells and regulating histone methylation, histone acetylation, DNA methylation, etc., pig embryos have high lipid content and are species-specific, and there is an urgent need to improve the developmental capacity of pig embryos in vitro. Summary of the Invention

[0004] The purpose of the present invention is to improve the pig embryonic development ability in vitro.

[0005] The present invention provides an application of a hormone-sensitive lipase HSL protein in improving the in vitro embryonic development ability of pigs. The amino acid sequence of the hormone-sensitive lipase HSL protein is shown in SEQ ID NO.3.

[0006] The present invention provides an application of an overexpressed hormone-sensitive lipase HSL gene in improving the in vitro embryonic development ability of pigs. The sequence of the hormone-sensitive lipase HSL gene is shown in SEQ ID NO.4.

[0007] The present invention provides an application of overexpressing hormone-sensitive lipase HSL mRNA in improving the developmental ability of pig embryos in vitro. The sequence of the hormone-sensitive lipase HSL mRNA is shown as SEQ ID NO.7.

[0008] The present invention provides an application of a recombinant vector containing an overexpressed hormone-sensitive lipase HSL gene in improving the in vitro embryonic development ability of pigs. The sequence of the hormone-sensitive lipase HSL gene is shown in SEQ ID NO.4.

[0009] It is further defined that the backbone of the recombinant vector is pcDNA3.1_EGFP.

[0010] It is further defined and characterized in that the recombinant vector also includes a Kozak sequence.

[0011] The present invention provides an application of a recombinant microbial cell containing an overexpressed hormone-sensitive lipase HSL gene in improving the in vitro embryonic development ability of pigs. The sequence of the hormone-sensitive lipase HSL gene is shown in SEQ ID NO.4.

[0012] The present invention provides a method for improving pig early embryonic development in vitro, which overexpresses hormone-sensitive lipase HSL in pig parthenogenetic embryos; the sequence of the hormone-sensitive lipase HSL is shown in SEQ ID NO.3.

[0013] It is further defined that overexpressing hormone-sensitive lipase HSL is to use a recombinant vector containing the hormone-sensitive lipase HSL gene as a template to perform reverse transcription to obtain the hormone-sensitive lipase HSL mRNA sequence.

[0014] The present invention provides a pig embryo obtained by the above method.

[0015] Beneficial effects: The present invention constructs an HSL gene mRNA vector for in vitro transcription, and the obtained mRNA is injected into pig parthenogenetic embryos, which can significantly improve the pig in vitro embryonic development ability. The mRNA injected into the pig parthenogenetic embryos of the present invention can fuse the EGFP gene and the HSL gene for expression, and can be quickly screened after microinjection. The vector constructed by the present invention has a Kozak sequence, which can promote stable translation after mRNA injection and improve the subsequent pig in vitro embryonic development ability. The HSL overexpression vector constructed by the present invention with pcDNA3.1_EGFP as the skeleton contains a T7 promoter and is suitable for in vitro transcription of a large amount of mRNA. The mRNA containing the HSL gene target sequence obtained by the in vitro transcription reaction of the present invention is injected into the pig parthenogenetic embryo by microinjection, which can make the pig in vitro embryo overexpress HSL protein. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 . -blunt-HSL carrier information;

[0017] Figure 2 .Information of HSL overexpression vector using pcDNA3.1_EGFP as the backbone;

[0018] Figure 3 .pcDNA3.1_EGFP vector information;

[0019] Figure 4Green fluorescence images of embryos specifically overexpressing HSL (injected with 50 and 100 ng / μL HSL mRNA), as well as embryo survival rate, EGFP positivity rate, EGFP fluorescence intensity, and HSL mRNA and protein expression levels. A represents green fluorescent protein expression in embryos; B represents embryo survival rate; C and D represent embryonic EGFP positivity rate and fluorescence intensity, respectively; and E and F represent HSL mRNA and protein expression levels in 2-cell embryos, respectively.

[0020] Figure 5 Effects of specific HSL overexpression on the developmental capacity of porcine parthenogenetic embryos (injection of 100 ng / μL HSL mRNA). Where A represents the cleavage rate, B represents the blastocyst rate, and C represents the number of blastocyst cells.

[0021] Figure 6 Effects of specific overexpression of HSL (injection of 100 ng / μL HSL mRNA) on lipid droplets, fatty acids, and ATP in early porcine embryos; A, B, C, and D show staining of HSL, lipid droplets, fatty acids, and ATP in early porcine embryos, E shows HSL mRNA expression levels, F shows HSL protein expression levels, G shows lipid droplet fluorescence intensity analysis, H shows lipid droplet size analysis, I shows fatty acid fluorescence intensity analysis, and J shows ATP fluorescence intensity analysis. DETAILED DESCRIPTION

[0022] In order to make the purpose, technical solutions and advantages of the present invention more clearly understood, the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.

[0023] Example 1. Construction of recombinant vector

[0024] 1. Acquisition of HSL gene with restriction enzyme sites (BamH I and EcoR I)

[0025] RNA was extracted from pig parthenogenetic embryos using an RNA extraction kit, and the corresponding cDNA was obtained by reverse transcription PCR. The cDNA product was PCR-generated using primers with restriction enzyme sites and KOZAK sequences (GCCACCAUGG) (reaction conditions: 50 μL system, containing KOD One TM PCR Master Mix 25 μL, 1.5 μL each of 10 μM HSL-F and HSL-R, 2 μL cDNA, 20 μL deionized water; reaction conditions: denaturation at 98°C for 10 s, 68°C for 30 s, for a total of 45 cycles), the obtained 2310 bp fragment was -blunt Simple Cloning Kit (Trans, CB111) for T vector ligation, construction -blunt-HSL vector ( Figure 1 for -blunt-HSL vector information) and transformed into Escherichia coli Trans-T1 for sequencing verification. The sequencing results had a consistency rate of 99.99%, indicating that the PCR product was the desired HSL gene fragment.

[0026] According to the CDS sequence of HSL in NCBI (Gene ID: 397583), primer sequences with restriction enzyme sites (BamH I and EcoR I) and KOZAK sequences were designed:

[0027] HSL-F: GGATCCGCCACCATGGACCTGCGCACAATGACA; (SEQ ID NO. 1);

[0028] HSL-R: GAATTCGACTGGCGGCGTGGTGGG; (SEQ ID NO. 2);

[0029] HSL amino acid sequence: (SEQ ID NO.3)

[0030] MDLRTMTQSLVTLAEDNMAFFSGQGPGETARRLSGVFAGIREQALGLEPALGRLLSVAHL

[0031] FDLDAETPANGYRSLVHTARCCLAHLLHKSRYVASNRRSIFFRTSHNLAELEAYLAALTQ

[0032] LRALAYYAQRLLAINRPGKLFFEGDEGVTADFLREYVTLHKGCFYGRCLGFQFTPAIRPFL

[0033] QTISIGLVSFGEHYKRNETGLSVTASSLFTSGRFAIDPELRGAEFERIIQNLDVHFWKAFWNI

[0034] TEIEVLSSLANMASATVRVSRLLSLPPKAFEMPLTADPKLTTVTISPPLAHTGPGPVLVRLISY

[0035] DLREGQDSEELSSLVRSEGPRGLELRPRPQQAPRSRSLVVHIHGGGFVAQTSKSHEPYLKS

[0036] WAQELGVPILSIDYSLAPEAPFPRALEECFYAYCWAVKHCGLLGSTGERICLAGDSAGGN

[0037] LCFTVSLRAAAYGVRVPDGIMAAYPATMLQSAASPSRLLSLMDPLLPLSVLSKCVSAYAG

[0038] GEMEDHSDSDQKALGMMGLVRRDTALLFRDLRLGASSWLNSFLELSGHKSRPNLVPTEE

[0039] PMRRSVSEAALAQPEGPLGTDSLKYLTLHDLSLSSETQDTPELSLSAETLGPTTPSAVNFLF

[0040] RPEDAPEEAEARDEISTKEEKVYSVRAAFPEGFHPRRSSQGAIQMPLYSAPIVKNPFMSPLL

[0041] APDSMLQTLPPVHIVACALDPMLDDSVMFARRLRSLGQPVTLHVVEDLPHGFLSLAALCRETRQAAALCVDRIRFILNPPGPATPAGPTTPPV;

[0042] HSL CDS序列:(SEQ ID NO.4)

[0043] ATGGACCTGCGCACAATGACACAGTCACTGGTGACCCTGGCGGAGGACAACATGGCCT

[0044] TCTTCTCTGGCCAGGGCCCTGGGGAGACAGCACGGCGGCTGTCAGGTGTCTTTGCGGG

[0045] TATTCGGGAACAGGCACTGGGGCTGGAGCCGGCCCTGGGCCGCCTGCTGAGTGTGGCA

[0046] CACCTCTTCGACCTGGATGCAGAGACGCCAGCCAACGGGTACCGCAGCCTGGTACACA

[0047] CAGCCCGCTGCTGCCTGGCACACCTGCTGCACAAATCACGCTACGTGGCCTCCAACCG

[0048] CCGCAGCATCTTCTTCCGCACAAGCCACAACCTGGCCGAACTCGAGGCCTACCTAGCT

[0049] GCTCTCACCCAGCTCCGCGCTCTGGCCTACTATGCCCAGCGCCTGCTGGCCATCAACCG

[0050] GCCCGGGAAGCTCTTCTTCGAGGGTGACGAAGGCGTCACTGCTGACTTCCTGCGCGAG

[0051] TATGTCACTCTACACAAGGGCTGCTTCTACGGCCGCTGCCTGGGCTTCCAGTTCACGCC

[0052] CGCCATCCGGCCTTTCCTGCAGACCATCTCCATCGGGCTGGTGTCCTTCGGGGAGCATT

[0053] ACAAACGCAATGAAACAGGCCTCAGTGTGACAGCCAGCTCCCTCTTCACCAGTGGCCG

[0054] CTTCGCCATCGACCCCGAGCTGCGTGGGGCCGAGTTTGAGCGGATCATACAGAACCTG

[0055] GATGTGCACTTCTGGAAAGCTTTCTGGAATATCACCGAGATTGAGGTGCTATCGTCTCTA

[0056] GCAAACATGGCATCGGCCACCGTGAGGGTAAGCCGCCTGCTCAGCCTACCGCCCAAGG

[0057] CCTTTGAAATGCCACTGACTGCTGACCCCAAGCTCACGGTCACCATTTCACCCCCGCTG

[0058] GCCCACACAGGCCCCGGGCCCGTCCTCGTCAGGCTCATCTCCTATGACCTGCGTGAAG

[0059] GACAGGACAGTGAGGAGCTCAGCAGCCTGGTGAGGTCTGAGGGCCCCAGGGGCCTAG

[0060] AGCTGCGGCCCCGCCCCCAGCAGGCACCCCGCTCCAGGTCCCTGGTCGTGCACATCCA

[0061] TGGTGGCGGCTTCGTGGCCCAGACGTCCAAATCCCACGAGCCTTACCTCAAGAGCTGG

[0062] GCCCAGGAGCTGGGGGTCCCCATCCTCTCCATCGACTACTCCCTAGCGCCCGAGGCCCC

[0063] CTTCCCCCGTGCGCTGGAGGAGTGCTTCTACGCCTACTGCTGGGCTGTCAAGCACTGC

[0064] GGTCTCCTCGGTTCCACAGGTGAGCGGATATGCCTCGCAGGAGACAGCGCAGGCGGGA

[0065] ACCTCTGCTTCACTGTGTCCCTTCGGGCAGCAGCCTATGGAGTGCGGGTGCCAGATGGC

[0066] ATCATGGCAGCCTACCCGGCCACAATGCTGCAGTCTGCCGCCTCCCCCTCCCGCCTTCT

[0067] CAGCCTCATGGACCCCCTGCTGCCCCTCAGCGTGCTCTCCAAGTGTGTCAGTGCCTATG

[0068] CTGGCGGGGAGATGGAGGATCACTCTGACTCAGACCAGAAGGCGCTGGGCATGATGG

[0069] GACTGGTGCGGCGGGACACAGCCCTGCTCTTCCGAGACCTCCGCCTGGGCGCCTCCTC

[0070] GTGGCTCAACTCCTTCCTGGAGCTGAGTGGGCACAAGTCCCGACCCAACTTGGTGCCC

[0071] ACAGAAGAGCCAATGCGGCGCAGTGTGTCTGAAGCAGCACTGGCCCAGCCTGAGGGC

[0072] CCACTGGGAACGGACTCCCTCAAGTACCTGACACTGCATGACCTGAGCCTAAGCTCCG

[0073] AGACACAGGACACCCCGGAGCTGTCACTGTCAGCTGAGACGCTTGGCCCCACCACAC

[0074] CCTCGGCTGTCAACTTCTTATTTCGACCTGAGGATGCACCTGAAGAGGCTGAGGCCCG

[0075] AGACGAGATTAGCACCAAGGAGGAGAAAGTCTACAGTGTGAGGGCCGCCTTCCCTGA

[0076] GGGTTTCCACCCAAGGCGCTCCAGCCAAGGTGCAATACAGATGCCCCTCTACTCAGCT

[0077] CCCATCGTCAAGAATCCCTTCATGTCACCACTGCTGGCACCTGACAGCATGCTGCAGAC

[0078] CCTCCCACCTGTGCACATTGTGGCCTGCGCGCTGGACCCCATGCTGGACGACTCGGTCA

[0079] TGTTCGCTAGGCGGCTACGCAGCCTGGGCCAGCCCGTGACGCTGCACGTGGTGGAGGA

[0080] CCTGCCGCACGGCTTCCTGAGCCTGGCGGCGCTGTGCCGGGAGACCCGGCAGGCCGC

[0081] GGCGCTGTGCGTGGACCGCATTCGCTTTATCCTCAATCCGCCGGGCCCTGCCACACCGGCTGGGCCCACCACGCCGCCAGTCTGA。

[0082] 2.以pcDNA3.1_EGFP作为骨架的HSL过表达载体的构建

[0083] For gene fragments with restriction enzyme cutting sites ( -blunt-HSL) and vector plasmid (pcDNA3.1_EGFP, Figure 3 (pcDNA3.1_EGFP vector information) was double-digested with BamH I and EcoR I enzymes to obtain sticky ends. After enzyme digestion, the target gene and plasmid were subjected to agarose electrophoresis and gel recovery. The recovered gel concentration was determined, and the target gene and plasmid were ligated using T4 ligase. The product was transformed into competent Escherichia coli DH5α and plated on a culture medium containing ampicillin for screening. Single colonies were picked for culture and identified by PCR and sequencing to obtain the HSL overexpression vector (pcDNA3.1_HSL_EGFP). Figure 2 This is the information of the HSL overexpression vector using pcDNA3.1_EGFP as the backbone.

[0084] 3. In vitro transcription

[0085] Using pcDNA3.1_HSL_EGFP as template, PCR reaction was performed using primers of T7 promoter sequence (reaction conditions: 50 μL system, containing KOD One TM PCR Master Mix (25 μL), 1.5 μL each of 10 μM HSL-F and HSL-R, 2 μL of cDNA, and 20 μL of deionized water (reaction conditions: denaturation at 98°C for 10 s, 68°C for 40 s, for a total of 45 cycles) were used. A 3232 bp HSL_EGFP fragment containing the T7 promoter sequence was obtained by agarose gel electrophoresis and gel recovery. Using NEB's mRNA in vitro transcription kit E2060, a capping transcription process and Poly(A) tailing reaction were performed to obtain a large amount of mRNA containing the porcine HSL gene transcript. The product was then purified using the OMEGA MicroElute RNA Clean Up Kit. Finally, the mRNA was diluted to 0-100 ng / μL in RNase-free triple-distilled water for subsequent microinjection.

[0086] T7 promoter primer sequence:

[0087] T7-F:5'-TAATACGACTCACTATAGGG-3'; (SEQ ID NO.5);

[0088] T7-R: 5'-TAGAAGGCACAGTCGAGG-3'; (SEQ ID NO. 6).

[0089] HSL mRNA sequence: (SEQ ID NO.7)

[0090] UAAUACGACUCACUAUAGGGAGACCCAAGCUGGCUAGCGUUUAAACUUAAGCUUG

[0091] GUACCGAGCUCGGAUCCGCCACCAUGGACCUGCGCACAAUGACACAGUCACUGGUG

[0092] ACCCUGGCGGAGGACAACAUGGCCUUCUUCUCUGGCCAGGGCCCUGGGGAGACAGC

[0093] ACGGCGGCUGUCAGGUGUCUUUGCGGGUAUUCGGGAACAGGCACUGGGGCUGGAG

[0094] CCGGCCCUGGGCCGCCUGCUGAGUGUGGCACACCUCUUCGACCUGGAUGCAGAGAC

[0095] GCCAGCCAACGGGUACCGCAGCCUGGUACACACAGCCCGCUGCUGCCUGGCACACC

[0096] UGCUGCACAAAUCACGCUACGUGGCCUCCAACCGCCGCAGCAUCUUCUUCCGCACA

[0097] AGCCACAACCUGGCCGAACUCGAGGCCUACCUAGCUGCUCUCACCCAGCUCCGCGC

[0098] UCUGGCCUACUAUGCCCAGCGCCUGCUGGCCAUCAACCGGCCCGGGAAGCUCUUCU

[0099] UCGAGGGUGACGAAGGCGUCACUGCUGACUUCCUGCGCGAGUAUGUCACUCUACAC

[0100] AAGGGCUGCUUCUACGGCCGCUGCCUGGGCUUCCAGUUCACGCCCGCCAUCCGGCC

[0101] UUUCCUGCAGACCAUCUCCAUCGGGCUGGUGUCCUUCGGGGAGCAUUACAAACGCA

[0102] AUGAAACAGGCCUCAGUGUGACAGCCAGCUCCCUCUUCACCAGUGGCCGCUUCGCC

[0103] AUCGACCCCGAGCUGCGUGGGGCCGAGUUUGAGCGGAUCAUACAGAACCUGGAUGU

[0104] GCACUUCUGGAAAGCUUUCUGGAAUAUCACCGAGAUUGAGGUGCUAUCGUCUCUA

[0105] GCAAACAUGGCAUCGGCCACCGUGAGGGUAAGCCGCCUGCUCAGCCUACCGCCCAA

[0106] GGCCUUUGAAAUGCCACUGACUGCUGACCCCAAGCUCACGGUCACCAUUUCACCCC

[0107] CGCUGGCCCACACAGGCCCCGGGCCCGUCCUCGUCAGGCUCAUCUCCUAUGACCUGC

[0108] GUGAAGGACAGGACAGUGAGGAGCUCAGCAGCCUGGUGAGGUCUGAGGGCCCCAG

[0109] GGGCCUAGAGCUGCGGCCCCGCCCCCAGCAGGCACCCCGCUCCAGGUCCCUGGUCGU

[0110] GCACAUCCAUGGUGGCGGCUUCGUGGCCCAGACGUCCAAAUCCCACGAGCCUUACC

[0111] UCAAGAGCUGGGCCCAGGAGCUGGGGGUCCCCAUCCUCUCCAUCGACUACUCCCUA

[0112] GCGCCCGAGGCCCCCUUCCCCCGUGCGCUGGAGGAGUGCUUCUACGCCUACUGCUG

[0113] GGCUGUCAAGCACUGCGGUCUCCUCGGUUCCACAGGUGAGCGGAUAUGCCUCGCAG

[0114] GAGACAGCGCAGGCGGGAACCUCUGCUUCACUGUGUCCCUUCGGGCAGCAGCCUAU

[0115] GGAGUGCGGGUGCCAGAUGGCAUCAUGGCAGCCUACCCGGCCACAAUGCUGCAGUC

[0116] UGCCGCCUCCCCCUCCCGCCUUCUCAGCCUCAUGGACCCCCUGCUGCCCCUCAGCGU

[0117] GCUCUCCAAGUGUGUCAGUGCCUAUGCUGGCGGGGAGAUGGAGGAUCACUCUGACU

[0118] CAGACCAGAAGGCGCUGGGCAUGAUGGGACUGGUGCGGCGGGACACAGCCCUGCUC

[0119] UUCCGAGACCUCCGCCUGGGCGCCUCCUCGUGGCUCAACUCCUUCCUGGAGCUGAG

[0120] UGGGCACAAGUCCCGACCCAACUUGGUGCCCACAGAAGAGCCAAUGCGGCGCAGUG

[0121] UGUCUGAAGCAGCACUGGCCCAGCCUGAGGGCCCACUGGGAACGGACUCCCUCAAG

[0122] UACCUGACACUGCAUGACCUGAGCCUAAGCUCCGAGACACAGGACACCCCGGAGCU

[0123] GUCACUGUCAGCUGAGACGCUUGGCCCCACCACACCCUCGGCUGUCAACUUCUUAU

[0124] UUCGACCUGAGGAUGCACCUGAAGAGGCUGAGGCCCGAGACGAGAUUAGCACCAAG

[0125] GAGGAGAAAGUCUACAGUGUGAGGGCCGCCUUCCCUGAGGGUUUCCACCCAAGGCG

[0126] CUCCAGCCAAGGUGCAAUACAGAUGCCCCUCUACUCAGCUCCCAUCGUCAAGAAUC

[0127] CCUUCAUGUCACCACUGCUGGCACCUGACAGCAUGCUGCAGACCCUCCCACCUGUG

[0128] CACAUUGUGGCCUGCGCGCUGGACCCCAUGCUGGACGACUCGGUCAUGUUCGCUAG

[0129] GCGGCUACGCAGCCUGGGCCAGCCCGUGACGCUGCACGUGGUGGAGGACCUGCCGC

[0130] ACGGCUUCCUGAGCCUGGCGGCGCUGUGCCGGGAGACCCGGCAGGCCGCGGCGCUG

[0131] UGCGUGGACCGCAUUCGCUUUAUCCUCAAUCCGCCGGGCCCUGCCACACCGGCUGG

[0132] GCCCACCACGCCGCCAGUCGAAUUCUGCAGAUAUCCAGCACAGUGGCGGCCGCUCG

[0133] AGGCCACCGGUGGCGGCGGUUCGGGAGGUGGCGGUUCUGGCGGAGGUGGCUCAAA

[0134] GCUUGUGAGCAAGGGCGAGGAGCUGUUCACCGGGGUGGUGCCCAUCCUGGUCGAGC

[0135] UGGACGGCGACGUAAACGGCCACAAGUUCAGCGUGUCCGGCGAGGGCGAGGGCGAU

[0136] GCCACCUACGGCAAGCUGACCCUGAAGUUCAUCUGCACCACCGGCAAGCUGCCCGU

[0137] GCCCUGCCCACCCUCGUGACCACCCUGACCUACGGCGUGCAGUGCUUCAGCCGCU

[0138] ACCCCGACCACAUGAAGCAGCACGACUUCUUCAAGUCCGCCAUGCCCGAAGGCUAC

[0139] GUCCAGGAGCGCACCAUCUUCUUCAAGGACGACGGCAACUACAAGACCCGCGCCGA

[0140] GGUGAAGUUCGAGGGCGACACCCUGGUGAACCGCAUCGAGCUGAAGGGCAUCGACU

[0141] UCAAGGAGGACGGCAACAUCCUGGGGCACAAGCUGGAGUACAACUACAACAGCCAC

[0142] AACGUCUAUAUCAUGGCCGACAAGCAGAAGAACGGCAUCAAGGUGAACUUCAAGA

[0143] UCCGCCACAACAUCGAGGACGGCAGCGUGCAGCUCGCCGACCACUACCAGCAGAAC

[0144] ACCCCCAUCGGCGACGGCCCCGUGCUGCUGCCCGACAACCACUACCUGAGCACCCAG

[0145] UCCGCCCUGAGCAAAGACCCCAACGAGAAGCGCGAUCACAUGGUCCUGCUGGAGUU

[0146] CGUGACCGCCGCCGGGAUCACUCUCGGCAUGGACGAGCUGUACAAGUAAUCUAGAGGGCCCGUUUAAACCCGCUGAUCAGCCUCGACUGUGCCUUCUA。

[0147] 实施例2.获得转基因胚胎

[0148] 1.猪卵母细胞的获取

[0149] Porcine ovaries were obtained from a local slaughterhouse and transported to the laboratory in 30°C saline. Cumulus-oocyte complexes (COCs) were extracted from follicles 3-6 mm in diameter using an 18G syringe. The COCs were cultured in MAT for 40 hours at 38.5°C in a 5% CO2 saturated humidity environment. Cumulus cells were eluted from the COCs in 0.1% hyaluronidase. Denuded oocytes (DOs) were then placed in MAN working solution (embryo manipulation medium). Mature oocytes with a first polar body and homogeneous cytoplasm were selected under a stereomicroscope.

[0150] 2. Parthenogenetic Embryo Culture

[0151] Preheat the FM fusion solution for parthenogenetic embryos in a 37°C incubator. Mount the FM-rinsed fusion chamber on a 37°C warming platform. Connect the electrodes to the chamber, ensuring a stable connection. Add 600 μL of FM fusion solution to the chamber. Transfer the mature oocytes to a small dish containing FM fusion solution. Allow the oocytes to fully settle to the bottom of the solution. Then, transfer the mature oocytes to the fusion chamber and arrange them in a straight line, parallel to each other, without floating in the liquid or overlapping. Apply two DC electric shocks at 1.2 kV / cm for 30 μs. After electrical activation, transfer the mature oocytes to a pre-equilibrated droplet of PZM3 working solution. After four thorough washes, place 50 oocytes per well in a PZM3 embryo culture plate and store them at 38.5°C in a saturated humidity environment with 5% CO2.

[0152] 3. Transform the in vitro transcription product into pig parthenogenetic embryos by microinjection

[0153] Microinjection droplets were prepared using MAN working solution and covered with mineral oil. 10 μL of HSL mRNA at an effective concentration of 50-100 ng / μL was injected into the cytoplasm of embryos cultured for 4-6 hours after parthenogenetic activation. After the injection, the embryos were moved to pre-equilibrated PZM3 wash drops for further culture. Embryonic development was observed in a 38.5°C, 5% CO2, saturated humidity environment.

[0154] 4. Detection of oocyte survival rate, EGFP positive rate, fluorescence intensity, HSL mRNA expression level, and HSL protein expression level

[0155] Different concentrations of HSL-EGFP mRNA (control group 0 ng / μL, 50 ng / μL, 100 ng / μL) were injected into the cytoplasm of parthenogenetic embryos that had been cultured for 4-6 hours. In addition, a blank control group and a negative control group (water injection group, C-Water) were set up. The embryo survival rate (number of surviving embryos / total number of embryos cultured × 100%), EGFP positivity rate (number of embryos with green fluorescence after injection / total number of injected embryos × 100%), fluorescence intensity, mRNA and protein expression levels were calculated.

[0156] like Figure 4 As shown in Figure A, after injection of 50 ng / μL and 100 ng / μL HSL mRNA, green fluorescent protein was expressed in the embryos, but not in the control group, indicating that the injection concentration was effective;

[0157] like Figure 4 As shown in Figure B, there was no significant difference in embryo survival rate among the negative control group, 50 ng / μL injection group, and 100 ng / μL injection group;

[0158] like Figure 4 As shown in Figures CD, the EGFP positive rate and fluorescence intensity of the 100 ng / μL group were significantly higher than those of the 50 ng / μL group (P < 0.05);

[0159] like Figure 4 As shown in Figure E, the HSL mRNA level in the 2-cell stage of the 100 ng / μL group was significantly higher than that in the control group (P < 0.05), and protein detection results showed that EGFP-HSL fusion protein was expressed.

[0160] HSL-EGFP-mRNA sequence:

[0161] HSL-EGFP-F:5'-CCCTCGGCTGTCAACTTCTTATTT-3'; (SEQ ID NO.8)

[0162] HSL-EGFP-R:5'-GCCCTCACACTGTAGACTTTCTCCT-3'; (SEQ ID NO.9)

[0163] The above results demonstrate that the method of this example has successfully established a porcine parthenogenetic embryo HSL overexpression system, and subsequent verification experiments were performed using an injection concentration of 100 ng / μL.

[0164] 5. Effect of specific overexpression of HSL on embryonic developmental capacity

[0165] To ensure the accuracy of the experiment, a blank control group (injected with the same dose of water as the experimental group), a negative control group (injected with 100 ng / μL of EGFP mRNA, i.e., adEGFP), and an experimental group (overexpression group, injected with 100 ng / μL of adHSL, overexpressed HSL mRNA) were set up in this step to test the cleavage rate, blastocyst rate, and blastocyst cell number. EGFP mRNA sequence: SEQ ID NO. 10

[0166] ;

[0167] Blastocyst cell number detection: The injected embryos were cultured in PZM-3 embryo culture medium for 168 hours. The blastocysts were then stained in 10 μg / mL Hoechst 33342 at room temperature in the dark for 5 minutes, and then washed three times in 0.2% PVA-PBS, each time for 8-10 minutes. An anti-fluorescence quencher was dropped on the center of the slide, and the blastocyst was moved into the droplet. A coverslip was gently placed on the droplet, and the slide was sealed. The slides were observed and photographed using a fluorescence microscope, and the blastocyst cell number was counted using ImageJ software.

[0168] like Figure 5 As shown, there were no significant differences in cleavage rate (Control: 91.8±0.7%, adEGFP: 85.8±2.3%, adHSL: 88.4±1.3%) and blastocyst cell number (Control: 57.0±0.6%, adEGFP: 53.7±1.9%, adHSL: 52.0±1.7%, P<0.05) among the groups. However, compared with the blank control group and the adEGFP group, the adHSL group significantly increased the blastocyst rate (Control: 28.1±1.8%, adEGFP: 25.2±2.2% vs. adHSL: 35.5±0.4%, P<0.05). These results indicate that specific overexpression of HSL can improve the developmental capacity of early porcine embryos.

[0169] 6. Effects of specific overexpression of HSL on lipid droplets, fatty acids, and ATP in early porcine embryos

[0170] Lipid droplet, fatty acid and ATP detection: embryos were moved from PZM-3 medium to 4% paraformaldehyde and fixed for more than 30 minutes; 500 mL of MAN operating solution was taken into a 24-well plate, and the embryos were placed in the MAN operating solution and washed three times, 5 minutes each time; after the embryos were washed, they were placed in 10 μg / mL BODIPY-LD, 6 μM BODIPY-FA, and 7 μM Biotracker ATP, and stained at room temperature in the dark for 30 minutes; 500 μL of MAN operating solution was taken into a 24-well plate, and washed three times, 5 minutes each time; the embryos were placed in 50 ng / mL Hoechst33342, and stained at room temperature in the dark for 8 minutes; 500 μL of MAN operating solution was taken into a 24-well plate, and washed three times, 5 minutes each time; the anti-fluorescence quencher was dropped in the center of the slide, the embryos were moved to the droplet, the coverslip was gently placed on the droplet, the slide was sealed, and the fluorescence microscope was used to observe, and the images of the control group and the experimental group were acquired using the same operating procedures and exposure times. J analyzed lipid droplet content and morphology, fatty acids, and ATP content.

[0171] To verify the effect of specific overexpression of HSL on lipid metabolism in pig embryos, lipid droplets, fatty acids, and ATP were detected in pig embryos. The expression levels of HSL mRNA and protein in 1-cell (embryos before the first cleavage), 2-cell (embryos cultured for 24 hours), and 4-cell (embryos cultured for 48 hours) embryos in the adHSL group were significantly increased ( Figure 6 A, E, and F in the adHSL group, P < 0.05). Compared with the blank control group, the lipid droplet content in 1-cell, 2-cell, and 4-cell embryos in the adHSL group was significantly reduced, and the lipid droplet diameter was reduced ( Figure 6 B, G, H, P < 0.05, fatty acid release and ATP content increased significantly ( Figure 6 C, D, I, and J, P < 0.05). In summary, during early porcine embryonic development, promoting HSL activity effectively reduced lipid droplet accumulation, increased fatty acid release and ATP levels, and thus enhanced in vitro embryonic development.

[0172] The above results indicate that the method of the present invention for improving the in vitro development ability of pig early embryos based on overexpression of the hormone-sensitive lipase gene can improve the in vitro development ability of pig embryos, has important economic value for improving the reproductive capacity of sow groups, and provides a theoretical basis for the research and development of related technical products.

[0173] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be based on the definition of the claims.

Claims

1. The application of hormone-sensitive lipase HSL protein in improving the development ability of pig embryos in vitro, characterized in that: The amino acid sequence of the hormone-sensitive lipase HSL protein is shown in SEQ ID NO. 3; the hormone-sensitive lipase HSL protein is overexpressed in pig parthenogenetic embryos.

2. The application of hormone-sensitive lipase HSL gene in improving the development ability of pig embryos in vitro, characterized in that: The hormone-sensitive lipase HSL gene sequence is shown in SEQ ID NO. 4; the hormone-sensitive lipase HSL gene is overexpressed in pig parthenogenetic embryos.

3. The application of hormone-sensitive lipase HSL mRNA in improving the development ability of pig embryos in vitro, characterized in that: The hormone-sensitive lipase HSL mRNA sequence is shown as SEQ ID NO.

7.

4. The use of a recombinant vector containing the hormone-sensitive lipase HSL gene in improving the developmental capacity of pig embryos in vitro, characterized in that: The hormone-sensitive lipase HSL gene sequence is shown in SEQ ID NO.

4.

5. The use according to claim 4, characterized in that The backbone of the recombinant vector is pcDNA3.1_EGFP.

6. The use according to claim 4, characterized in that The recombinant vector also includes a Kozak sequence.

7. Use of recombinant microbial cells containing the hormone-sensitive lipase HSL gene in improving the in vitro embryonic development ability of pigs, characterized in that: The hormone-sensitive lipase HSL gene sequence is shown in SEQ ID NO.

4.

8. A method for improving pig early embryonic development in vitro, characterized in that: Hormone-sensitive lipase HSL is overexpressed in pig parthenogenetic embryos; the sequence of the hormone-sensitive lipase HSL is shown in SEQ ID NO.

3.

9. The method according to claim 8, characterized in that Overexpression of hormone-sensitive lipase HSL involves using a recombinant vector containing the hormone-sensitive lipase HSL gene as a template for reverse transcription to obtain a hormone-sensitive lipase HSL mRNA sequence.

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