Application of hormone-sensitive lipase HSL gene in improving in-vitro embryonic developmental capacity of pigs

Abstract

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

Description

Technical Field

[0001] The invention belongs to the technical field of embryo development, and in particular relates to the application of hormone-sensitive lipase HSL gene in improving the in vitro embryo development ability of pigs. Background Art

[0002] In vitro production (IVP) mainly includes: in vitro maturation (IVM), in vitro fertilization (IVF) and in vitro embryo culture (IVC). Through IVP technology, we can fully tap the genetic potential of my country's excellent pig breeds, expand excellent breeds, promote genetic improvement, and support the preparation of disease model pigs and organ donor pigs. However, the development capacity of mammalian embryos in vitro is much 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 modifications. Although the developmental capacity of in vitro embryos can be improved by effectively removing excess ROS in cells and regulating histone methylation, histone acetylation, DNA methylation, etc., pig embryos have a high lipid content and are species-specific, and there is an urgent need to improve the developmental capacity of pig in vitro embryos. Summary of the invention

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

[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 in vitro embryonic development ability of pigs. 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 hormone-sensitive lipase HSL gene sequence is shown in SEQ ID NO.4.

[0012] The invention provides a method for improving the early embryonic development of pigs in vitro, which involves overexpressing 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 for 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 embryo development ability. The mRNA injected into the pig parthenogenetic embryos of the present invention can fuse and express the EGFP gene and the HSL gene, 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 embryo 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, so that the pig in vitro embryo can overexpress the HSL protein. BRIEF DESCRIPTION OF THE DRAWINGS

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

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

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

[0019] Figure 4.Green fluorescence images of embryos specifically overexpressing HSL (injected with 50, 100 ng / μL HSL mRNA), as well as embryo survival rate, EGFP positive rate, EGFP fluorescence intensity, HSL mRNA expression level, and HSL protein expression level; A is the expression of green fluorescent protein in embryos; B is the embryo survival rate; CD are the embryo EGFP positive rate and fluorescence intensity, respectively; EF are the HSL mRNA and protein expression levels in 2-cell embryos, respectively;

[0020] Figure 5 .The effect of specific overexpression of HSL on the developmental ability of pig parthenogenetic embryos (injection of 100ng / μL HSL mRNA). A is the cleavage rate, B is the blastocyst rate, and C is the number of blastocyst cells;

[0021] Figure 6 .The effect of specific overexpression of HSL (injection of 100 ng / μL HSL mRNA) on lipid droplets, fatty acids and ATP in early pig embryos; AD is the staining of HSL, lipid droplets, fatty acids and ATP in early pig embryos, E is the expression level of HSL mRNA, F is the expression level of HSL protein, G is the analysis of lipid droplet fluorescence intensity, H is the analysis of lipid droplet size, I is the analysis of fatty acid fluorescence intensity, and J is the analysis of ATP fluorescence intensity. DETAILED DESCRIPTION

[0022] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with 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 used to limit the present invention.

[0023] Example 1. Construction of recombinant vector

[0024] 1. Obtaining HSL gene with restriction 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-treated with primers containing restriction sites and KOZAK sequences (GCCACCAUGG) (reaction conditions: 50 μL system, containing KOD One TM PCR Master Mix 25 μL, 10 μM HSL-F and HSL-R 1.5 μL each, cDNA 2 μL, deionized water 20 μL; reaction conditions: denaturation 98 ° C 10 s, 68 ° C 30 s, a total of 45 cycles), the obtained 2310 bp fragment was -blunt Simple Cloning Kit (Trans, CB111) for T vector ligation and 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 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 sequence: (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. Construction of the HSL overexpression vector with pcDNA3.1_EGFP as the backbone

[0083] For gene fragments with restriction sites ( -blunt-HSL) and vector plasmid (pcDNA3.1_EGFP, Figure 3 The target gene and plasmid were subjected to agarose electrophoresis and gel recovery, and the concentration of the recovered gel was determined. The target gene and plasmid were connected by T4 ligase, and the product was transformed into competent Escherichia coli DH5α, coated on a culture medium plate containing ampicillin for screening, and a single colony was selected for culture, and PCR and sequencing were performed to identify the HSL overexpression vector (pcDNA3.1_HSL_EGFP). Figure 2 This is the HSL overexpression vector information with 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, 10 μM HSL-F and HSL-R 1.5 μL each, cDNA 2 μL, deionized water 20 μL; reaction conditions: denaturation at 98°C for 10 s, 68°C for 40 s, for a total of 45 cycles), a 3232 bp HSL_EGFP fragment containing a T7 promoter sequence was obtained by agarose gel electrophoresis and gel recovery, and a large amount of mRNA containing the porcine HSL gene transcription product was obtained by capping transcription and Poly (A) tailing reaction using NEB's mRNA in vitro transcription kit E2060, and then the product was purified using OMEGA's RNA purification kit MicroElute RNA Clean Up Kit, and finally the mRNA was diluted to 0-100 ng / μL using RNase-free triple distilled water for subsequent microinjection operations.

[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] GCCUGGCCCACCCUCGUGACCACCCUGACCUACGGCGUGCAGUGCUUCAGCCGCU

[0138] ACCCCGACCACAUGAAGCAGCACGACUUCUUCAAGUCCGCCAUGCCCGAAGGCUAC

[0139] GUCCAGGAGCGCACCAUCUUCUUCAAGGACGACGGCAACUACAAGACCCGCGCCGA

[0140] GGUGAAGUUCGAGGGCGACACCCUGGUGAACCGCAUCGAGCUGAAGGGCAUCGACU

[0141] UCAAGGAGGACGGCAACAUCCUGGGGCACAAGCUGGAGUACAACUACAACAGCCAC

[0142] AACGUCUAUAUCAUGGCCGACAAGCAGAAGAACGGCAUCAAGGUGAACUUCAAGA

[0143] UCCGCCACAACAUCGAGGACGGCAGCGUGCAGCUCGCCGACCACUACCAGCAGAAC

[0144] ACCCCCAUCGGCGACGGCCCCGUGCUGCUGCCCGACAACCACUACCUGAGCACCCAG

[0145] UCCGCCCUGAGCAAAGACCCCAACGAGAAGCGCGAUCACAUGGUCCUGCUGGAGUU

[0146] CGUGACCGCCGCCGGGAUCACUCUCGGCAUGGACGAGCUGUACAAGUAAUCUAGAGGGCCCGUUUAAACCCGCUGAUCAGCCUCGACUGUGCCUUCUA。

[0147] Example 2. Obtaining transgenic embryos

[0148] 1. Acquisition of porcine oocytes

[0149] Pig ovaries were obtained from a local slaughterhouse and transported back to the laboratory in 30°C saline. A syringe equipped with an 18G needle was used to extract cumulus-oocyte complexes (COCs) from follicles with a diameter of 3-6 mm. The obtained COCs were cultured in MAT for 40 h at 38.5°C and 5% CO2 saturated humidity. The COCs cultured for 40 h were placed in 0.1% hyaluronidase to elute the cumulus cells, and the eluted naked oocytes (DOs) were placed in the embryo manipulation fluid MAN working fluid, and mature oocytes with the first polar body and uniform cytoplasm were selected under a stereomicroscope.

[0150] 2. Parthenogenetic Embryo Culture

[0151] Preheat the FM fusion solution of the parthenogenetic embryo in a 37°C incubator in advance, fix the FM rinsed fusion tank on a 37°C warm platform, connect the electrode to the fusion tank to ensure stable connection, and add 600μL FM fusion solution to the tank. Transfer the mature oocytes to a small dish containing FM fusion solution, allow the oocytes to fully settle to the bottom of the liquid, move the mature oocytes to the fusion tank, and place them in a row in parallel, without floating liquid or stacking together, use direct current shock twice, 1.2Kv / cm, pulse time 30μs, after the completion of electrical activation, move the mature oocytes to the PZM3 working solution wash drop that has been balanced in advance, and after fully washing 4 times, place one hole for every 50 oocytes in a PZM3 embryo culture plate in a 38.5°C, 5% CO2 saturated humidity environment.

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

[0153] Use MAN working solution to make microinjection operation drops, cover the surface with mineral oil, and inject 10pL of HSL mRNA with an effective concentration of 50-100ng / μL into the cytoplasm of embryos cultured for 4-6h after parthenogenetic activation. After the injection, move the embryos to the pre-balanced PZM3 wash drops for continued culture, and observe the embryonic development. The culture conditions are 38.5℃, 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 0ng / μL, 50ng / μL, 100ng / μL) were injected into the cytoplasm of parthenogenetic embryos that had been cultured for 4-6h. In addition, a blank control group and a negative control group (water injection group, C-Water) were set up, and 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 A, after injection of 50ng / μL and 100ng / 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 B, there was no significant difference in embryo survival rate among the negative control group, 50ng / μL injection group and 100ng / μL injection group;

[0158] like Figure 4 As shown in CD, the EGFP positive rate and fluorescence intensity of the 100ng / μL group were significantly higher than those of the 50ng / 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 the protein detection results showed that the 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 prove that, through the method of this example, a porcine parthenogenetic embryo HSL overexpression system has been successfully established, and a subsequent verification experiment was carried out 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 EGFP mRNA at a concentration of 100 ng / μL, i.e., adEGFP), and an experimental group (overexpression group, injected with adHSL at a concentration of 100 ng / μL, injected with overexpressed HSL mRNA) were set up in this step to detect 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, and then the blastocysts were placed in 10μg / mL Hoechst 33342 for staining in the dark for 5 minutes at room temperature, and then washed three times in 0.2% PVA-PBS, each time for 8-10 minutes; the anti-fluorescence quencher was dropped on the center of the slide, the blastocyst was 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 photograph, and the blastocyst cell number was counted using ImageJ software.

[0168] like Figure 5 As shown, there was no significant difference in the cleavage rate (Control: 91.8±0.7%, adEGFP: 85.8±2.3%, adHSL: 88.4±1.3%) and the number of blastocyst cells (Control: 57.0±0.6%, adEGFP: 53.7±1.9%, adHSL: 52.0±1.7%, P<0.05) among the groups; 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). The above results show that specific overexpression of HSL can improve the developmental ability of early pig 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 placed in a 24-well plate, and the embryos were placed in the MAN operating solution and washed three times, each time for 5 minutes; 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 placed in a 24-well plate, and washed three times, each time for 5 minutes; 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 placed in a 24-well plate, and washed three times, each time for 5 minutes; 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 obtained using the same operating procedures and exposure time, and the images were obtained using Image J analyzed lipid droplet content and morphology, fatty acids, and ATP content.

[0171] In order 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 figure, P<0.05). Compared with the blank control group, the lipid droplet content of 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 in the figure, P < 0.05). In summary, during the early embryonic development of pigs, promoting HSL activity can effectively reduce lipid droplet accumulation, increase fatty acid release and ATP levels, and thus enhance the ability of in vitro embryonic development.

[0172] The above results indicate that the method of the present invention for improving the in vitro early embryonic development ability of pigs 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 as above in the preferred embodiment, 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 in vitro embryonic development ability of pigs, characterized in that: The amino acid sequence of the hormone-sensitive lipase HSL protein is shown in SEQ ID NO.

3.

2. The application of 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.

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

7.

4. The use of a recombinant vector 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.

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 a recombinant microbial cell containing a 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 early embryonic development in vitro in pigs, 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 is to use a recombinant vector containing the hormone-sensitive lipase HSL gene as a template for reverse transcription to obtain the hormone-sensitive lipase HSL mRNA sequence.

10. A pig embryo obtained by the method according to claim 8 or claim 9.

Images