Application of hormone-sensitive lipase HSL gene in improving in-vitro maturation rate of porcine oocytes

Abstract

The invention provides application of a hormone-sensitive lipase HSL gene in improving the in-vitro maturation rate of porcine oocytes, and belongs to the technical field of oocyte development. The invention aims to improve the in-vitro maturation rate of the porcine oocytes. The invention provides application of an overexpressed hormone-sensitive lipase HSL gene in improving in-vitro maturation rate, fatty acid content and ATP (adenosine triphosphate) content of porcine oocytes. The amino acid sequence of the hormone-sensitive lipase HSL is shown as SEQ ID NO.3. The invention further provides a preparation method of the overexpressed hormone-sensitive lipase HSL gene. The invention proves that the overexpression hormone-sensitive lipase HSL gene can provide technical support for improving the in-vitro maturation rate of pig oocytes and improving the reproductive capacity of pigs, provides a brand new way for the breeding work of improved pigs, and has good application value.

Description

Technical Field

[0001] The invention belongs to the technical field of oocyte development, and particularly relates to application of hormone sensitive lipase HSL gene in improving in vitro maturation rate of pig oocytes. Background Art

[0002] In vitro maturation (IVM) of oocytes is a key step in in vitro production (IVP) of embryos. Through IVP technology, we can fully tap the genetic potential of excellent pig breeds in my country, expand excellent breeds, promote genetic improvement, and support the preparation of disease model pigs and organ donor pigs. The quality and maturation rate of oocytes largely determine the fate of embryonic development. Improving the in vitro development potential of oocytes is conducive to the development of the IVP system, has an important impact on embryonic development, implantation, and fetal growth and development, and directly determines the output efficiency of IVP. Therefore, how to improve the efficiency and quality of IVM will still be an important task for a certain period of time in the future. Summary of the invention

[0003] The purpose of the invention is to improve the in vitro maturation rate of pig oocytes.

[0004] The present invention provides an application of a hormone-sensitive lipase HSL protein in improving the in vitro maturation rate, fatty acid content and ATP content of pig oocytes. The amino acid sequence of the hormone-sensitive lipase HSL protein is shown in SEQ ID NO.3.

[0005] The present invention provides an application of a hormone-sensitive lipase HSL gene in improving the in vitro maturation rate, fatty acid content and ATP content of pig oocytes. The sequence of the hormone-sensitive lipase HSL gene is shown in SEQ ID NO.4.

[0006] The present invention provides an application of overexpressed hormone-sensitive lipase HSL mRNA in improving the in vitro maturation rate, fatty acid content and ATP content of pig oocytes. The hormone-sensitive lipase HSL mRNA sequence is shown as SEQ ID NO.7.

[0007] The present invention provides an application of a recombinant vector containing an overexpressed hormone-sensitive lipase HSL gene in improving the in vitro maturation rate, fatty acid content and ATP content of pig oocytes. The hormone-sensitive lipase HSL gene sequence is shown in SEQ ID NO.4.

[0008] It is further defined that the backbone of the recombinant vector is pcDNA3.1_EGFP.

[0009] It is further defined that the recombinant vector further comprises a Kozak sequence.

[0010] The present invention provides an application of a recombinant microbial cell containing an overexpressed hormone-sensitive lipase HSL gene in improving the in vitro maturation rate, fatty acid content and ATP content of pig oocytes. The hormone-sensitive lipase HSL gene sequence is shown in SEQ ID NO.4.

[0011] The present invention provides a method for improving the in vitro maturation rate, fatty acid content or ATP content of pig oocytes, and overexpressing hormone-sensitive lipase HSL protein in pig oocytes; the sequence of the hormone-sensitive lipase HSL protein is shown in SEQ ID NO.3.

[0012] It is further defined that the recombinant vector containing the hormone sensitive lipase HSL gene is used as a template for reverse transcription to obtain the hormone sensitive lipase HSL mRNA sequence.

[0013] The present invention provides a pig oocyte obtained by the above method.

[0014] Beneficial effects: The present invention constructs an mRNA vector for overexpressing HSL gene for in vitro transcription, and the obtained mRNA is injected into pig oocytes, which can significantly improve the in vitro maturation rate of pig oocytes.

[0015] The mRNA injected into the porcine oocyte 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 in vitro maturation rate of porcine oocytes. The HSL overexpression vector constructed by the present invention with pcDNA3.1_EGFP as a skeleton contains a T7 promoter and is suitable for in vitro transcription of a large amount of mRNA. The mRNA transcribed from the target sequence obtained by the in vitro transcription reaction of the present invention, the target sequence includes the HSL gene, and then the mRNA is injected into the porcine oocyte by microinjection, so that the oocyte 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 .PCR results of HSL overexpression vector;

[0020] Figure 5 .Green fluorescence images of oocytes that specifically overexpress HSL, as well as oocyte 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 oocytes; B is the oocyte survival rate; CD are the oocyte GFP positive rate and fluorescence intensity, respectively; EF are the HSL mRNA and protein expression levels in oocytes, respectively;

[0021] Figure 6 Effects of specific overexpression of HSL on lipid droplets, fatty acids and ATP in porcine oocytes; AB is the expression level of HSL protein, CE is the content and size of oocyte lipid droplets, FG is the content of oocyte fatty acids, and HI is the content of oocyte ATP. 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 intended to limit the present invention.

[0023] The specific implementation of the present invention is described in detail below in conjunction with specific embodiments.

[0024] Example 1. Construction of recombinant vector

[0025] 1. Obtaining HSL gene with restriction sites (BamH I and EcoR I)

[0026] 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.

[0027] 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:

[0028] HSL-F: GGATCCGCCACCATGGACCTGCGCACAATGACA; (SEQ ID NO. 1);

[0029] HSL-R: GAATTCGACTGGCGGCGTGGTGGG; (SEQ ID NO. 2);

[0030] HSL amino acid sequence: (SEQ ID NO.3)

[0031] MDLRTMTQSLVTLAEDNMAFFSGQGPGETARRLSGVFAGIREQALGLEPALGRLLSVAHL

[0032] FDLDAETPANGYRSLVHTARCCLAHLLHKSRYVASNRRSIFFRTSHNLAELEAYLAALTQ

[0033] LRALAYYAQRLLAINRPGKLFFEGDEGVTADFLREYVTLHKGCFYGRCLGFQFTPAIRPFL

[0034] QTISIGLVSFGEHYKRNETGLSVTASSLFTSGRFAIDPELRGAEFERIIQNLDVHFWKAFWNI

[0035] TEIEVLSSLANMASATVRVSRLLSLPPKAFEMPLTADPKLTTVTISPPLAHTGPGPVLVRLISY

[0036] DLREGQDSEELSSLVRSEGPRGLELRPRPQQAPRSRSLVVHIHGGGFVAQTSKSHEPYLKS

[0037] WAQELGVPILSIDYSLAPEAPFPRALEECFYAYCWAVKHCGLLGSTGERICLAGDSAGGN

[0038] LCFTVSLRAAAYGVRVPDGIMAAYPATMLQSAASPSRLLSLMDPLLPLSVLSKCVSAYAG

[0039] GEMEDHSDSDQKALGMMGLVRRDTALLFRDLRLGASSWLNSFLELSGHKSRPNLVPTEE

[0040] PMRRSVSEAALAQPEGPLGTDSLKYLTLHDLSLSSETQDTPELSLSAETLGPTTPSAVNFLF

[0041] RPEDAPEEAEARDEISTKEEKVYSVRAAFPEGFHPRRSSQGAIQMPLYSAPIVKNPFMSPLL

[0042] APDSMLQTLPPVHIVACALDPMLDDSVMFARRLRSLGQPVTLHVVEDLPHGFLSLAALCRETRQAAALCVDRIRFILNPPGPATPAGPTTPPV;

[0043] HSL CDS sequence: (SEQ ID NO.4)

[0044] ATGGACCTGCGCACAATGACACAGTCACTGGTGACCCTGGCGGAGGACAACATGGCCT

[0045] TCTTCTCTGGCCAGGGCCCTGGGGAGACAGCACGGCGGCTGTCAGGTGTCTTTGCGGG

[0046] TATTCGGGAACAGGCACTGGGGCTGGAGCCGGCCCTGGGCCGCCTGCTGAGTGTGGCA

[0047] CACCTCTTCGACCTGGATGCAGAGACGCCAGCCAACGGGTACCGCAGCCTGGTACACA

[0048] CAGCCCGCTGCTGCCTGGCACACCTGCTGCACAAATCACGCTACGTGGCCTCCAACCG

[0049] CCGCAGCATCTTCTTCCGCACAAGCCACAACCTGGCCGAACTCGAGGCCTACCTAGCT

[0050] GCTCTCACCCAGCTCCGCGCTCTGGCCTACTATGCCCAGCGCCTGCTGGCCATCAACCG

[0051] GCCCGGGAAGCTCTTCTTCGAGGGTGACGAAGGCGTCACTGCTGACTTCCTGCGCGAG

[0052] TATGTCACTCTACACAAGGGCTGCTTCTACGGCCGCTGCCTGGGCTTCCAGTTCACGCC

[0053] CGCCATCCGGCCTTTCCTGCAGACCATCTCCATCGGGCTGGTGTCCTTCGGGGAGCATT

[0054] ACAAACGCAATGAAACAGGCCTCAGTGTGACAGCCAGCTCCCTCTTCACCAGTGGCCG

[0055] CTTCGCCATCGACCCCGAGCTGCGTGGGGCCGAGTTTGAGCGGATCATACAGAACCTG

[0056] GATGTGCACTTCTGGAAAGCTTTCTGGAATATCACCGAGATTGAGGTGCTATCGTCTCTA

[0057] GCAAACATGGCATCGGCCACCGTGAGGGTAAGCCGCCTGCTCAGCCTACCGCCCAAGG

[0058] CCTTTGAAATGCCACTGACTGCTGACCCCAAGCTCACGGTCACCATTTCACCCCCGCTG

[0059] GCCCACACAGGCCCCGGGCCCGTCCTCGTCAGGCTCATCTCCTATGACCTGCGTGAAG

[0060] GACAGGACAGTGAGGAGCTCAGCAGCCTGGTGAGGTCTGAGGGCCCCAGGGGCCTAG

[0061] AGCTGCGGCCCCGCCCCCAGCAGGCACCCCGCTCCAGGTCCCTGGTCGTGCACATCCA

[0062] TGGTGGCGGCTTCGTGGCCCAGACGTCCAAATCCCACGAGCCTTACCTCAAGAGCTGG

[0063] GCCCAGGAGCTGGGGGTCCCCATCCTCTCCATCGACTACTCCCTAGCGCCCGAGGCCCC

[0064] CTTCCCCCGTGCGCTGGAGGAGTGCTTCTACGCCTACTGCTGGGCTGTCAAGCACTGC

[0065] GGTCTCCTCGGTTCCACAGGTGAGCGGATATGCCTCGCAGGAGACAGCGCAGGCGGGA

[0066] ACCTCTGCTTCACTGTGTCCCTTCGGGCAGCAGCCTATGGAGTGCGGGTGCCAGATGGC

[0067] ATCATGGCAGCCTACCCGGCCACAATGCTGCAGTCTGCCGCCTCCCCCTCCCGCCTTCT

[0068] CAGCCTCATGGACCCCCTGCTGCCCCTCAGCGTGCTCTCCAAGTGTGTCAGTGCCTATG

[0069] CTGGCGGGGAGATGGAGGATCACTCTGACTCAGACCAGAAGGCGCTGGGCATGATGG

[0070] GACTGGTGCGGCGGGACACAGCCCTGCTCTTCCGAGACCTCCGCCTGGGCGCCTCCTC

[0071] GTGGCTCAACTCCTTCCTGGAGCTGAGTGGGCACAAGTCCCGACCCAACTTGGTGCCC

[0072] ACAGAAGAGCCAATGCGGCGCAGTGTGTCTGAAGCAGCACTGGCCCAGCCTGAGGGC

[0073] CCACTGGGAACGGACTCCCTCAAGTACCTGACACTGCATGACCTGAGCCTAAGCTCCG

[0074] AGACACAGGACACCCCGGAGCTGTCACTGTCAGCTGAGACGCTTGGCCCCACCACAC

[0075] CCTCGGCTGTCAACTTCTTATTTCGACCTGAGGATGCACCTGAAGAGGCTGAGGCCCG

[0076] AGACGAGATTAGCACCAAGGAGGAGAAAGTCTACAGTGTGAGGGCCGCCTTCCCTGA

[0077] GGGTTTCCACCCAAGGCGCTCCAGCCAAGGTGCAATACAGATGCCCCTCTACTCAGCT

[0078] CCCATCGTCAAGAATCCCTTCATGTCACCACTGCTGGCACCTGACAGCATGCTGCAGAC

[0079] CCTCCCACCTGTGCACATTGTGGCCTGCGCGCTGGACCCCATGCTGGACGACTCGGTCA

[0080] TGTTCGCTAGGCGGCTACGCAGCCTGGGCCAGCCCGTGACGCTGCACGTGGTGGAGGA

[0081] CCTGCCGCACGGCTTCCTGAGCCTGGCGGCGCTGTGCCGGGAGACCCGGCAGGCCGC

[0082] GGCGCTGTGCGTGGACCGCATTCGCTTTATCCTCAATCCGCCGGGCCCTGCCACACCGGCTGGGCCCACCACGCCGCCAGTCTGA。

[0083] 2. Construction of the HSL overexpression vector with pcDNA3.1_EGFP as the backbone

[0084] For the gene fragment with restriction enzyme 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.

[0085] 3. In vitro Transcription

[0086] 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 transcription product of the porcine HSL gene 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.

[0087] T7 promoter primer sequence:

[0088] T7-F:5'-TAATACGACTCACTATAGGG-3'; (SEQ ID NO.5);

[0089] T7-R: 5'-TAGAAGGCACAGTCGAGG-3'; (SEQ ID NO. 6).

[0090] HSL mRNA sequence: (SEQ ID NO.7)

[0091] UAAUACGACUCACUAUAGGGAGACCCAAGCUGGCUAGCGUUUAAACUUAAGCUUG

[0092] GUACCGAGCUCGGAUCCGCCACCAUGGACCUGCGCACAAUGACACAGUCACUGGUG

[0093] ACCCUGGCGGAGGACAACAUGGCCUUCUUCUCUGGCCAGGGCCCUGGGGAGACAGC

[0094] ACGGCGGCUGUCAGGUGUCUUUGCGGGUAUUCGGGAACAGGCACUGGGGCUGGAG

[0095] CCGGCCCUGGGCCGCCUGCUGAGUGUGGCACACCUCUUCGACCUGGAUGCAGAGAC

[0096] GCCAGCCAACGGGUACCGCAGCCUGGUACACACAGCCCGCUGCUGCCUGGCACACC

[0097] UGCUGCACAAAUCACGCUACGUGGCCUCCAACCGCCGCAGCAUCUUCUUCCGCACA

[0098] AGCCACAACCUGGCCGAACUCGAGGCCUACCUAGCUGCUCUCACCCAGCUCCGCGC

[0099] UCUGGCCUACUAUGCCCAGCGCCUGCUGGCCAUCAACCGGCCCGGGAAGCUCUUCU

[0100] UCGAGGGUGACGAAGGCGUCACUGCUGACUUCCUGCGCGAGUAUGUCACUCUACAC

[0101] AAGGGCUGCUUCUACGGCCGCUGCCUGGGCUUCCAGUUCACGCCCGCCAUCCGGCC

[0102] UUUCCUGCAGACCAUCUCCAUCGGGCUGGUGUCCUUCGGGGAGCAUUACAAACGCA

[0103] AUGAAACAGGCCUCAGUGUGACAGCCAGCUCCCUCUUCACCAGUGGCCGCUUCGCC

[0104] AUCGACCCCGAGCUGCGUGGGGCCGAGUUUGAGCGGAUCAUACAGAACCUGGAUGU

[0105] GCACUUCUGGAAAGCUUUCUGGAAUAUCACCGAGAUUGAGGUGCUAUCGUCUCUA

[0106] GCAAACAUGGCAUCGGCCACCGUGAGGGUAAGCCGCCUGCUCAGCCUACCGCCCAA

[0107] GGCCUUUGAAAUGCCACUGACUGCUGACCCCAAGCUCACGGUCACCAUUUCACCCC

[0108] CGCUGGCCCACACAGGCCCCGGGCCCGUCCUCGUCAGGCUCAUCUCCUAUGACCUGC

[0109] GUGAAGGACAGGACAGUGAGGAGCUCAGCAGCCUGGUGAGGUCUGAGGGCCCCAG

[0110] GGGCCUAGAGCUGCGGCCCCGCCCCCAGCAGGCACCCCGCUCCAGGUCCCUGGUCGU

[0111] GCACAUCCAUGGUGGCGGCUUCGUGGCCCAGACGUCCAAAUCCCACGAGCCUUACC

[0112] UCAAGAGCUGGGCCCAGGAGCUGGGGGUCCCCAUCCUCUCCAUCGACUACUCCCUA

[0113] GCGCCCGAGGCCCCCUUCCCCCGUGCGCUGGAGGAGUGCUUCUACGCCUACUGCUG

[0114] GGCUGUCAAGCACUGCGGUCUCCUCGGUUCCACAGGUGAGCGGAUAUGCCUCGCAG

[0115] GAGACAGCGCAGGCGGGAACCUCUGCUUCACUGUGUCCCUUCGGGCAGCAGCCUAU

[0116] GGAGUGCGGGUGCCAGAUGGCAUCAUGGCAGCCUACCCGGCCACAAUGCUGCAGUC

[0117] UGCCGCCUCCCCCUCCCGCCUUCUCAGCCUCAUGGACCCCCUGCUGCCCCUCAGCGU

[0118] GCUCUCCAAGUGUGUCAGUGCCUAUGCUGGCGGGGAGAUGGAGGAUCACUCUGACU

[0119] CAGACCAGAAGGCGCUGGGCAUGAUGGGACUGGUGCGGCGGGACACAGCCCUGCUC

[0120] UUCCGAGACCUCCGCCUGGGCGCCUCCUCGUGGCUCAACUCCUUCCUGGAGCUGAG

[0121] UGGGCACAAGUCCCGACCCAACUUGGUGCCCACAGAAGAGCCAAUGCGGCGCAGUG

[0122] UGUCUGAAGCAGCACUGGCCCAGCCUGAGGGCCCACUGGGAACGGACUCCCUCAAG

[0123] UACCUGACACUGCAUGACCUGAGCCUAAGCUCCGAGACACAGGACACCCCGGAGCU

[0124] GUCACUGUCAGCUGAGACGCUUGGCCCCACCACACCCUCGGCUGUCAACUUCUUAU

[0125] UUCGACCUGAGGAUGCACCUGAAGAGGCUGAGGCCCGAGACGAGAUUAGCACCAAG

[0126] GAGGAGAAAGUCUACAGUGUGAGGGCCGCCUUCCCUGAGGGUUUCCACCCAAGGCG

[0127] CUCCAGCCAAGGUGCAAUACAGAUGCCCCUCUACUCAGCUCCCAUCGUCAAGAAUC

[0128] CCUUCAUGUCACCACUGCUGGCACCUGACAGCAUGCUGCAGACCCUCCCACCUGUG

[0129] CACAUUGUGGCCUGCGCGCUGGACCCCAUGCUGGACGACUCGGUCAUGUUCGCUAG

[0130] GCGGCUACGCAGCCUGGGCCAGCCCGUGACGCUGCACGUGGUGGAGGACCUGCCGC

[0131] ACGGCUUCCUGAGCCUGGCGGCGCUGUGCCGGGAGACCCGGCAGGCCGCGGCGCUG

[0132] UGCGUGGACCGCAUUCGCUUUAUCCUCAAUCCGCCGGGCCCUGCCACACCGGCUGG

[0133] GCCCACCACGCCGCCAGUCGAAUUCUGCAGAUAUCCAGCACAGUGGCGGCCGCUCG

[0134] AGGCCACCGGUGGCGGCGGUUCGGGAGGUGGCGGUUCUGGCGGAGGUGGCUCAAA

[0135] GCUUGUGAGCAAGGGCGAGGAGCUGUUCACCGGGGUGGUGCCCAUCCUGGUCGAGC

[0136] UGGACGGCGACGUAAACGGCCACAAGUUCAGCGUGUCCGGCGAGGGCGAGGGCGAU

[0137] GCCACCUACGGCAAGCUGACCCUGAAGUUCAUCUGCACCACCGGCAAGCUGCCCGU

[0138] GCCUGGCCCACCCUCGUGACCACCCUGACCUACGGCGUGCAGUGCUUCAGCCGCU

[0139] ACCCGACCACAUGAAGCAGCACGACUUCUUCAAGUCCGCCAUGCCCGAAGGCUAC

[0140] GUCCAGGAGCGCACCAUCUUCUUCAAGGACGACGGCAACUACAAGACCCGCGCCGA

[0141] GGUGAAGUUCGAGGGCGACACCCUGGUGAACCGCAUCGAGCUGAAGGGCAUCGACU

[0142] UCAAGGAGGACGGCAACAUCCUGGGGCACAAGCUGGAGUACAACUACAACAGCCAC

[0143] AACGUCUAUAUCAUGGCCGACAAGCAGAAGAACGGCAUCAAGGUGAACUUCAAGA

[0144] UCCGCCACAACAUCGAGGACCGGCAGCGUGCAGCUCGCCGACCACUACCAGCAGAAC

[0145] ACCCCCAUCGGCGACGGCCCCGUGCUGCUGCCCGACAACCACUACCUGAGCACCCAG

[0146] UCCGCCCUGAGCAAAGACCCCAACGAGAAGCGCGAUCACAUGGUCCUGCUGGAGUU

[0147] CGUGACCGCCGCCGGGAUCACUCUCGGCAUGGACGAGCUGUACAAGUAAUCUAGAGGGCCCGUUUAAACCCGCUGAUCAGCCUCGACUGUGCCUUCUA.

[0148] Example 2. Obtaining transgenic oocytes

[0149] 1. Obtaining pig oocytes

[0150] Pig ovaries were obtained from a local slaughterhouse and transported back to the laboratory in 30°C saline. Cumulus oocyte complexes (COCs) with a diameter of 3-6 mm were extracted from follicles using a syringe equipped with an 18G needle. The obtained COCs were placed in 0.1% hyaluronidase to elute the cumulus cells.

[0151] 2. Transform the in vitro transcription product into porcine oocytes by microinjection

[0152] The eluted naked oocytes (DOs) were placed in an embryo manipulation solution MAN solution with cytochalasin B added, and the mRNA (concentration of 0-100 ng / μL) containing the target sequence of the HSL gene obtained by in vitro transcription in step 3 was injected into the cytoplasm of the GV stage pig oocytes by microinjection at a dose of 10 pL. After the injection, the pig oocytes injected with mRNA were transferred to the IVM culture medium for continuous culture for 42 hours.

[0153] 3. Detection of oocyte survival rate, EGFP positive rate, fluorescence intensity, HSL mRNA expression level, and HSL protein expression level

[0154] Different concentrations of HSL-EGFP mRNA (0 ng / μL, 50 ng / μL, 100 ng / μL in the control group) were injected into the cytoplasm of GV stage oocytes. After continuous culture in IVM culture medium for 42 hours, the survival rate of oocytes (number of surviving oocytes / total number of oocytes cultured × 100%), EGFP positive rate (number of oocytes with green fluorescence after injection / total number of oocytes injected × 100%), fluorescence intensity and mRNA expression level were calculated.

[0155] HSL-EGFP-mRNA sequence:

[0156] HSL-EGFP-F:5'-CCCTCGGCTGTCAACTTCTTATTT-3'; (SEQ ID NO.8)

[0157] HSL-EGFP-R:5'-GCCCTCACACTGTAGACTTTCTCCT-3'; (SEQ ID NO.9);

[0158] like Figure 5 As shown in A, after injection of 50 ng / μL and 100 ng / μL HSL mRNA, green fluorescent protein was expressed in oocytes, but not in the control group, indicating that the injection concentration was effective.

[0159] like Figure 5 As shown in B, there was no significant difference in the oocyte survival rate between the control group and the 50ng / μL group, but the survival rate of the 100ng / μL group was significantly decreased compared with the control group and the 50ng / μL group (P<0.05);

[0160] like Figure 5 As shown in CD, although there was no significant difference in EGFP fluorescence intensity between the 50ng / μL and 100ng / μL groups ( Figure 5D), but the EGFP positive rate of the 50ng / μL group was significantly higher than that of the 100ng / μL group ( Figure 5 C);

[0161] like Figure 5 As shown in E, the HSL mRNA expression level in the 50 ng / μL group was 511 times higher than that in the control group, and the HSL-EGFP protein expression level was significantly increased ( Figure 5 F).

[0162] The above results prove that, through this embodiment, a porcine oocyte HSL overexpression system has been successfully established, and a subsequent verification experiment was carried out using an injection concentration of 50 ng / μL.

[0163] 4. Detection of the in vitro maturation rate of oocytes and subsequent embryonic development by specific overexpression of HSL

[0164] To ensure the accuracy of the experiment, a blank control group (injected with water), a negative control group (injected with EGFP mRNA at a concentration of 50 ng / μL, injected with adEGFP), and an experimental group (overexpression group, injected with adHSL at a concentration of 50 ng / μL, injected with overexpressed HSL mRNA) were set up in this step. The in vitro maturation rate of porcine oocytes was evaluated by observing the discharge of the first polar body (the number of oocytes discharged with the first polar body / the total number of oocytes cultured × 100%). As shown in Table 1, compared with the blank control group and adEGFP, the in vitro maturation rate of porcine oocytes in the adHSL group was significantly improved (blank control group: 74.0±0.7%, adEGFP: 73.3±0.7% vs. adHSL: 82.5±0.4%, P<0.05).

[0165] SEQ ID NO.10:

[0166] UAAUACGACUCACUAUAGGAUGAGCAAGGGCGAGGAGCUGUUCACCGGGGUGGUGCCCAUCCUGGUCGAGCUGGACGGCGACGUAAACGGCCACAAGUUCAGCGUGUCCGGCGAGGGCGAGGGCGAUGCCACCUACGGCAAGCUGACCCUGAAGUUCAUCUGCACCACCGGCAAGCUGCCCGUGCCCUGGCCCACCCUCGUGACCACCCUGACCUACGGCGUGCAGUGCUUCAGCCGCUACCCCGACCACAUGAAGCAGCACGACUUCUUCAAGUCCGCCAUGCCCGAAGGCUACGTCCAGGAGCGCACCAUCUUCUUCAAGGACGACGGCAACUACAAGACCCGCGCCGAGGUGAAGUUCGAGGGCGACACCCUGGUGAACCGCAUCGAGCUGAAGGGCAUCGACUUCAAGGAGGACGGCAACAUCCUGGGGCACAAGCUGGAGUACAACUACAACAGCCACAACGUCUAUAUCAUGGCCGACAAGCAGAAGAACGGCAUCAAGGUGAACUUCAAGAUCCGCCACAACAUCGAGGACGGCAGCGUGCAGCUCGCCGACCACUACCAGCAGAACACCCCCAUCGGCGACGGCCCCGUGCUGCUGCCCGACAACCACUACCUGAGCACCCAGUCCGCCCUGAGCAAAGACCCCAACGAGAAGCGCGAUCACAUGGUCCUGCUGGAGUUCGUGACCGCCGCCGGGAUCACUCUCGGCAUGGACGAGCUGUACAAGUAAUCUAGAGGGCCCGUUUAAACCCGCUGAUCAGCCUCGACUGUGCCUUCUA;

[0167] Table 1. Effects of specific overexpression of HSL on in vitro maturation of porcine oocytes

[0168]

[0169] Subsequently, in order to further clarify the effect of HSL on the maturation quality of porcine oocytes, a parthenogenetic activation experiment was carried out, and the cleavage rate, blastocyst rate, blastocyst cell number and blastocyst diameter of parthenogenetic embryos were counted at 48h and 168h, respectively. As shown in Table 2, there were no significant differences in cleavage rate (blank control group: 98.6±0.8%, adEGFP: 91.9±4.4%, adHSL: 95.0±2.8%), blastocyst cell number (blank control group: 46.4±3.9, adEGFP: 46.2±4.6, adHSL: 43.1±2.9) and blastocyst diameter (blank control group: 224.8±6.1μm, adEGFP: 224.9±7.1μm, adHSL: 215.8±5.0μm) among the groups; however, compared with the blank control group and adEGFP group, the adHSL group significantly increased the blastocyst rate (blank control group: 44.9±0.6%, adEGFP: 44.8±1.0% vs. adHSL: 58.5±0.3%, P<0.05). In summary, specific overexpression of HSL can improve the in vitro maturation of porcine oocytes and their subsequent embryonic development ability.

[0170] Table 2. Effect of specific overexpression of HSL on the developmental capacity of pig parthenogenetic embryos

[0171]

[0172] 5. Effects of specific overexpression of HSL on lipid droplets, fatty acids and ATP in porcine oocytes

[0173] 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.

[0174] In order to verify the effect of specific overexpression of HSL on the lipid metabolism level of porcine oocytes, HSL, lipid droplets, fatty acids and ATP in porcine oocytes were detected. Compared with the blank control group and adEGFP group, the expression level of HSL protein in the adHSL group was significantly increased ( Figure 6 AB), lipid droplet content and lipid droplet size were significantly reduced ( Figure 6 CE), fatty acid release increased significantly ( Figure 6 FG), ATP content increased significantly ( Figure 6 HI).

[0175] The above results indicate that the method for improving the in vitro maturation rate of pig oocytes by overexpressing the hormone-sensitive lipase gene (HSL) in pig oocytes described in the present invention can improve the in vitro maturation rate of pig oocytes, 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.

[0176] 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 maturation rate, fatty acid content and ATP content of porcine oocytes, 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 maturation rate, fatty acid content and ATP content of porcine oocytes, characterized in that: The hormone-sensitive lipase HSL gene sequence is shown in SEQ ID NO.

4.

3. The use of hormone-sensitive lipase HSL mRNA in improving the in vitro maturation rate, fatty acid content and ATP content of porcine oocytes, characterized in that: The hormone-sensitive lipase HSL mRNA sequence is shown as SEQ ID NO.

7.

4. Use of a recombinant vector containing the hormone-sensitive lipase HSL gene in improving the in vitro maturation rate, fatty acid content, and ATP content of porcine oocytes, 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 the hormone-sensitive lipase HSL gene in improving the in vitro maturation rate, fatty acid content, and ATP content of porcine oocytes, characterized in that: The hormone-sensitive lipase HSL gene sequence is shown in SEQ ID NO.

4.

8. A method for increasing the in vitro maturation rate, fatty acid content or ATP content of porcine oocytes, characterized in that: Hormone-sensitive lipase HSL protein is overexpressed in porcine oocytes; the sequence of the hormone-sensitive lipase HSL protein is shown in SEQ ID NO.

3.

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

10. Pig oocyte obtained by the method according to claim 8 or claim 9.

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