CN117511884A - Rtl1在抑制牦牛成肌细胞分化中的应用 - Google Patents
Rtl1在抑制牦牛成肌细胞分化中的应用 Download PDFInfo
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Abstract
本发明涉及基因工程技术领域,特别是涉及RTL1在抑制牦牛成肌细胞分化中的应用。本发明利用免疫荧光实验检测RTL1‑Si和RTL‑OE对肌管形成的影响,结果显示,RTL‑OE会显著抑制肌管的形成,而RTL‑Si会显著促进肌管的形成。上述实验结果表明RTL1有抑制成肌细胞分化的作用。
Description
技术领域
本发明涉及基因工程技术领域,特别是涉及RTL1在抑制牦牛成肌细胞分化中的应用。
背景技术
由于牦牛肉具有鲜美的口感、丰富的营养成分、多种氨基酸的含量以及高蛋白低脂肪的特点,因此成为了备受大众喜爱的绿色食品。然而,牦牛生长和增重存在波浪式消长的问题,这主要归因于其特殊的生存环境和长达7个月的枯草期,这些问题对其经济价值和使用价值产生了影响。家畜的生长速度和产肉性能与骨骼肌的生长发育密切相关。牦牛的生产性状主要表现在骨骼肌的生长发育过程中,而骨骼肌的生长快慢和数量多少直接影响了牦牛肉的产量。骨骼肌的发育是一个复杂的生理过程,其中包括纤维数量的增加(在出生前)和纤维体积的增大(在出生后)。在出生后的阶段,动物通过肌纤维体积的增大来实现骨骼肌的生长,其中主要依赖于肌成肌细胞的增殖、分化和蛋白质的周转等途径。肌成肌细胞是一种特殊的成肌干细胞,参与了两个生物过程:一是再生功能,当成熟的肌纤维细胞受到损伤时,肌成肌细胞会激活,然后增殖、迁移和分化形成新的肌细胞,最终与其他细胞融合形成肌肉纤维;另一个功能是维持肌肉纤维细胞核的数量,从而保证肌纤维细胞核和细胞质的比例相对平衡。
在发育过程之中,Dlk1-Dio3印记基因簇发挥了十分必要的功能,当甲基化出现异常的时候,可能会对整个发育过程产生影响,比如:发育停滞、过早成熟等现象。于此时,此区域的印记状态发生变化之后,干细胞的多样化也会受到相应影响,现今,在这一区域研究较多的主要是Dlk1、Rtl1、Gtl2、Dio3等印记基因。
RTL1基因也称为PEG1I(paternally expressed gene11),是一种类似Sushi的逆转座子,失去了自主编码与Gag和Pol蛋白同源的蛋白的能力(Youngson etal.,2005)。该基因位于DLK1和III型碘甲状腺激素脱碘酶基因(dlk1-dio3)的印迹基因位点,研究发现该基因在胚胎和胎盘组织中高表达,并在母体胎盘和胎儿营养输送中发挥重要作用。小鼠中RTL1的缺失或过表达导致胎儿生长发育或新生儿发育延迟。还发现RTL1与绵羊的肌肉肥大有关。但是现有技术还未有关于RTL1对于成肌细胞具有何种影响的报道。
发明内容
为了解决上述问题,本发明提供了RTL1在抑制牦牛成肌细胞分化中的应用,证明了RTL1基因具有抑制牦牛成肌细胞分化的作用。
为了实现上述目的,本发明提供如下技术方案:
本发明提供了RTL1基因在抑制牦牛成肌细胞分化中的应用。
本发明还提供了RTL1基因在抑制牦牛肌管形成中的应用。
本发明的有益效果:
本发明使用RTL1-Si和RTL-OE超表达载体转染成肌细胞来进一步的进行实验,于转染后24h收取细胞,进行检测。结果显示,RTL-OE会显著促进RTL1的表达,同时会显著降低MYOG、MYF5 mRNA和蛋白水平的表达,对MYOD,MYF5的蛋白和mRNA表达水平产生较显著的影响,RTL1-Si显著降低了RTL1的表达,同时会显著降低分化标记基因MYOG、MYF5 mRNA和蛋白水平的表达。本发明利用免疫荧光实验检测RTL1-Si和RTL-OE对肌管形成的影响,结果显示,RTL-OE会显著抑制肌管的形成,而RTL-Si会显著促进肌管的形成。上述实验结果表明RTL1有抑制成肌细胞分化的作用。
附图说明
为了更清楚地说明本发明实施例或现有技术中的技术方案,下面将对实施例中所需要使用的附图作简单地介绍。
图1为双荧光载体质粒pmirGLO示意图;
图2为miR-22-3p-3p与MEG8/RTL1结合位点;
图3为双荧光素酶实验验证MEG8/miR-22-3p,RTL1/miR-22-3p的靶向调控关系;
图4为miR-22-3p在成肌细胞不同分化时期表达水平;
图5为miR-22-3p mimic促进成肌细胞分化;
图6为miR-22-3p inhibitor抑制成肌细胞分化;
图7为RTL1-OE抑制成肌细胞分化;
图8为RTL1-Si促进成肌细胞分化;
图9为MEG8-RTL1-miR-22-3p之间的调控关系。
具体实施方式
本发明提供了RTL1基因在抑制牦牛成肌细胞分化中的应用。在本发明中,所述RTL1基因在GenBank中的登录号为NM_001195020.1。
本发明还提供了RTL1基因在抑制牦牛肌管形成中的应用。在本发明中,所述RTL1基因在GenBank中的登录号为NM_001195020.1。
为了进一步说明本发明,下面结合实施例对本发明进行详细地描述,但不能将它们理解为对本发明保护范围的限定。
实施例1
1实验方法
1.1腺病毒介导的RTL1过表达载体的构建
本实施例采用AdEasy腺病毒表达载体系统,以GenBank公布的牛RTL1基因mRNA序列(NM_001195020.1)为模板,委托通用生物(安徽)股份有限公司合成RTL1基因CDS(cording sequence)全长序列。将CDS序列经BglII/HindIII限制性内切酶酶切并连接到pShuttle-CMV(购自丰晖生物,货号BR009)穿梭载体上,并在BJ5183感受态细胞内将pShuttle-CMV-RTL1与pAdEasy-1骨架载体同源重组。重组质粒经PacI限制性内切酶酶切线性化后转染至Hek-293A细胞中进行腺病毒的包装和扩繁。本实施例将RTL1重组过表达腺病毒命名为RTL1-OE。本实施例所用的阴性对照(negative control,NC)病毒常规购买即可,在本实施例中将其命名为OE-NC。腺病毒介导的RTL1过表达载体的表达效率分别用RT-PCR方法和Westernblot方法鉴定。
1.2腺病毒介导的RNA干扰载体的构建
为构建腺病毒介导的RNA干扰载体,首先,本实施例以牛RTL1基因mRNA序列为模板设计RTL1基因的干扰靶点,并委托通用生物(安徽)股份有限公司合成干扰序列(SEQ IDNo.1)ACTAAAGATATTTGCATGTCGCTATGTGTTCTGGGAAATCACCATAAACGTG AAATGTCTTTGGATTTGGGAATCTTATAAGTTCTGTATGAGACCACTCAGAC GATGATGGAGCGTAAGAttcaagagaTCTTACGCTCCATCATCGTCTttttt,同时将shRNA通过BglII/SalI酶切连接到pShuttle-CMV载体上。在BJ5183感受态细胞内将pShuttle-CMV-shRTL1与pAdEasy-1骨架载体同源重组。提取重组质粒并经PacI限制性内切酶酶切线性化后转染至Hek-293A细胞中进行腺病毒的包装和扩繁。本实施例分别将重组shRNA干扰腺病毒和对照腺病毒命名为sh-RTL1和sh-NC。腺病毒介导的RNA干扰载体的干扰效率分别用RT-PCR方法和Westernblot方法鉴定。
1.3miR-22-3p慢病毒载体构建
为构建miRNA过表达/干扰慢病毒载体,首先委托通用生物(安徽)股份有限公司合成干扰或过表达序列并将其连接至慢病毒载体pLVX-shRNA1上。连接完成后,将重组载体转化至大肠杆菌进行扩增和提取,并通过测序确认插入序列已经成功连接到慢病毒载体上。将验证正确的载体转染至HEK293T细胞系。辅助载体可协助包装和扩增慢病毒。完成包装后,收集慢病毒液并用于进一步的miRNA干扰或过表达实验。实验过程中,确保使用适当的实验条件和控制组来验证miRNA干扰或过表达的效果,并进行进一步的功能研究。
1.4miR-22-3p/MEG8结合位点、RTL1/miR-22-3p结合位双荧光素酶报告基因载体构建
使用RNAhybrid、Starbase预测RTL1/miR-22-3p,miR-22-3p/MEG8结合位点,根据NCBI数据库中结合位点序列进行PCR扩增,同时扩增结合位点突变片段,扩增片段回收后连接于pmirGLO载体,连接成功后使用去内毒素质粒提取试剂盒进行质粒提取。
双荧光素酶报告基因载体的核苷酸序列如SRQ ID No.2所示:
CATGCAAGCTGATCCGGCTGCTAACAAAGCCCGAAAGGAAGCTGAGTTGGCTGCTGCCACCGCTGAGCAATAACTAGCATAACCCCTTGGGGCGGCCGCTTCGAGCAGACATGATAAGATACATTGATGAGTTTGGACAAACCACAACTAGAATGCAGTGAAAAAAATGCTTTATTTGTGAAATTTGTGATGCTATTGCTTTATTTGTAACCATTATAAGCTGCAATAAACAAGTTAACAACAACAATTGCATTCATTTTATGTTTCAGGTTCAGGGGGAGATGTGGGAGGTTTTTTTAAGCAAGTAAAACCTCTACAAATGTGGTAAAATCGAATTTTAACAAAATATTAACGCTTACAATTTCCTGATGCGGTATTTTCTCCTTACGCATCTGTGCGGTATTTCACACCGCATACGCGGATCTGCGCAGCACCATGGCCTGAAATAACCTCTGAAAGAGGAACTTGGTTAGGTACCTTCTGAGGCGGAAAGAACCAGCTGTGGAATGTGTGTCAGTTAGGGTGTGGAAAGTCCCCAGGCTCCCCAGCAGGCAGAAGTATGCAAAGCATGCATCTCAATTAGTCAGCAACCAGGTGTGGAAAGTCCCCAGGCTCCCCAGCAGGCAGAAGTATGCAAAGCATGCATCTCAATTAGTCAGCAACCATAGTCCCGCCCCTAACTCCGCCCATCCCGCCCCTAACTCCGCCCAGTTCCGCCCATTCTCCGCCCCATGGCTGACTAATTTTTTTTATTTATGCAGAGGCCGAGGCCGCCTCGGCCTCTGAGCTATTCCAGAAGTAGTGAGGAGGCT
TTTTTGGAGGCCTAGGCTTTTGCAAAAAGCTTGATTCTTCTGACACAACAG
TCTCGAACCAAAGGCTGGAGCCACCATGGCTTCCAAGGTGTACGACCCCG
AGCAACGCAAACGCATGATCACTGGGCCTCAGTGGTGGGCTCGCTGCAAG
CAAATGAACGTGCTGGACTCCTTCATCAACTACTATGATTCCGAGAAGCAC
GCCGAGAACGCCGTGATTTTTCTGCATGGTAACGCTGCCTCCAGCTACCTG
TGGAGGCACGTCGTGCCTCACATCGAGCCCGTGGCTAGATGCATCATCCCT
GATCTGATCGGAATGGGTAAGTCCGGCAAGAGCGGGAATGGCTCATATCGC
CTCCTGGATCACTACAAGTACCTCACCGCTTGGTTCGAGCTGCTGAACCTT
CCAAAGAAAATCATCTTTGTGGGCCACGACTGGGGGGCTTGTCTGGCCTTT
CACTACTCCTACGAGCACCAAGACAAGATCAAGGCCATCGTCCATGCTGA
GAGTGTCGTGGACGTGATCGAGTCCTGGGACGAGTGGCCTGACATCGAGG
AGGATATCGCCCTGATCAAGAGCGAAGAGGGCGAGAAAATGGTGCTTGAG
AATAACTTCTTCGTCGAGACCATGCTCCCAAGCAAGATCATGCGGAAACTG
GAGCCTGAGGAGTTCGCTGCCTACCTGGAGCCATTCAAGGAGAAGGGCGA
GGTTAGACGGCCTACCCTCTCCTGGCCTCGCGAGATCCCTCTCGTTAAGGG
AGGCAAGCCCGACGTCGTCCAGATTGTCCGCAACTACAACGCCTACCTTC
GGGCCAGCGACGATCTGCCTAAGATGTTCATCGAGTCCGACCCTGGGTTCT
TTTCCAACGCTATTGTCGAGGGAGCTAAGAAGTTCCCTAACACCGAGTTCG
TGAAGGTGAAGGGCCTCCACTTCAGCCAGGAGGACGCTCCAGATGAAATG
GGTAAGTACATCAAGAGCTTCGTGGAGCGCGTGCTGAAGAACGAGCAGAC
CGGTGGTGGGAGCGGAGGTGGCGGATCAGGTGGCGGAGGCTCCGGAGGG
ATTGAACAAGATGGATTGCACGCAGGTTCTCCGGCCGCTTGGGTGGAGAG
GCTATTCGGCTATGACTGGGCACAACAGACAATCGGCTGCTCTGATGCCGC
CGTGTTCCGGCTGTCAGCGCAGGGGCGCCCGGTTCTTTTTGTCAAGACCG
ACCTGTCCGGTGCCCTGAATGAACTGCAGGACGAGGCAGCGCGGCTATCG
TGGCTGGCCACGACGGGCGTTCCTTGCGCAGCTGTGCTCGACGTTGTCAC
TGAAGCGGGAAGGGACTGGCTGCTATTGGGCGAAGTGCCGGGGCAGGATC
TCCTGTCATCTCACCTTGCTCCTGCCGAGAAAGTATCCATCATGGCTGATGC
AATGCGGCGGCTGCATACGCTTGATCCGGCTACCTGCCCATTCGACCACCA
AGCGAAACATCGCATCGAGCGAGCACGTACTCGGATGGAAGCCGGTCTTG
TCGATCAGGATGATCTGGACGAAGAGCATCAGGGGCTCGCGCCAGCCGAA
CTGTTCGCCAGGCTCAAGGCGCGCATGCCCGACGGCGAGGATCTCGTCGT
GACCCATGGCGATGCCTGCTTGCCGAATATCATGGTGGAAAATGGCCGCTT
TTCTGGATTCATCGACTGTGGCCGGCTGGGTGTGGCGGACCGCTATCAGGA
CATAGCGTTGGCTACCCGTGATATTGCTGAAGAGCTTGGCGGCGAATGGGC
TGACCGCTTCCTCGTGCTTTACGGTATCGCCGCTCCCGATTCGCAGCGCATC
GCCTTCTATCGCCTTCTTGACGAGTTCTTCTGAGCGGGACTCTGGGGTTCG
AAATGACCGACCAAGCGACGCCCAACCTGCCATCACGATGGCCGCAATAA
AATATCTTTATTTTCATTACATCTGTGTGTTGGTTTTTTGTGTGAATCGATAG
CGATAAGGATCCTCTTTGCGCTTGCGTTTTCCCTTGTCCAGATAGCCCAGTA
GCTGACATTCATCCGGGGTCAGCACCGTTTCTGCGGACTGGCTTTCTACGT
AATGGTTTCTTAGACGTCAGGTGGCACTTTTCGGGGAAATGTGCGCGGAA
CCCCTATTTGTTTATTTTTCTAAATACATTCAAATATGTATCCGCTCATGAGA
CAATAACCCTGATAAATGCTTCAATAATATTGAAAAAGGAAGAGTATGAGTA
TTCAACATTTCCGTGTCGCCCTTATTCCCTTTTTTGCGGCATTTTGCCTTCCT
GTTTTTGCTCACCCAGAAACGCTGGTGAAAGTAAAAGATGCTGAAGATCA
GTTGGGTGCACGAGTGGGTTACATCGAACTGGATCTCAACAGCGGTAAGA
TCCTTGAGAGTTTTCGCCCCGAAGAACGTTTTCCAATGATGAGCACTTTCA
AAGTTCTGCTATGTGGCGCGGTATTATCCCGTATTGACGCCGGGCAAGAGC
AACTCGGTCGCCGCATACACTATTCTCAGAATGACTTGGTTGAGTACTCAC
CAGTCACAGAAAAGCATCTTACGGATGGCATGACAGTAAGAGAATTATGC
AGTGCTGCCATAACCATGAGTGATAACACTGCGGCCAACTTACTTCTGACA
ACTATCGGAGGACCGAAGGAGCTAACCGCTTTTTTGCACAACATGGGGGA
TCATGTAACTCGCCTTGATCGTTGGGAACCGGAGCTGAATGAAGCCATACC
AAACGACGAGCGTGACACCACGATGCCTGTAGCAATGGCAACAACGTTGC
GCAAACTATTAACTGGCGAACTACTTACTCTAGCTTCCCGGCAACAATTAAT
AGACTGGATGGAGGCGGATAAAGTTGCAGGACCACTTCTGCGCTCGGCCC
TTCCGGCTGGCTGGTTTATTGCTGATAAATCTGGAGCCGGTGAGCGTGGGT
CTCGCGGTATCATTGCAGCACTGGGGCCAGATGGTAAGCCCTCCCGTATCG
TAGTTATCTACACGACGGGGAGTCAGGCAACTATGGATGAACGAAATAGAC
AGATCGCTGAGATAGGTGCCTCACTGATTAAGCATTGGTAATTCGAAATGA
CCGACCAAGCGACGCCCAACCGGTATCAGCTCACTCAAAGGCGGTAATAC
GGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAA
GGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTT
CCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTC
AGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCT
GGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATAC
CTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGC
TGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTG
CACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGT
CTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCAC
TGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTT
GAAGTGGTGGCCTAACTACGGCTACACTAGAAGGACAGTATTTGGTATCTG
CGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATC
CGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGC
AGATTACGCGCAGAAAAAAAGGATTTCAAGAAGATCCTTTGATCTTTTCTA
CGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTC
ATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTATAGTCCGGAAATA
CAGGAACGCACGCTGGATGGCCCTTCGCTGGGATGGTGAAACCATGAAAA
ATGGCAGCTTCAGTGGATTAAGTGGGGGTAATGTGGCCTGTACCCTCTGGT
TGCATAGGTATTCATACGGTTAAAATTTATCAGGCGCGATTGCGGCAGTTTT
TCGGGTGGTTTGTTGCCATTTTTACCTGTCTGCTGCCGTGATCGCGCTGAAC
GCGTTTTAGCGGTGCGTACAATTAAGGGATTATGGTAAATCCACTTACTGTC
TGCCCTCGTAGCCATCGAGATAAACCGCAGTACTCCGGCCACGATGCGTCC
GGCGTAGAGGATCGAGATCTACCGGGTAGGGGAGGCGCTTTTCCCAAGGC
AGTCTGGAGCATGCGCTTTAGCAGCCCCGCTGGGCACTTGGCGCTACACA
AGTGGCCTCTGGCCTCGCACACATTCCACATCCACCGGTAGGCGCCAACC
GGCTCCGTTCTTTGGTGGCCCCTTCGCGCCACCTTCTACTCCTCCCCTAGTC
AGGAAGTTCCCCCCCGCCCCGCAGCTCGCGTCGTGCAGGACGTGACAAAT
GGAAGTAGCACGTCTCACTAGTCTCGTGCAGATGGACAGCACCGCTGAGC
AATGGAAGCGGGTAGGCCTTTGGGGCAGCGGCCAATAGCAGCTTTGCTCC
TTCGCTTTCTGGGCTCAGAGGCTGGGAAGGGGTGGGTCCGGGGGCGGGCT
CAGGGGCGGGCTCAGGGGCGGGGCGGGCGCCCGAAGGTCCTCCGGAGGC
CCGGCATTCTGCACGCTTCAAAAGCGCACGTCTGCCGCGCTGTTCTCCTCT
TCCTCATCTCCGGGCCTTTCGACCTGCAGCCCAAGCTTGGCAATCCGGTAC
TGTTGGTAAAGCCACCATGGAAGATGCCAAAAACATTAAGAAGGGCCCAG
CGCCATTCTACCCACTCGAAGACGGGACCGCCGGCGAGCAGCTGCACAAA
GCCATGAAGCGCTACGCCCTGGTGCCCGGCACCATCGCCTTTACCGACGCA
CATATCGAGGTGGACATTACCTACGCCGAGTACTTCGAGATGAGCGTTCGG
CTGGCAGAAGCTATGAAGCGCTATGGGCTGAATACAAACCATCGGATCGTG
GTGTGCAGCGAGAATAGCTTGCAGTTCTTCATGCCCGTGTTGGGTGCCCTG
TTCATCGGTGTGGCTGTGGCCCCAGCTAACGACATCTACAACGAGCGCGA
GCTGCTGAACAGCATGGGCATCAGCCAGCCCACCGTCGTATTCGTGAGCA
AGAAAGGGCTGCAAAAGATCCTCAACGTGCAAAAGAAGCTACCGATCATA
CAAAAGATCATCATCATGGATAGCAAGACCGACTACCAGGGCTTCCAAAG
CATGTACACCTTCGTGACTTCCCATTTGCCACCCGGCTTCAACGAGTACGA
CTTCGTGCCCGAGAGCTTCGACCGGGACAAAACCATCGCCCTGATCATGA
ACAGTAGTGGCAGTACCGGATTGCCCAAGGGCGTAGCCCTACCGCACCGC
ACCGCTTGTGTCCGATTCAGTCATGCCCGCGACCCCATCTTCGGCAACCAG
ATCATCCCCGACACCGCTATCCTCAGCGTGGTGCCATTTCACCACGGCTTC
GGCATGTTCACCACGCTGGGCTACTTGATCTGCGGCTTTCGGGTCGTGCTC
ATGTACCGCTTCGAGGAGGAGCTATTCTTGCGCAGCTTGCAAGACTATAAG
ATTCAATCTGCCCTGCTGGTGCCCACACTATTTAGCTTCTTCGCTAAGAGCA
CTCTCATCGACAAGTACGACCTAAGCAACTTGCACGAGATCGCCAGCGGC
GGGGCGCCGCTCAGCAAGGAGGTAGGTGAGGCCGTGGCCAAACGCTTCC
ACCTACCAGGCATCCGCCAGGGCTACGGCCTGACAGAAACAACCAGCGCC
ATTCTGATCACCCCCGAAGGGGACGACAAGCCTGGCGCAGTAGGCAAGGT
GGTGCCCTTCTTCGAGGCTAAGGTGGTGGACTTGGACACCGGTAAGACAC
TGGGTGTGAACCAGCGCGGCGAGCTGTGCGTCCGTGGCCCCATGATCATG
AGCGGCTACGTTAACAACCCCGAGGCTACAAACGCTCTCATCGACAAGGA
CGGCTGGCTGCACAGCGGCGACATCGCCTACTGGGACGAGGACGAGCACT
TCTTCATCGTGGACCGGCTGAAGAGCCTGATCAAATACAAGGGCTACCAG
GTAGCCCCAGCCGAACTGGAGAGCATCCTGCTGCAACACCCCAACATCTT
CGACGCCGGGGTCGCCGGCCTGCCCGACGACGATGCCGGCGAGCTGCCCG
CCGCAGTCGTCGTGCTGGAACACGGTAAAACCATGACCGAGAAGGAGATC
GTGGACTATGTGGCCAGCCAGGTTACAACCGCCAAGAAGCTGCGCGGTGG
TGTTGTGTTCGTGGACGAGGTGCCTAAAGGACTGACCGGCAAGTTGGACG
CCCGCAAGATCCGCGAGATTCTCATTAAGGCCAAGAAGGGCGGCAAGATC
GCCGTGTAATTCTAGTTGTTTAAACGAGCTCGCTAGCCTCGAGTCTAGAGTCGACCTGCAGG。
1.5双荧光素酶报告基因质粒转染及活性检测
1)细胞接种和培养
(1)培养细胞至密度为90%左右,PBS清洗2次。
(2)弃去上清,加入适当体积的0.25%胰酶消化细胞,待细胞变圆后,加入完全培养基终止反应。
(3)用5ml枪头吹打各处细胞,收集混合液至15ml试管,150g离心3min。
(4)去上清,并进行细胞计数,按实验内容将细胞接种至12孔培养板中,将培养板置于37℃,5%CO2的培养箱内培养过夜。
(5)24h后,根据实验要求,对其进行转染处理。
2)细胞转染
(1)细胞密度大约在70%左右进行转染。
(2)准备转染试剂,溶液1和溶液2,下面以12孔板中的三个孔为一组为例:
溶液1:150μl优化液+9μl脂质体3000。
溶液2:150μl优化液+1.5μg质粒+6μl P3000+45pmol片段。
(3)将溶液1与溶液2混合,室温下放置15min。
(4)将溶液1与溶液2的混合液逐滴加入孔中,摇动培养板,轻轻混匀,37℃、5%CO2继续培养24h。
3)荧光素酶活性的检测
(1)吸尽细胞培养液后用1×PBS润洗2遍,加入250μl细胞裂解液。
(2)将样本、萤火虫荧光素酶检测试剂、Renilla荧光素酶检测试剂平衡至室温后再进行以下操作:
(3)取20μl细胞裂解样本,加至酶标板中。
(4)每孔加入100μl萤火虫荧光素酶检测试剂,用移液器轻柔吹打混匀后读数。
(5)每孔加入100μl Renilla荧光素酶检测试剂,酶标仪震荡混匀后读数。
根据结果计算荧光素酶的比值。
2实验结果
2.1miR-22-3p直接靶向MEG8和RTL1
MEG8可以影响牦牛成肌细胞的增殖和分化,这与前期测得的lncRNA中预测的结果一致,同时发现在MvsE和AvsE两组中还存在一个值得关注的ceRNA调控机制,因此本发明推测在成肌细胞中存在一个MEG8-RTL1-miR22的调控网络并同时影响的成肌细胞的分化。
由差异LncRNA分析以及差异mRNA-lncRNA cis/trans网络结果发现,RT L1在两个比较组中存在顺势(cis)调控靶基因-MEG8(XR_314844.1),为了进一步探究RTL1-MEG8之间的互作关系,本发明以MEG8为基点,运用生物信息学分析筛选出差异表达novel211_mature;bta-miR-22-3p为MEG8/RTL1的潜在靶点。同时利用在线软件TargetScan与RNAhybrid共同预测发现MEG8/RTL1存在诸多靶向miRNA,其中miR-22-3p引起关注,在MEG8、miR-22-3p及miR-22-3p靶基因RTL1之间存在相同的碱基互补配对结合DNA序列(图2)。
为了进一步验证miR-22-3p与MEG8、RTL1之间的调控关系,本实施例通过碱基合成miR-22-3p在RTL1、MEG8基因上的结合位点,分别构建野生型(结合位点未突变)和突变型(结合位点定点突变)的双荧光素酶报告基因载体,同时公司(生物(安徽)股份有限公司)合成miR-22-3p突变mimic,采用双荧光素实验检测miR-22-3p与MEG8、RTL1之间的互作关系。结果显示,miR-22-3pmimic可以显著降低野生型RTL1双荧光素酶报告基因载体(MEG8-pmirGLO)的活性,而miR-22-3p mimic对突变型MEG8双荧光素酶报告基因载体(MEG8-mut-pmirGLO)无影响,当miR-22-3p mimic突变后,对野生型和突变型MEG8载体都不会产生影响(图3中的A)。同理,miR-22-3p mimic也可以显著降低野生型RTL1双荧光素酶报告基因载体(RTL1-pmirGLO)的活性,而miR-22-3p mimic则对突变型RTL1双荧光素酶报告基因载体(RTL1-mut-pmirGLO)无影响,当miR-22-3p mimic突变后,对野生型和突变型RTL1载体都不会产生影响(图3中的B)。上述结果表明,MEG8与miR-22-3p、miR-22-3p与RTL1之间存在直接靶向关系。
2.2miR-22-3p促进成肌细胞分化
在上述结果中,miR-22-3p直接靶向MEG8和RTL1,说明这三者之间存在直接调控关系,因此本实施例进一步研究了miR-22-3p在卫星细胞分化过程中的作用。首先在成肌细胞吧分化不同时间点检测了miR-22-2p的表达水平,结果表明miR-22-3p在分化过程中呈持续上升状态(图4)
为了进一步研究miR-22-3p在成肌细胞分化过程中发挥的作用,本实施例分别使用miR-22-3p mimic和miR-22-3p inhibitor转染成肌细胞,细胞转染后诱导分化,于诱导分化24h收取细胞进行mRNA和蛋白水平的检测。QPCR和Western blot结果显示,miR-22-3pmimic会显著促进miR-22-3p的表达,同时会显著提升分化标记基因MYF5、MYOD mRNA和蛋白水平的表达(图5)。miR-22-3pinhibitor会显著抑制miR-22-3p的表达,同时会显著促进MYF5、MYOD mRNA和蛋白水平的表达(图6)。我们利用免疫荧光实验检测miR-22-3p mimic和inhibitor对肌管形成的影响,结果显示,miR-22-3p mimic会显著促进肌管的形成(图5中的D,F),而miR-22-3p inhibitor会显著抑制肌管的形成(图6中的D,F)。上述结果表明miR-22-3p在成肌细胞分化过程中有促进作用。
2.2RTL1抑制成肌细胞分化
本实施例使用RTL1-Si和RTL-OE超表达载体转染成肌细胞来进一步的进行实验,同上述一致,于转染后24h收取细胞,进行检测。结果显示,RTL-OE会显著促进RTL1的表达,同时会显著降低MYOG、MYF5 mRNA和蛋白水平的表达(图7),对MYOD,MYF5的蛋白和mRNA表达水平产生较显著的影响,RTL1-Si显著降低了RTL1的表达,同时会显著降低分化标记基因MYOG、MYF5 mRNA和蛋白水平的表达(图7)。本发明利用免疫荧光实验检测RTL1-Si和RTL-OE对肌管形成的影响,结果显示,RTL-OE会显著抑制肌管的形成(图7中的D),而RTL-Si会显著促进肌管的形成(图7中的G,图8)。上述实验结果表明RTL1有抑制成肌细胞分化的作用。
2.3MEG8通过与RTL1竞争性结合miR-22-3p影响成肌细胞分化
为了进一步探究MEG8、miR-22-3p、RTL1这三者之间的关系,本实施例首先检测了MEG8、miR-22-3p、RTL1三个基因之间互相的调控关系。结果表明,过表达MEG8会显著上调miR-22-3p的表达,同时会显著下调RTL1mRNA和蛋白水平的表达,敲低MEG8会显著下调miR-22-3p的表达(图9中的AC),同时会显著上调RTL1mRNA和蛋白水平的表达(图9中的BC)。而miR-22-3pmimic会显著抑制RTL1的表达(图9中的CD),相反miR-22-3p inhibitor会显著促进RTL1的表达(图9中的CE)。此结果表明了MEG8与RTL1之间的正调控关系,以及miR-22-3p与RTL1之间的负调控关系。
尽管上述实施例对本发明做出了详尽的描述,但它仅仅是本发明一部分实施例,而不是全部实施例,人们还可以根据本实施例在不经创造性前提下获得其他实施例,这些实施例都属于本发明保护范围。
Claims (2)
1.RTL1基因在抑制牦牛成肌细胞分化中的应用。
2.RTL1基因在抑制牦牛肌管形成中的应用。
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