CN111944033B - Rbp4蛋白或其编码基因在调控成肌细胞分化和融合中的应用 - Google Patents
Rbp4蛋白或其编码基因在调控成肌细胞分化和融合中的应用 Download PDFInfo
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Abstract
本发明涉及基因工程技术领域，具体公开了RBP4蛋白或其编码基因在调控成肌细胞分化和融合中的应用。本发明发现RBP4基因的过表达能够抑制成肌细胞分化和融合，进而提出RBP4蛋白或其编码基因、或含有其编码基因的生物材料在调控成肌细胞分化和融合、肌肉发育中的应用。本发明明确了RBP4基因具有抑制成肌细胞的分化和融合的作用，为今后进一步研究RBP4基因在细胞分化和融合的作用机制及其在家畜肉质性状改良方面的应用研究奠定了基础。
Description
技术领域
本发明涉及基因工程技术领域，具体地说，涉及一种RBP4蛋白或其编码基因在调控成肌细胞分化和融合中的应用。
背景技术
C2C12成肌细胞系是从小鼠C3H细胞中分离得到的成肌细胞系，体外培养时分化的能力较强，可以通过改变培养基中存在血清的量来促进其肌向分化，较常被选为在体外研究骨骼肌成肌细胞分化的模型，在体外条件下模拟骨骼肌细胞的生长发育过程以供研究骨骼肌发育的具体调控机制。
视黄醇结合蛋白(Retinol-binding proteins,RBPs)属疏水小分子结合蛋白家族成员，是重要的维生素A转运蛋白，协助视黄酸/视黄醇在胞内和血清视中转运。RBP4是一种重要的胞外视黄醇结合蛋白，其促进转化生长因子的表达，参与胚胎的早期发育。此外，RBP4还可以转运维生素A并影响繁殖性能和脂肪分化。目前尚无RBP4在肌肉发育调控作用的研究报道，其在肌细胞分化和肌纤维形成中的作用尚不清楚。
对于肉用家养动物，清楚地了解其RBP4基因对肌肉发育的作用，有利于加快动物品种培育。因此，明确RBP4基因在肌细胞中的作用，不仅能为研究肌肉发育的表达调控机制提供新思路，更为今后进一步研究RBP4基因在家畜肉质性状方面发挥的调控作用奠定了基础。
发明内容
本发明的目的是提供一种RBP4蛋白或其编码基因在调控成肌细胞分化和融合中的应用。
本发明从不同生长速度和脂肪沉积能力的猪种的转录组数据中，均筛选到了RBP4基因，发现其不仅在肝脏中表达，在肌肉中也有表达，且在生长速度不同的猪种的肌肉组织中表达差异显著，说明RBP4可能还参与肌肉发育调控。并由此进一步研究从而提出本发明方案。
具体地，本发明的技术方案如下：
第一方面，本发明提供RBP4蛋白或其编码基因、或含有其编码基因的生物材料在调控成肌细胞分化和融合中的应用。
第二方面，本发明提供RBP4蛋白或其编码基因、或含有其编码基因的生物材料在调控肌肉发育中的应用。
本发明中通过提高RBP4基因的表达量，抑制成肌细胞的分化和融合。
第三方面，本发明提供RBP4蛋白或其编码基因、或含有其编码基因的生物材料在动物品种选育中的应用。
本发明中，所述生物材料为表达盒、载体、宿主细胞或重组菌。
优选所述生物材料为超表达载体pCDH-RBP4。
构建超表达载体pCDH-RBP4时，根据猪RBP4基因序列设计一对PCR引物，所述PCR引物的序列如SEQ ID NO.1和SEQ ID NO.2所示。
具体地，所述RBP4基因的超表达载体pCDH-RBP4的构建方法如下：
S1、根据猪RBP4基因序列设计一对PCR引物，在两个引物的5'端分别引入BamHI酶切位点和NotI酶切位点，利用该对引物从猪cDNA中扩增出RBP4基因的CDS区序列，长度为606bp；
S2、用BamHI和NotI双酶切步骤S1中所得序列及pCDH载体，通过连接酶连接，使所述序列***质粒载体pCDH的BamHI和NotI酶切位点，构建出猪RBP4基因的表达载体pCDH-RBP4。
本发明中，所述RBP4基因的CDS序列如SEQ ID NO.5所示。
第四方面，本发明提供调控动物肌肉发育的方法，通过转基因的方法，调节动物体内RBP4基因的表达。
本发明的有益效果至少在于：
1、本发明首次构建了超表达猪RBP4基因的表达载体pCDH-RBP4，可以显著提高RBP4基因的表达，填补了目前没有超表达猪RBP4基因表达载体的空白，为探究猪RBP4基因生物学功能提供了研究工具；
2、本发明证明了超表达载体pCDH-RBP4转染进细胞超表达RBP4后可以抑制小鼠成肌细胞C2C12的分化与融合。明确了猪RBP4基因在肌细胞分化和融合过程中具有重要调控作用，不仅能为研究肌肉发育的表达调控机制提供新思路，更为今后进一步研究RBP4基因在家畜肉质性状方面发挥的调控作用奠定了基础。
附图说明
图1为RBP4-CDS电泳图，泳道1和2分别为2000plus DNAmarker和RBP4-CDS；
图2为pCDH载体骨架图；
图3为双酶切电泳图，泳道1-4依次为2000plus DNA marker，pCDH环状质粒，双酶切后的线性pCDH质粒，双酶切后的RBP4-CDS；
图4为超表达载体效率检测图,***P<0.001；
图5为成肌分化标志基因的mRNA表达检测图，*P<0.05,**P<0.01,***P<0.001；
图6为免疫荧光染色检测肌细胞分化图，其中，中间上下两图显示的是DAPI染色的细胞核，最左侧上下两图显示的是被染色的MyHC阳性细胞，最右侧上下两图(Merge)为二者叠加图；
图7为成肌融合标志基因的mRNA表达检测图，N.S.代表差异不显著，**P<0.01；
图8为免疫荧光染色检测肌细胞融合图，其中，中间上下两图显示的是DAPI染色的细胞核，最左侧上下两图显示的是被染色的MyHC阳性细胞，最右侧上下两图(Merge)为二者叠加图；箭头标注的为发生融合的多核肌管。
具体实施方式
下面将结合实施例对本发明的优选实施方式进行详细说明。需要理解的是以下实施例的给出仅是为了起到说明的目的，并不是用于对本发明的范围进行限制。本领域的技术人员在不背离本发明的宗旨和精神的情况下，可以对本发明进行各种修改和替换。
下述实施例中所使用的实验方法如无特殊说明，均为常规方法。下述实施例中所用的材料、试剂等，如无特殊说明，均可从商业途径得到。
实施例1猪RBP4基因超表达载体的构建
1、猪RBP4基因CDS区序列的获得
根据猪RBP4基因序列(Gene ID:397124)设计一对PCR引物，在两个引物的5'端分别引入BamHI酶切位点CGGGATCC(SEQ IDNO.3)和NotI酶切位点ATTTGCGGCCGC(SEQ IDNO.4)，上、下游引物序列分别如SEQ ID NO.1和SEQ ID NO.2所示。利用该对引物以猪cDNA为模板扩增出RBP4基因的CDS区序列RBP4-CDS(如SEQ ID NO.5所示)，长度为606bp(参见图1所示的RBP4-CDS电泳图)，并对扩增产物进行胶回收。PCR扩增使用20uL反应体系：10μL 2×Power Taq PCR Master Mix，8μL ddH2O，上下游引物各0.5μL，cDNA模板1μL；PCR反应条件：94℃预变性5分钟；30个循环：94℃变性30秒，60℃退火30秒，72℃延伸60秒；最后72℃终延伸5分钟。
2、pCDH-RBP4超表达载体的构建
(1)pCDH载体骨架如图2所示(购自优宝生物)，用BamHI和NotI双酶切步骤1中所得PCR产物RBP4-CDS及pCDH环状质粒。按照表1配制酶切体系，配制完成以后将体系置于37℃的水浴锅中酶切2h。酶切结束后进行1％琼脂糖凝胶电泳(双酶切电泳图如图3所示)，根据Tiangen胶回收试剂盒回收纯化含粘性末端的酶切产物。
表1双酶切体系试剂配制表
(2)连接两种胶回收酶切产物，按照表2配置连接体系，于16℃连接过夜。
表2连接体系
(3)将重组载体转化进DH5α大肠杆菌感受态细胞中，混匀后冰浴30分钟，然后在42℃热激转化90秒，随后冰浴2分钟，加入37℃温育好的无菌LB培养基(1％胰蛋白胨、0.5％酵母提取物、1％氯化钠)950μL，在37℃，180转/分的条件下震荡培养2小时，取100μL菌液均匀涂布在氨苄青霉素抗性固体LB培养基(含100μg/mL的氨苄青霉素)上，37℃培养箱过夜倒置培养12～16小时。
(4)测序：取单菌落，在1mL含100μg/mL氨苄青霉素的液体LB培养基中培养，150rpm摇菌至浑浊，菌液送往测序公司进行测序验证，最终确定载体构建成功，命名为pCDH-RBP4。
3、超表达效率检测
待六孔板中C2C12细胞生长至70％～90％的汇合度时，使用转染试剂lipo2000将超表达质粒pCDH-RBP4转染进细胞中，以转染空载体pCDH的为对照，转染6h后换液。利用Trizo1法从转染48h的成肌细胞中提取总RNA，再用Tiangen反转录试剂盒将RNA反转录为cDNA，以cDNA为模板，采用Bio-Rad公司的SYBR Green荧光染料，Roche公司LightCycler480荧光定量PCR仪，以β-actin作为内参，进行荧光定量PCR实验，用2-ΔΔCt法计算每个基因的相对mRNA表达量，每组实验做3个平行，每个实验重复三次：反应体系为20μL；反应条件：95℃预变性5分钟，95℃变性15秒，56℃退火15秒，72℃，15秒循环40次。荧光定量PCR检测RBP4基因所用的上游引物、下游引物分别如SEQ ID NO.6和SEQ ID NO.7所示。
结果如图4超表达载体效率检测图所示，转染超表达载体后肌卫星细胞中RBP4表达量显著高于对照组，说明pCDH-RBP4超表达载体能提高目标基因的表达量。
实施例2 RBP4基因超表达载体在小鼠成肌细胞分化中的作用
利用转染试剂lipo2000将超表达载体pCDH-RBP4转染进小鼠成肌细胞C2C12中，以转染空载体pCDH的为对照，6小时后换成分化培养基(含2％马血清的DMEM培养基)，对细胞进行诱导分化。分化4天后，一部分细胞用于提取RNA并反转录成cDNA，利用荧光定量PCR检测成肌分化标志基因MyHC、MyoG、MyoD的表达变化；对另一部分细胞进行免疫荧光染色，检测成肌分化标志基因MyHC的变化。
图5所示为采用荧光定量PCR检测成肌细胞分化标志基因MyHC、MyoG、MyoD的mRNA表达水平变化，发现超表达组基因表达量显著低于对照组，说明pCDH-RBP4通过超表达RBP4基因而抑制成肌细胞的分化。
图6所示为采用免疫荧光染色检测成肌细胞分化标志基因MyHC的变化，转染了pCDH-RBP4的细胞中MyHC阳性细胞少于转染对照pCDH空载体的细胞，说明RBP4基因具有抑制成肌细胞分化的作用。
实施例3 RBP4基因超表达载体在小鼠成肌细胞融合中的作用
利用转染试剂lipo2000将超表达载体pCDH-RBP4转染进小鼠成肌细胞C2C12中，以转染空载体pCDH的为对照，6小时后换成分化培养基(含2％马血清的DMEM培养基)，对细胞进行诱导分化。分化4天后，一部分细胞用于提取RNA并反转录成cDNA，利用荧光定量PCR检测成肌融合标志基因β-1integrin和Myomaker的mRNA表达变化；对另一部分细胞进行免疫荧光染色，检测成肌细胞的肌管融合情况。
荧光定量PCR结果如图7成肌融合标志基因的mRNA表达检测图所示，超表达组融合标志基因表达量低于对照组，说明pCDH-RBP4通过超表达RBP4基因而抑制成肌细胞融合。
免疫荧光染色结果如图8免疫荧光染色检测肌细胞融合图所示，对照组肌管发生了多核融合情况，而超表达组为单细胞核肌管，说明超表达RBP4基因能抑制成肌细胞融合。
综上所述，本发明首次构建了超表达猪RBP4基因的表达载体pCDH-RBP4，可以显著提高RBP4基因的表达，填补了目前没有超表达猪RBP4基因表达载体的空白，为探究猪RBP4基因生物学功能提供了研究工具；
本发明证明了超表达载体pCDH-RBP4转染进细胞超表达RBP4后可以抑制小鼠成肌细胞C2C12的分化与融合。明确了猪RBP4基因在肌细胞分化和融合过程中具有重要调控作用，不仅能为研究肌肉发育的表达调控机制提供新思路，更为今后进一步研究RBP4基因在家畜肉质性状方面发挥的调控作用奠定了基础。
虽然，上文中已经用一般性说明及具体实施方案对本发明作了详尽的描述，但在本发明基础上，可以对之作一些修改或改进，这对本领域技术人员而言是显而易见的。因此，在不偏离本发明精神的基础上所做的这些修改或改进，均属于本发明要求保护的范围。
序列表
<110> 中国农业大学
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Claims (10)
1.RBP4蛋白或其编码基因、或含有其编码基因的生物材料在调控成肌细胞分化和融合中的应用，所述RBP4蛋白的编码基因的CDS序列如SEQ ID NO.5所示。
2.RBP4蛋白或其编码基因、或含有其编码基因的生物材料在调控肌肉发育中的应用，所述RBP4蛋白的编码基因的CDS序列如SEQ ID NO.5所示。
3.根据权利要求1或2所述的应用，其特征在于，通过提高RBP4基因的表达量，抑制成肌细胞的分化和融合。
4.RBP4蛋白或其编码基因、或含有其编码基因的生物材料在动物品种选育中的应用，所述RBP4蛋白的编码基因的CDS序列如SEQ ID NO.5所示。
5.根据权利要求1、2、4任一项所述的应用，其特征在于，所述生物材料为表达盒、载体、宿主细胞或重组菌。
6.根据权利要求3所述的应用，其特征在于，所述生物材料为表达盒、载体、宿主细胞或重组菌。
7.根据权利要求5所述的应用，其特征在于，所述生物材料为超表达载体pCDH-RBP4。
8.根据权利要求6所述的应用，其特征在于，所述生物材料为超表达载体pCDH-RBP4。
9.根据权利要求7或8所述的应用，其特征在于，构建超表达载体pCDH-RBP4时，根据猪RBP4基因序列设计一对PCR引物，所述PCR引物的序列如SEQ ID NO.1和SEQ ID NO.2所示。
10.调控动物肌肉发育的方法，其特征在于，通过转基因的方法，调节动物体内RBP4基因的表达，所述RBP4基因的CDS序列如SEQ ID NO.5所示。
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104404133A (zh) * | 2014-10-20 | 2015-03-11 | 中国农业大学 | 与猪生长速度相关的snp标记及其应用 |
| CN104673818A (zh) * | 2015-03-25 | 2015-06-03 | 吉林大学 | 猪rbp4蛋白体外表达及其抗体制备 |
| CN111748615A (zh) * | 2020-05-14 | 2020-10-09 | 南京医科大学附属逸夫医院 | 一种rbp4作为肌少症治疗靶点的检测方法 |
Family Cites Families (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2006015209A2 (en) * | 2004-07-29 | 2006-02-09 | Stem Cell Innovations, Inc. | Differentiation of stem cells |
| US9662314B2 (en) * | 2011-10-21 | 2017-05-30 | Tufts Medical Center, Inc. | Compounds and methods for the treatment of muscular disease, and related screening methods |
| US20180163178A1 (en) * | 2015-05-29 | 2018-06-14 | Robert J. Schneider | Auf1 encoding compositions for muscle cell uptake, satellite cell populations, and satellite cell mediated muscle generation |
-
2020
- 2020-08-06 CN CN202010784784.7A patent/CN111944033B/zh active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104404133A (zh) * | 2014-10-20 | 2015-03-11 | 中国农业大学 | 与猪生长速度相关的snp标记及其应用 |
| CN104673818A (zh) * | 2015-03-25 | 2015-06-03 | 吉林大学 | 猪rbp4蛋白体外表达及其抗体制备 |
| CN111748615A (zh) * | 2020-05-14 | 2020-10-09 | 南京医科大学附属逸夫医院 | 一种rbp4作为肌少症治疗靶点的检测方法 |
Non-Patent Citations (5)
| Title |
|---|
| Posttranscriptional regulation of cyclin D1 by ARE-binding proteins AUF1 and HuR in cycling myoblasts;Utpalendu Ghosh等;《Journal of Biosciences》;pubmed;20181231;第43卷;第685-691页 * |
| retinol-binding protein 4 precursor [Sus scrofa];GenBank DataBase;《GenBank DataBase》;GenBank DataBase;20200701;Accession NO：NP_999222 * |
| Sequencing and characterization of miRNAs and mRNAs from the longissimus dorsi of Xinjiang brown cattle and Kazakh cattle;NaLi等;《Gene》;ELSEVIER;20200530;第741卷(第30期);第144537页 * |
| Sus scrofa retinol binding protein 4 (RBP4), mRNA;GenBank DataBase;《GenBank DataBase》;GenBank DataBase;20200701;Accession NO： NM_214057 * |
| 视黄醇结合蛋白及其基因的分子生物学;郭晓红等;《遗传》;CNKI;20041231;第26卷(第2期);第257-262页 * |
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