CN116218846B - 一种调控猪Ptrf基因表达的增强子序列的鉴定及其应用 - Google Patents
一种调控猪Ptrf基因表达的增强子序列的鉴定及其应用 Download PDFInfo
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Abstract
本发明公开了一种调控猪Ptrf基因表达的增强子序列的鉴定及其应用，猪Ptrf基因增强子，其核苷酸序列如SEQ ID No.1所示。利用双荧光素酶报告系统在293FT细胞中证实了该增强子活性，能显著增强Ptrf启动子的转录活性。后续，公开了在猪间充质干细胞中稳定转染dCas9‑KRAB‑sgRNA，构建Ptrf基因增强子表达抑制细胞株的方法，本发明基于CRIS PR/dCas9‑KRAB系统实现对Ptrf基因增强子的表达抑制，具有操作便利、干扰效率高的优势。这一Ptrf基因增强子序列可为后续制备调控脂肪含量的基因编辑猪提供技术支持。
Description
技术领域
本发明属于基因工程领域，具体涉及一种调控猪Ptrf基因表达的增强子序列的鉴定及其应用。
背景技术
Ptrf基因：在脂肪细胞中，小窝结构约占细胞表面的30％，通过调节脂肪酸的转移或甘油三脂的生物合成以及作为过量游离脂肪酸的汇合参与到脂质分解和合成的调节。聚合酶I和转录释放因子(Ptrf)，作为小泡的外衣蛋白，调节脂肪细胞分化，并决定脂肪组织的可扩张性。PTRF蛋白结构位于细胞膜小窝结构的核心，是质膜表面脂质微区形成的重要因素，被认为是小窝结构的典型组织者。值得注意的是，人类Ptrf基因中的先天性突变导致先天性全身性脂肪营养不良4型(CGL4)，这是一种罕见的常染色体隐性遗传疾病，其特征是体脂几乎完全丢失。Ptrf纯合敲除小鼠比野生型的体脂出现明显减少，以及胰岛素抵抗。同时，过表达Ptrf的小鼠体脂则大量积累，组织切片实验显示脂肪细胞直径和体积显著增加，脂肪细胞出现肥大，肝脏中脂肪积累程度更高。我们可以预期，Ptrf是一个理想的靶基因，以调节全身脂肪沉积的性状。
增强子：作为典型的顺式调节元件(CREs)，增强子是非编码基因组中的重要序列，可在细胞发育和分化中调节靶基因的精确时空表达。增强子作为一种顺式调控元件，由转录因子结合位点(TFBs)的密集簇组成，与细胞类型特异性的转录因子、染色质修饰物、内聚蛋白、凝集蛋白和CTCF等结构蛋白结合，通过增强子和启动子之间的染色质循环调节目标基因的转录。与其他调控因子相比，活性增强子是在许多生物过程中协调基因表达调控的关键元素，可以通过修改基因的表达模式进而引起更精细的改变，研究已证实通过靶向增强子能更好地调节基因的表达水平。
CRISPR/dCas9-KRAB系统：研究人员在CRISPR/Cas技术系统的基础上对链球菌中的Cas9核酸酶结构域进行了改造，从而产生了核酸内切酶活性缺失的Cas9，又称dCas9。dCas9不再能够切割基因组，但是任然能够在SgRNA的引导下，以高精度的靶向基因组DNA位置。KRAB可以与dCas9协作，实现特异性DNA甲基化调控。dCas9-KRAB系统更能够以时空特异性的方式沉默靶区表达，重塑表观遗传修饰，避免或延迟不可逆后果。目前，dCas9-KRAB被广泛用于阻断转录调控元件窗口附近的转录。dCas9-KRAB比传统方法有明显的优势，dCas9-KRAB可以利用顺式调控DNA元件在不同组织中编排基因特异性表达，可作为多种生物体特定基因表达调控的有效工具，实现简单灵活的基因表达调控。
综上，Ptrf基因是调控猪脂肪沉积的重要基因靶标之一，是猪育种过程中决定脂肪含量，肉质口感等相关性状的重要候选位点。Ptrf基因的表达量能够直接的影响个体的代谢，而目前尚未见对Ptrf基因增强子元件的相关研究。
发明内容
本发明所要解决的技术问题为：第一是提供一个猪Ptrf基因增强子，第二是提供一种该增强子表达抑制的细胞模型。
本发明的技术方案为：猪Ptrf基因增强子，其核苷酸序列如SEQ ID No.1所示。
扩增上述所述的猪Ptrf基因增强子的引物对，所述引物对的核苷酸序列如SEQ IDNo.2和SEQ ID No.3所示。
扩增上述所述的猪Ptrf基因增强子的方法，以猪基因组DNA 为模板，以SEQ IDNo.2和SEQ ID No.3所示的引物对进行PCR扩增，得到SEQ ID No.1所示的猪Ptrf基因增强子。
一种生物材料，含有上述所述的猪Ptrf基因增强子，所述生物材料为重组DNA、载体或工程菌等。
上述所述的猪Ptrf基因增强子或上述所述的生物材料在增强基因启动子转录活性上的应用。
进一步地，所述基因为猪Ptrf基因。
一种猪Ptrf基因增强子表达抑制的细胞模型的制备方法，包括如下步骤：
(1)设计靶向猪Ptrf基因增强子的sgRNA，并将该sgRNA对应的DNA序列连接到CRISPR/dCas9-KRAB慢病毒载体上，所述猪Ptrf基因增强子的核苷酸序列如SEQ ID No.1所示；
(2)将步骤(1)得到的慢病毒载体在293FT细胞中进行包装，收集慢病毒液；
(3)将步骤(2)得到的慢病毒液进行超滤浓缩，得到慢病毒浓缩液；
(4)用步骤(3)得到的慢病毒浓缩液感染处于细胞增殖期的猪间充质干细胞，筛选阳性细胞，获得猪Ptrf基因增强子表达抑制的细胞株。
进一步地，步骤(1)中，sgRNA对应的DNA序列如SEQ ID No.4、SEQ ID No.5或SEQID No.6所示。
进一步地，步骤(3)中，超滤浓缩的方法为：将慢病毒液转移到超滤管，4℃，15000r/min离心30min，浓缩至1mL或更少体积。
进一步地，步骤(4)中，筛选阳性细胞的方法为嘌呤霉素筛选。
与现有技术相比，本发明具有以下有益效果：
1、本发明提供一种新的猪Ptrf基因增强子，利用双荧光素酶报告系统证实了该增强子能够在293FT细胞中显著增强启动子的转录活性。
2、本发明通过慢病毒转染法、超滤管浓缩慢病毒和嘌呤霉素筛选相结合，率先制备了Ptrf增强子表达抑制细胞株，克服了CRISPR/dCas9-KRAB系统系统难转染、不能长时间稳定表达的缺陷。实现了Cas9-KRAB系统的稳定表达。同时，构建的Ptrf增强子表达抑制细胞株同样可以为今后制备增强子基因编辑猪提供技术支持。
附图说明
图1将Ptrf启动子与增强子克隆至PGL3-Basic质粒后的质粒图谱。
图2本发明中利用双荧光素酶验证增强子活性的相对荧光柱状图。
图3本发明提供的Ptrf基因增强子表达抑制细胞株构建方法流程示意图。
图4体外培养的猪原代骨髓间充质干细胞形态学观察图。
图5将Ptrf增强子打靶sgRNA克隆至CRISPR-dCas9-puro质粒的质粒图谱。
图6经过嘌呤霉素筛选的阳性细胞株形态学观察图。
图7Ptrf基因增强子表达抑制细胞株成脂诱导效果对比图。
图8本发明实施例得到的Ptrf基因增强子表达抑制细胞株Ptrf mRNA表达量检测结果图。
具体实施方式
下述实施例中的实验方法，如无特殊说明，均为常规方法。下述实施例中所用的试验材料，如无特殊说明，均为从商业渠道购买得到的。
1、Ptrf增强子序列的克隆和活性分析，包括以下步骤：
将Ptrf启动子序列(SEQ ID No.7所示)克隆到报告基因上游，通过检测报告基因的表达检测启动子的活性。将pGL3-basic载体和Ptrf基因启动子序列由擎科生物科技公司进行连接，连接成功载体命名为pGL3-promoter载体。进一步抽提巴马猪的耳源组织DNA，扩增Ptrf增强子序列(SEQ ID No.1所示)。随后，利用SalⅠ酶切位点将增强子序列转入pGL3-promoter载体，构建的质粒见图1。Ptrf增强子区域PCR扩增使用的引物对的核苷酸序列PE-F/PE-R如下：
PE-F:5’-TGGTTCTAGGACTTCAAGATGAG-3’(SEQ ID No.2)
PE-R:5’-CAGGGATAGAACCCGCAATCT-3’(SEQ ID No.3)
同时，由于插入片段的长度为1kb，我们选取了一段长度近似的(SEQ ID No.8所示)确定不含有增强子活性的序列作为对照，对照组引物对的核苷酸序列如下：
Con-F：5’-CCAAGCTGTTGGTCAGACTCC-3’
Con-R：5’-TGGATGGCTCTCCCTTAGGA-3’
分别将片段插入荧光素酶报告载体中，随后将构建好的载体分别转染到293细胞中，同时转入的还有內源对照载体pRL-TK。转染48小时后，使用Luciferase Assay System(Promega)试剂盒，按其说明书进行检测。
实验结果如图2所示，插入增强子之后，与负对照相比，增强子对应序列的载体显示出了增强子活性，而且显著高于pGL3-promoter载体活性。
2、供稳定转染CRISPR/dCas9系统的单克隆细胞株的构建方法，包括如下步骤，如图3所示：
2.1采集巴马猪骨髓，通过组织贴壁法培养猪原代骨髓间充质细胞，并观察了BMSCs的细胞形态学，细胞形态正常，见图4。
2.2慢病毒dCas9-KRAB-SgRNA载体的构建，设计靶向猪Ptrf增强子(Ptrf-E2)的sgRNA，设计的sgRNA将退火后的双链sgRNA连接到dCas9-KRAB载体BbsI酶切位点，dCas9-KRAB-puro载体见图5质粒图谱所示。所述sgRNA对应的核苷酸序列如SEQ ID No.4、SEQ IDNo.5或SEQ ID No.6所示。
2.3质粒以CRISPR-dCas9-puro-sgRNA(14906bp)：psPAX2(10668bp)：VSVG(6507bp)摩尔质量为1:1:1的比列，选择了磷酸钙转染法进行共转染。在T175培养瓶种植293FT细胞，以293FT细胞作为宿主进行慢病毒的生产，待细胞汇合度达到70％时，使用磷酸钙细胞转染试剂盒进行转染，在转染后的24h，48h，72h收集病毒液。
2.4再使用超滤浓缩分离技术提取慢病毒浓缩液。含慢病毒上清液转移到超滤管，4℃，15000r/min离心30min，浓缩至1mL或更少体积，收集后过滤除菌用于后续实验。该纯化
方法的特点：设备要求低，实验成本低，病毒纯度较高，毒性较高。
2.5将慢病毒浓缩液用以感染处于细胞增殖期的猪间充质干细胞。细胞经过慢病毒感染三天后，使用2μg/mL嘌呤霉素筛选巴马猪BMSCs阳性细胞，获得Ptrf-E2增强子表达抑制细胞株，经过嘌呤霉素筛选后的细胞形态正常，细胞活力正常，见图6。
2.6进行细胞株的成脂诱导，在成脂12天通过荧光定量实验检测脂肪细胞中的Ptrf mRNA表达量，对比两种处理的细胞株，在成脂诱导12天后，细胞内甘油三脂含量差异显著，见图7。
2.7猪Ptrf基因在脂肪细胞中的表达量分析，本发明利用实时定量PCR对Ptrf基因表达量进行分析。提取不同时间节点下细胞的棕RNA，反转录成cDNA。将cDNA稀释后作为模板，按照试剂盒说明加入到定量PCR 96孔板中，放入定量PCR仪中进行反应。
2.8实验结果如图8所示，与负对照相比，成功构建增强子表达抑制的细胞株，其Ptrf基因mRNA表达量显示出来明显的表达量下调，证明Ptrf-E2增强子对于Ptrf基因有显著的调控作用。
Claims (8)
1.猪Ptrf基因增强子，其特征在于，所述增强子的核苷酸序列如SEQ ID No.1所示。
2. 扩增权利要求1所述的猪Ptrf基因增强子的引物对，其特征在于，所述引物对的核苷酸序列如SEQ ID No.2和SEQ ID No.3所示。
3. 扩增权利要求1所述的猪Ptrf基因增强子的方法，其特征在于，以猪基因组DNA 为模板，以SEQ ID No.2和SEQ ID No.3所示的引物对进行PCR 扩增，得到SEQ ID No.1所示的猪Ptrf基因增强子。
4.含有权利要求1所述的猪Ptrf基因增强子的生物材料，所述生物材料为重组DNA、载体或工程菌。
5.权利要求1所述的猪Ptrf基因增强子或权利要求4所述的生物材料在增强猪Ptrf基因启动子转录活性上的应用。
6.一种猪Ptrf基因增强子表达抑制的细胞模型的制备方法，其特征在于，包括如下步骤：
（1）设计靶向猪Ptrf基因增强子的sgRNA，并将该sgRNA对应的DNA序列连接到CRISPR/dCas9-KRAB慢病毒载体上，所述猪Ptrf基因增强子的核苷酸序列如SEQ ID No.1所示；
（2）将步骤（1）得到的慢病毒载体在293FT细胞中进行包装，收集慢病毒液；
（3）将步骤（2）得到的慢病毒液进行超滤浓缩，得到慢病毒浓缩液；
（4）用步骤（3）得到的慢病毒浓缩液感染处于细胞增殖期的猪间充质干细胞，筛选阳性细胞，获得猪Ptrf基因增强子表达抑制的细胞株；
步骤（1）中，所述sgRNA对应的DNA序列如SEQ ID No.4、SEQ ID No.5或SEQ ID No.6所示。
7. 根据权利要求6所述的制备方法，其特征在于，步骤（3）中，超滤浓缩的方法为：将慢病毒液转移到超滤管，4℃，15000 r/min离心 30 min，浓缩至 1 mL 或更少体积。
8.根据权利要求6所述的制备方法，其特征在于，步骤（4）中，筛选阳性细胞的方法为嘌呤霉素筛选。
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