CN113621621A - 一种靶向奶山羊CIDEa基因的小干扰RNA及其应用 - Google Patents
一种靶向奶山羊CIDEa基因的小干扰RNA及其应用 Download PDFInfo
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Abstract
本发明涉及一种针对CIDEa基因的小干扰RNA及其在奶山羊乳腺上皮细胞调控乳脂代谢中的应用，设计合成了一个针对CIDEa基因的siRNA，将该小干扰RNA转染体外培养的奶山羊乳腺上皮细胞，有效抑制了CIDEa基因的表达，同时抑制了细胞中脂滴积累，降低细胞甘油三酯含量，但增加了细胞中游离脂肪酸的含量；小干扰RNA显著上调脂肪酸从头合成基因脂肪酸合酶、脂肪分解基因激素敏感脂酶表达，同时促进转录因子固醇调节元件结合蛋白1表达。CIDEa基因小干扰RNA改变了细胞中脂肪酸含量，脂肪酸组成是影响羊乳营养价值和风味的主要因素，为改善羊奶营养价值和风味提供重要依据。
Description
技术领域
本发明涉及一种靶向奶山羊CIDEa基因的小干扰RNA及其应用，属于生物工程技术领域。
背景技术
诱导细胞凋亡DNA片段化因子45(DFF45)样效应因子a(Cell death inducing DNAfragmentation factor 45-like effectors a，CIDEa)属于CIDE家族重要一员，CIDE家族基因参与调控多个脂代谢通路，包括脂滴的生长和融合、极低密度脂蛋白的成熟以及脂解过程。研究表明，CIDEa过表达能促进肝脏细胞大脂滴的形成，并且能够促进肝脏中的脂质累积。在脂肪肝中沉默CIDEa基因，可缓解脂肪肝程度，脂合成基因表达下调，线粒体活性增加。在小鼠体内沉默CIDEa基因，小鼠肝脏中脂滴变小，脂质累积减少；过表达CIDEa基因，小鼠肝脏中形成大脂滴，脂质累积增加。CIDEa基因是参与脂肪代谢的一种重要调控因子。研究还发现，CIDEa在小鼠妊娠以及哺乳期的乳腺组织中高度表达，在牛的泌乳期乳腺组织中CIDEa表达明显高于干奶期和非妊娠期。推测CIDEa基因可能参与乳腺组织乳脂代谢过程。
羊乳具有丰富的营养价值，特别是在乳脂方面具有独特优势。由于山羊奶乳脂滴较牛奶更小，易被人体消化吸收，且山羊奶短中链脂肪酸含量丰富。脂肪含量及脂肪酸组成是影响羊乳营养价值和风味的主要原因，因此，深入研究奶山羊CIDEa基因的功能及其在乳腺乳脂合成代谢调控中的机制对改善羊乳品质具有重要意义。为了进一步提高羊乳品质，本发明尝试在奶山羊乳腺细胞中转染CIDEa基因的小干扰RNA序列，以期对活细胞中乳脂滴和甘油三酯含量产生影响，研究成果为改善羊乳品质提供理论依据。
发明内容
针对现有技术的不足，本发明的目的是提供一种针对CIDEa基因的小干扰RNA及其在奶山羊乳腺上皮细胞调控乳脂代谢中的应用，其中小干扰RNA转染体外培养的奶山羊乳腺上皮细胞，可靶向抑制CIDEa基因表达，调控活细胞中脂质的代谢，为改善羊乳品质提供理论依据。
为了实现上述目的，本发明所采用的技术方案是：
一种靶向奶山羊CIDEa基因的小干扰RNA，所述RNA的正向及反向序列分别为：
F：5’-GCAUCGCGAAAGUCACCUUTT-3’，
R：5’-AAGGUGACUUUCGCGAUGCTT-3’。
所述的小干扰RNA在调控奶山羊乳腺上皮细胞脂质合成中的应用。
所述脂质包括甘油三酯、游离脂肪酸和脂滴。
所述的小干扰RNA在调控奶山羊乳腺上皮细胞脂质代谢相关基因表达中的应用。
所述基因包括ACACA、FASN、ATGL、HSL、CD36、FABP3、SREBP1、PPARG、INSIG1、DGAT1和DGAT2。
本发明有益效果：
本发明设计合成了一个针对CIDEa基因的siRNA，将该小干扰RNA转染体外培养的奶山羊乳腺上皮细胞，有效抑制了CIDEa基因的表达，同时抑制了细胞中脂滴积累，降低细胞甘油三酯含量，但增加了细胞中游离脂肪酸的含量；小干扰RNA显著上调脂肪酸从头合成基因脂肪酸合酶(FASN)、脂肪分解基因激素敏感脂酶(HSL)表达，同时促进转录因子固醇调节元件结合蛋白1(SREBP1)表达。CIDEa基因小干扰RNA改变了细胞中脂肪酸含量，脂肪酸组成是影响羊乳营养价值和风味的主要因素，因此CIDEa基因小干扰RNA为改善羊奶营养价值和风味提供重要依据。
附图说明
图1为RT-qPCR检测si-CIDEa干扰效率的结果示意图；
其中，*代表P<0.05；**代表P<0.01，下同；
图2为干扰CIDEa基因对山羊乳腺上皮细胞脂质代谢相关基因表达的影响；
图3为干扰CIDEa基因对山羊乳腺上皮细胞脂滴(A)及甘油三酯含量(B)的影响；
图4为干扰CIDEa基因对细胞沉淀中游离脂肪酸(A)和细胞培养液中游离脂肪酸(B)含量的影响。
具体实施方式
以下结合实施例对本发明的具体实施方式作进一步详细说明。
本发明实施例中实验数据运用SPSS22.0统计学软件进行数据分析，采用t检验和One-way ANOVA进行方差分析，结果以平均值±标准差表示，以P＜0.05表示差异显著性判断标准。
实施例1 CIDEa基因siRNA设计与合成
根据实验室前期克隆得到的奶山羊CIDEa基因编码区序列(序列如下所示)，利用ThermoFisher设计网站(https://rnaidesigner.thermofisher.com/rnaiexpress/setOption.do？designO)对CIDEa基因的siRNA进行设计，筛选靶点相差25bp以上、GC含量在30％～52％之间、预测分值较高且与靶基因特异性结合的3对siRNA，其序列分别为：
si-CIDE60,
F：5’-GCAGACAAAGAAAGUGCUGTT-3’(SEQ ID NO.1)；
R：5’-CAGCACUUUCUUUGUCUGCTT-3’(SEQ ID NO.2)；
si-CIDE374,
F：5’-GCAUCGCGAAAGUCACCUUTT-3’(SEQ ID NO.3)；
R：5’-AAGGUGACUUUCGCGAUGCTT-3’(SEQ ID NO.4)；
si-CIDE554,
F：5’-GCCAGUGUCUUGUCCACAUTT-3’(SEQ ID NO.5)；
R：5’-AUGUGGACAAGACACUGGCTT-3’(SEQ ID NO.6)。
同时设置阴性对照，其序列为，
F：5'-UUCUCCGAACGUGUCACGUTT-3'(SEQ ID NO.7)；
R：5'-ACGUGACACGUUCGGAGAATT-3'(SEQ ID NO.8)。
所有siRNA序列均由生工生物工程(上海)股份有限公司合成。
奶山羊CIDEa基因编码区序列如下：
ATGGGGTCGCAGACAAAGAAAGTGCTGTTCACACCCCTCATGCATCCCGCTCGCCCCTTCCGTGTCTCCAACCATGACCGGAGCAGCCGCCGAGGGGTGATGGCCAGTAGCCTGCAGGAGCTCCTCAGCAAGACCCTGGATGCGCTGGTGGTCCCCAGCCAACTGGTCACCTTGGTGCTGGAGGAGGATGGCACTGTGGTGGACACAGAGGAGTTCTTCCAGACCCTGGGGGACAACACACACCTCATGGTCCTGGAGCAGGGGCAGAAATGGACACCGGCTGGCAGCCACACCCCAGCCCGCTGGCCACCTCAGAGACGGGGCATCGCGAAAGTCACCTTCGACTTGTACAAGCTGAGCCCCAAGGATGTCATTGGCTGCCTCAATGTGAAGGCCACCATGTACGAGATGTATTCCGTGTCCTACGACATCCACTGCACCGGGTTCAAGGCCATGCTCAGGAGCCTGCTGCGGTTCCTGTCTCACGCTGCCCAGGTGACTGGCCAGTGTCTTGTCCACATGGGTACCTACATGCTCCGAGTGCTGGCTGAAACGGAGGAGCAGGCGGTGCCTGGCTCGCGCCCTCGGAGAGGGATCAAGAGTGGATAG(SEQ ID NO.9)。
实施例2奶山羊乳腺上皮细胞培养与转染
奶山羊乳腺上皮细胞采用胰酶消化乳腺组织块法进行分离细胞，通过高密度接种法进行传代培养，采用DMEM/F12细胞培养基，其含有10％FBS、100U/mL青霉素、100μg/mL链霉素、5μg/mL氢化可得松、5μg/mL胰岛素和10ng/mL表皮生长因子，于37℃、5％CO2培养箱中培养。待显微镜下观察细胞汇合度达70％～80％时，参照Invitrogen公司的LipofectamineTMRNAiMAX说明书，将实施例1所得的3对siRNA与不针对任何基因的阴性对照siRNA分别配制成终浓度为100μmol/L转染复合物，室温静置20min，将复合物分别添加于不同的细胞中。转染48h后，收集细胞，用于后续RNA提取。
实施例3细胞总RNA提取与cDNA的合成
siRNA转染48h后，收集细胞，采用北京天根生化科技有限公司细胞/组织总RNA提取试剂盒提取细胞总RNA，按照说明书进行操作。RNA样品用紫外分光光度计(NANODROP2000)测定RNA样品A260 nm/A280 nm吸收比率值，检测浓度大于200ng/μL及纯度为1.8～2.0时为合格样品，以提取的RNA为模板，按照Takara公司反转录试剂盒说明书步骤，将所有检测合格的RNA反转录成cDNA。
实施例4 CIDEa干扰效率的检测
山羊乳腺上皮细胞分别转染3条si-CIDEa后，采用RT-qPCR检测CIDEa基因mRNA水平。
实时荧光定量PCR：基因引物及内参基因引物见表1，各对引物扩增基因产物长度约200bp大小。试验以UXT和GAPDH基因作为内参。
PCR反应体系为：10.0μL的荧光定量PCR反应混合物(2×SYBR Premix Ex TaqMix)，1.0μL的实施例3中的反转录cDNA为模板，上、下游引物(10μmol/L)各0.6μL，加入无RNA酶的H2O补足20μL反应体系。于ABI 7500荧光定量PCR仪上进行反应，反应条件为：95℃30s，95℃5s，60℃32s，40个循环；添加熔解曲线。采用2－△△Ct法对数据进行分析(其中△Ct＝Ct目的基因-Ct内参；△△Ct＝△Ct试验组-△Ct对照组)。每个样品设置3个重复。数据采用SPSS22.0进行分析，采用t检验进行统计分析，P<0.05为差异显著，用*表示，P<0.01为差异极显著，用**表示。
表1实时定量PCR引物序列
结果发现，与对照组相比，si-CIDE60、si-CIDE374和si-CIDE554干扰效率分别为62％、88％和86％(P<0.01，图1)，说明siRNA干扰效果良好，可以显著抑制CIDEa基因的表达。选用干扰效率最好的si-CIDE374进行后续实验。
RT-qPCR检测结果还发现，干扰CIDEa基因，导致脂肪酸合成基因(FASN)表达量上调(图2A)，说明CIDEa影响乳腺上皮细胞中脂肪酸重新合成；脂肪分解基因(HSL)表达量显著增多(图2B)，表示干扰ATGL后促进脂解的增加；而脂肪酸结合蛋白3(Fatty acidbinding protein3,FABP3)(图2C)和二酰基甘油转移酶1(Diacylglycerolacyltransferase 1,DGAT1)(图2D)表达量显著下调(P<0.05)，则说明CIDEa导致的脂质合成减少，抑制了外源脂肪酸的转运和甘油三酯的重新合成；转录调控关键因子(SREBP1)(图2E)表达量显著上调(P<0.05)，表明CIDEa的减少能够促进SREBP1重新合成甘油三酯。
实施例5 CIDEa基因干扰对细胞中脂质合成的影响
(1)油红O染色
细胞转染siRNA 48h后，弃去培养基，用PBS清洗两遍；然后用10％中性甲醛固定细胞45min；每孔细胞(6孔板)加入1mL油红O染色30min；最后用PBS清洗3遍洗净剩余油红O，最后于倒置显微镜下进行观察拍照。
(2)细胞中甘油三酯和游离脂肪酸检测
细胞转染siRNA 48h后，用0.25％胰蛋白酶/EDTA消化液消化细胞，收集细胞沉淀，PBS清洗沉淀，然后加入PBS 200μL，用于细胞超声破碎，按照甘油三酯检测试剂盒操作说明检测细胞内甘油三酯的含量，简要流程为取适量裂解液水浴(70℃)加热10min，于室温下2000r/min离心5min，取上清液10μL与工作液190μL于37℃反应10min，最后用酶标仪于550nm处测其吸光度。
游离脂肪酸的检测中细胞转染、收集和裂解方法同用上述甘油三酯检测方法相同，待细胞转染siRNA48h后，收集细胞培养液和细胞沉淀，按照细胞游离脂肪酸检测试剂盒操作说明检测其含量变化。
油红O染色发现，干扰CIDEa基因后，细胞中脂滴积聚减少；同时，细胞中甘油三酯含量显著降低(P<0.05)，见图3；但干扰CIDEa基因后，细胞培养液和细胞沉淀中游离脂肪酸含量显著增加(P<0.05)见图4。表明CIDEa基因能够促进甘油三酯合成。
以上所述仅为本发明较佳的实施例，对于本领域的技术人员来说，本发明可以有各种更改和变化。凡在本发明的精神和原则之内，所作的任何修改、等同替换、改进等，均应包含在本发明的保护范围之内。
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<120> 一种靶向奶山羊CIDEa基因的小干扰RNA及其应用
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Claims (5)
1.一种靶向奶山羊CIDEa基因的小干扰RNA，其特征在于，所述RNA的正向及反向序列分别为：
F：5’-GCAUCGCGAAAGUCACCUUTT-3’，
R：5’-AAGGUGACUUUCGCGAUGCTT-3’。
2.如权利要求1所述的小干扰RNA在调控奶山羊乳腺上皮细胞脂质合成中的应用。
3.如权利要求2所述的应用，其特征在于，所述脂质包括甘油三酯、游离脂肪酸和脂滴。
4.如权利要求1所述的小干扰RNA在调控奶山羊乳腺上皮细胞脂质代谢相关基因表达中的应用。
5.如权利要求4所述的应用，其特征在于，所述基因包括ACACA、FASN、ATGL、HSL、CD36、FABP3、SREBP1、PPARG、INSIG1、DGAT1和DGAT2。
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