CN110305966B - 一种罗非鱼肝脏dgat2基因表达的调控方法 - Google Patents
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Abstract
本发明公开了一种罗非鱼肝脏DGAT2基因表达的调控方法，属于生物化学与分子生物学领域。本发明的方法包括：(1)注射雌二醇；(2)RNA逆转录；(3)实时定量PCR扩增。本发明以DGAT2‑F：5’‑CGTTTCACCACCTTCGTCATC‑3’，DGAT2‑R：5’‑CAGCTGGTAAAGGCCACCAT‑3’为特异性引物，采用β‑actin作为内参，进行实时定量PCR扩增反应，对罗非鱼DGAT2基因表达结果进行分析。本发明的方法操作简便，适合快速进行大量样本的操作，而且检测试剂价格低廉、易获得，不仅对今后有关罗非鱼脂质代谢研究提供依据，而且为人为调控罗非鱼脂代谢提供了新方法。
Description
技术领域
本发明属于生物化学与分子生物学领域，具体涉及一种罗非鱼肝脏DGAT2基因表达的调控方法。
背景技术
脂代谢参与多种鱼类生物学过程，如生长、生殖、抗病抗逆等。多项研究表明脂代谢与鱼类生长有关，例如，饲喂高脂的饲料可导致脂累积并影响尼罗罗非鱼的生长速度。脂进入体内后通过内源和外源两个途径进行运输，肝脏是脂代谢的关键部位，它是内源和外源两个途径的关键作用部位。最近的研究表明，脂代谢受多种因子调控，例如水体中的镉离子浓度可促进欧洲鳗鲡的脂吸收能力，除了环境因子和营养因素以外，新的研究也表明激素对脂代谢有普遍的调控作用。
类固醇类激素已经被证明可以调控鱼类的性腺发育以及控制能量代谢、渗透调节、体色、行为、学习和营养吸收等活动。在哺乳动物中，脂代谢被类固醇类激素调控已经得到了广泛研究。在人类中，雌二醇被认为是肥胖和脂代谢的调控因子，雌二醇还可以调节甘油三酯和脂蛋白a的水平，这些线索均表明雌二醇的处理对治疗心脏病和肥胖有意义。此外，雌二醇也可调节哺乳动物的食物摄入与能量平衡。而相对于哺乳类，鱼类类固醇类激素调节脂代谢的研究还较为缺乏。目前关于脂代谢的研究主要集中于饲料中的营养因子对其的调控作用，而雌二醇等激素的调控方法研究还相对缺乏。
二脂酰甘油酰基转移酶2(DGAT2)是内质网细胞微粒体酶，它催化甘油三酯生成，是甘油三酯生成过程中的最终限速酶。DGAT2与多种疾病以及生长发育相关，例如敲除DGAT2基因的小鼠会在出生不久后由于缺乏TG而死亡。在人体中，DGAT2高表达与高甘油三酯水平有关，可以引起肥胖症、高血糖、冠心病等脂代谢异常疾病。DAGT2可作为这些代谢疾病的治疗和诊断靶标之一。由于DGAT2在甘油三酯合成中的重要作用，近年来在水产动物中被广泛研究。
雌二醇是一种类固醇类性激素，有α和β两种构型，其中17β-雌二醇在动物体内活性最高。雌二醇在动物食物摄入和能量稳态调节等方面都具有重要意义。在哺乳动物中，雌二醇主要由性腺分泌且被认为与脂代谢有密切关系，例如它可以调节切除卵巢大鼠的甘油三酯水平以及绝经后女性脂代谢紊乱的状况。此外，雌二醇不单单存在于体内，也存在于环境中。环境中的雌二醇大多为脂溶性，可通过摄食作用最终富集到人体中，而这种雌二醇难以降解并且不易排出，并可与自身分泌的雌二醇竞争性地与雌二醇受体结合，最终导致细胞发生功能性改变。雌二醇不仅对哺乳动物产生影响，而且可作用于鱼类活动。它具有参与鱼体内能量代谢、氧化应激、异生素代谢和脂代谢等作用。有报道称用雌激素处理斑马鱼后，斑马鱼肝脏甘油三酯含量和脂肪酸合成酶基因的明显上调。这些结果都表明雌二醇可以调节脂代谢水平。
罗非鱼是联合国粮农组织向全世界推广养殖的优良品种之一，具有生长速度快，繁殖能力强，耐低氧等优良性状。罗非鱼现已成为我国主要淡水养殖鱼类之一，近年来我国罗非鱼产业发展较为迅速，已成为全球第一大罗非鱼养殖国。目前，我国罗非鱼产业主要集中于广东，广西和海南三省，并且以出口为主，内销为辅。然而近年来由于养殖条件改善，养殖罗非鱼出现了脂质过剩现象，引起罗非鱼代谢紊乱，出现高脂肪、高胆固醇等问题。因此，研究并调节罗非鱼脂代谢水平，对提高养殖鱼品质显得尤为重要。通过调节罗非鱼DGAT2基因从而控制罗非鱼脂代谢紊乱现象，不仅对今后有关罗非鱼脂质代谢研究提供依据，而且为罗非鱼人为调控脂代谢提供了新方法。
现有的调节DGAT2的手段包括寡核苷酸链干扰和天然产物调节DGAT2表达，这两种方法有以下缺点：1.寡核苷酸链干扰成本高，该方法依赖于合成的寡核苷酸链，试剂价格成本高；2.天然产物需要通过化学方法提纯，操作繁琐；3.无论是寡核苷酸链干扰还是天然产物调节，目前大部分应用于细胞实验，用于鱼类活体特别是养殖罗非鱼的方案还未有报道。
发明内容
针对现有技术存在的问题，本发明提供一种罗非鱼肝脏DGAT2基因表达的调控方法，可以快速调节罗非鱼DGAT2基因，而且操作简便，成本低。
本发明通过以下技术方案来实现：
一种检测罗非鱼肝脏DGAT2基因表达的引物，该引物为：
DGAT2-F：5’-CGTTTCACCACCTTCGTCATC-3’；
DGAT2-R：5’-CAGCTGGTAAAGGCCACCAT-3’。
一种利用上述引物进行的罗非鱼肝脏DGAT2基因表达的调控方法，包括如下步骤：
(1)注射雌二醇溶液：取健康罗非鱼，注射雌二醇溶液，获得待检罗非鱼；
(2)RNA逆转录：取出待检罗非鱼肝脏，提取肝脏的RNA，将提取的RNA逆转录为模板cDNA；
(3)实时定量PCR扩增：以步骤(2)得到的cDNA为模板，以DGAT2-F，DGAT2-R为特异性引物，采用β-actin作为内参，进行PCR扩增反应，对DGAT2基因表达结果进行分析；
所述DGAT2-F：5’-CGTTTCACCACCTTCGTCATC-3’；
所述DGAT2-R：5’-CAGCTGGTAAAGGCCACCAT-3’。
作为技术方案的优选，采用β-actin作为内参的β-actin-正向引物：5’-CCACAGCCGAGAGGGAAAT-3’；β-actin-反向引物：5’-CCATCTCCTGCTCGAAGTC-3’。
作为技术方案的优选，所述PCR扩增体系包含：
作为技术方案的进一步优选，所述PCR扩增体系包含的正向引物为DGAT2-F，反向引物为DGAT2-R；或正向引物为β-actin-正向引物，反向引物为β-actin-反向引物。
作为技术方案的优选，所述PCR扩增的条件为：先95℃预变性2min；随后进行循环95℃变性5s，60℃退火30s，如此循环40次。
作为技术方案的优选，将雌二醇溶解于二甲基亚砜中得到雌二醇溶液，所述雌二醇溶液的浓度为50-60mg/kg。
作为技术方案的进一步优选，所述雌二醇溶液的注射量为1-1.5ml。
本发明的有益效果：
(1)本发明设计的罗非鱼肝脏DGAT2基因引物特异性好、灵敏度高、稳定性强。
(2)本发明通过注射雌二醇，促进罗非鱼肝脏中DGAT2基因的表达，再通过实时定量PCR扩增快速检测罗非鱼肝脏中DGAT2基因的表达，操作简便，适合快速进行大量样本的操作，而且检测试剂价格低廉、易获得，适合推广应用。
(3)本发明将雌二醇运用到罗非鱼肝脏DGAT2基因表达的调控中，结果提示罗非鱼肝脏DGAT2的表达受到雌二醇的调控作用，生殖腺分泌的雌二醇对罗非鱼肝脏DGAT2表达有促进作用，这为人为调控罗非鱼脂代谢提供了新方法，对提高养殖罗非鱼品质显得尤为重要。
附图说明
图1为本发明实施例1的雌二醇注射组和对照组罗非鱼肝脏中DGAT2表达量比较图。
具体实施方式
本发明用下列实施例进行说明，但不是对本发明的使用范围的限制。
实施例1
奥尼罗非鱼肝脏DGAT2基因的表达分析
(1)注射雌二醇溶液：取10尾2-3kg的健康奥尼罗非鱼，随机挑选5尾，每尾注射1-1.5mL浓度为50-60mg/kg的雌二醇的二甲基亚砜溶液，获得待检奥尼罗非鱼；其余5尾每尾注射1-1.5mL二甲基亚砜作为对照。
(2)RNA逆转录：分别取出待检奥尼罗非鱼肝脏5mg，立刻放入液氮冷冻，然后用研钵磨碎，用RNA Trizol试剂裂解提取肝脏总RNA。RNA浓度和纯度用分光光度计检测，并通过1.2％琼脂糖凝胶电泳检验RNA是否降解。将提取的RNA样本用PrimeScriptTM RT reagentKit 047A(TakaRa)试剂盒进行反转录，得到模板cDNA。
(3)实时定量PCR扩增：将待检cDNA样品1:40稀释，取3.4μL双蒸水，0.4μL正向引物，0.4μL反向引物，0.8μL稀释后的cDNA和5μL TB Green Premix Ex Taq II(TliRNaseHplus)(2×)(Takara,Japan)混匀，采用β-actin作为内参，置于荧光定量96孔板中，每个样品做3个平行。用Pikoreal 96 Real-Time PCR system(Thermo Fisher Scientific,USA)检测DGAT2基因的表达量，PCR扩增的条件为：先95℃预变性2min；随后进行循环95℃变性5s，60℃退火30s，如此循环40次。
其中，正向引物为DGAT2-F：5’-CGTTTCACCACCTTCGTCATC-3’；
反向引物为DGAT2-R：5’-CAGCTGGTAAAGGCCACCAT-3’；
β-actin-正向引物为：5’-CCACAGCCGAGAGGGAAAT-3’；
β-actin-反向引物为：5’-CCATCTCCTGCTCGAAGTC-3’。
(4)荧光定量数据分析
用2-ΔΔCt方法对采集数据进行分析，用SPSS16.0进行T检验分析，结果如图1显示，从图中可以看出，对照组的奥尼罗非鱼肝脏DGAT2相对表达量为1.23±0.35，雌二醇注射组的奥尼罗非鱼肝脏DGAT2相对表达量为5.61±1.16，雌二醇注射组的奥尼罗非鱼肝脏DGAT2基因表达明显高于对照组，可见注射雌二醇后奥尼罗非鱼肝脏DGAT2的表达显著升高。该结果提示罗非鱼肝脏DGAT2的表达受到雌二醇的调控作用，生殖腺分泌的雌二醇对罗非鱼肝脏DGAT2表达有促进作用。因此，可以利用雌二醇人为刺激罗非鱼肝脏DGAT2的表达。
序列表
<110> 广西壮族自治区水产科学研究院
<120> 一种罗非鱼肝脏DGAT2基因表达的调控方法
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Claims (5)
1.一种提高奥尼罗非鱼肝脏DGAT2基因表达的调控方法，其特征在于，包括如下步骤：
（1）注射雌二醇溶液：取2-3kg的健康奥尼罗非鱼，注射雌二醇溶液，所述雌二醇溶液的注射量为1-1.5ml/尾；获得待检罗非鱼；
所述雌二醇溶液是将雌二醇溶解于二甲基亚砜中得到雌二醇溶液，雌二醇溶液的浓度为50-60 mg/kg；
（2）RNA逆转录：取出待检罗非鱼肝脏，提取肝脏的RNA，将提取的RNA逆转录为模板cDNA；
（3）实时定量PCR扩增：以步骤（2）得到的cDNA为模板，以DGAT2-F，DGAT2-R为特异性引物，采用β-actin作为内参，进行PCR扩增反应，对DGAT2基因表达结果进行分析；
所述DGAT2-F：5’-CGTTTCACCACCTTCGTCATC-3’；
所述DGAT2-R：5’-CAGCTGGTAAAGGCCACCAT-3’。
2.根据权利要求1所述的提高奥尼罗非鱼肝脏DGAT2基因表达的调控方法，其特征在于，采用β-actin作为内参的β-actin-正向引物：5’-CCACAGCCGAGAGGGAAAT-3’；β-actin-反向引物：5’-CCATCTCCTGCTCGAAGTC-3’。
3.根据权利要求1所述的提高奥尼罗非鱼肝脏DGAT2基因表达的调控方法，其特征在于，所述PCR扩增体系包含：
双蒸水 3.4 μL
cDNA 0.8 μL
TB Green 5 μL
正向引物 0.4μL
反向引物 0.4μL。
4.根据权利要求1所述的提高奥尼罗非鱼肝脏DGAT2基因表达的调控方法，其特征在于，所述PCR扩增体系包含的正向引物为DGAT2-F，反向引物为DGAT2-R；或正向引物为β-actin-正向引物，反向引物为β-actin-反向引物。
5.根据权利要求1所述的提高奥尼罗非鱼肝脏DGAT2基因表达的调控方法，其特征在于，所述PCR扩增的条件为：先95℃预变性2 min；随后进行循环95℃变性5 s，60℃退火30s，如此循环40次。
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| 鱼类脂肪组织核磁共振成像研究及斑马鱼(Danio rerio)脂代谢中的性别差异;吴俊琳;《中国优秀硕士论文全文数据库》;20150916;第4章 2.2-5 * |
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