CN108586597A - 鱼类促摄食成熟肽及其应用 - Google Patents
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Abstract
本发明公开了一种鱼类促摄食成熟肽及其应用，涉及基因工程领域，该鱼类促摄食成熟肽及其应用发明鱼类能量动态平衡相关基因编码基因在鱼类的神经系统高表达，并通过分子调控机制研究及生理实验证实该基因产生的成熟肽adropin具有调节鱼类摄食的功能，能够用于调节鱼类摄食以及用于制备促进鱼类摄食的饲料。
Description
技术领域
本发明涉及基因工程领域，具体来说，本发明涉及一种鱼类促摄食成熟肽及其应用。
背景技术
鱼类的摄食和能量代谢调节主要受下丘脑-靶腺轴的控制，其中下丘脑-肝脏轴能够感应机体的营养状况，并做出适当的食欲调节行为和代谢反应，以维持机体的能量稳态，而能量自我平衡机制的损坏将会导致食欲和能量代谢异常。近年来鱼类内分泌代谢调控机制的研究表明，神经肽是参与调控摄食和能量代谢的一类重要物质。因此，研究鱼类神经肽调控摄食和能量代谢的作用机制将为鱼类养殖业持续健康发展提供技术支持。
Adropin是由能量动态平衡相关基因编码的神经肽，主要表达于脑部、肝脏、肌肉、肾脏、心肌、胰腺等组织中，提示adropin可能具有广泛的生理作用。近期研究表明adropin在哺乳动物中参与了摄食行为的调控。腹腔注射adropin可使饮食诱导肥胖型(DIO)小鼠的摄食量降低和体重减轻，但脑室注射adropin对大鼠摄食无明显影响；然而，敲除adropin基因的小鼠则表现出食欲减退。由此可见，Adropin在哺乳动物中参与调控摄食的研究结论并不一致，具体调控机制亦未阐明。通过搜索EST数据库与鱼类的基因组数据库，并进行基因共线性分析，首次证实了鱼类能量动态平衡相关基因的存在，并证实了鱼类能量动态平衡相关基因编码的成熟肽adropin能增加罗非鱼的饵料摄入量，显示出了较强的促摄食功能，从而为开发一种新型诱食剂奠定了基础。现有诱食剂活性低，用量大，生产成本较高并且有一定的毒副作用。
发明内容
本发明所要解决的技术问题是提供一种鱼类促摄食成熟肽及其应用，基于罗非鱼的理论研究，鉴定了鱼类adripin神经肽基因编码的成熟肽adropin具有调节鱼类摄食的功能，并且可以和摄食调控关键因子orexin协同调控摄食。这为开发出高活性、无毒和成本低廉的诱食剂，作为饲料添加剂，促进养殖鱼类等的发展具有广泛的应用价值。
为实现上述目的，本发明提供以下的技术方案：
该鱼类促摄食成熟肽及其应用包括如下步骤：
(1)通过分析人、鸡及鱼类基因组和EST数据库，在鱼类中发现adropin基因的存在，如图1所示。根据这些序列设计引物，结合分子克隆技术，在罗非鱼中克隆得到adropincDNA序列480bp，其中开放阅读框(ORF)序列333bp，编码的adropin蛋白前体为110个氨基酸。蛋白前体等电点为7.67，分子量为12.72千道尔顿，其中N-端信号肽由22个氨基酸组成；
(2)采用实时荧光定量PCR检测罗非鱼adropin基因在各种组织中的表达，结果显示在脑、肝脏、脾脏、卵巢、垂体和肌肉中都有较高的表达；另外在心脏和肠的表达量较弱；在肾脏和鳃中检测不到表达信号。从中显示出adropin具有明显的组织表达特异性，如图2所示。Adropin基因在脑和垂体中的高表达预示着adropin在上游神经中枢系统中的调控作用；
(3)用实时荧光定量PCR检测罗非鱼基因在进食状态下的变化，结果表明：在进食1小时后，下丘脑中adropin基因的表达量显著高于对照组(P＜0.05)，表明adropin与摄食调控相关，如图3所示；
(4)鱼类adropin神经肽基因可以用于鱼类摄食调控。采用腹腔注射化学合成的adropin成熟肽，实际应用表明，adropin能够显著性增加罗非鱼的摄食量，如图4所示，并且可以和orexin一起叠加刺激摄食。因此，该鱼类adropin神经肽基因编码的成熟肽adropin可以用于诱导鱼类摄食。
采用以上技术方案的有益效果是：该鱼类促摄食成熟肽及其应用可以让鱼类adropin神经肽基因在鱼类下丘脑中大量表达，该基因产生的成熟肽具有调节鱼类摄食的功能，可以通过化学合成的方法进行开发利用，使用方便，效果显著，具有广泛的应用价值。
附图说明
下面结合附图对本发明的具体实施方式作进一步详细的描述。
图1是本发明鱼类adropin神经肽的序列图；
图2是本发明鱼类adropin神经肽基因在各个组织的表达分析图；
图3是本发明鱼类adropin神经肽基因在进食过程的表达变化图；
图4是本发明鱼类adropin神经肽腹腔注射对罗非鱼摄食的影响图。
具体实施方式
下面结合附图详细说明本发明鱼类促摄食成熟肽及其应用的优选实施方式。
图1至图4出示本发明鱼类促摄食成熟肽及其应用的具体实施方式：
实施例1：
1.罗非鱼脑总RNA的提取
取健康罗非鱼，解剖后立即分离出脑组织。采用Trizol试剂法提取获得罗非鱼脑总RNA，其OD260/280＝1.9，电泳结果显示，28S rRNA，18S rRNA条带清晰，28S条带亮度为18S的两倍，表明所得总RNA未被蛋白质、酚和基因组DNA污染，纯度高。
2.cDNA第一链的合成
取1μg罗非鱼鱼脑总RNA样品进行DNA酶处理以去除基因组DNA的污染，应用iScriptTM反转录试剂盒进行反转录，所得产物置于-20℃保存备用。
3.引物设计
通过分析罗非鱼基因组，我们在罗非鱼adropin基因保守区设计引物，扩增片段大小约为300bp。
引物序列为：F：5_-CTCAC (T/C) TCCAT (T/C) GC (A/C) GGAGG_3_
R：5_-GCTTTCG(A/C)TTTGG(C/G)AGGAAG-3。
4.罗非鱼adropin基因cDNA全序列的克隆
以合成的第一链cDNA作为模板，以引物进行PCR扩增，所得PCR产物上样至1.5％琼脂糖凝胶，以低电压电泳分离DNA片段，从凝胶中纯化回收目的产物。将纯化后的目的产物连接至载体，转化DH5 α大肠杆菌，挑选阳性克隆测序。根据已得到的cDNA片段序列设计特异性引物，利用cDNA末端快速扩增技术(Rapid Amplification of cDNAends，RACE)对目的基因的3’-和5’-末端进行PCR扩增。按照RACE试剂盒GeneRacerTM Kit说明书对罗非鱼脑总RNA进行去磷酸，去mRNA 5’帽子结构，与RNA Oligo连接，最终通过反转录合成cDNA第一链。用罗非鱼adropin cDNA的3’-和5’-RACE特异引物(AD-F2，AD-F3和AD-R2，AD-R3)和GeneRacerTM Kit的通用引物，以GeneRacerTM Kit反转录合成的第一链cDNA为第一轮PCR模板，以稀释的第一轮PCR产物为第二轮PCR模板，扩增罗非鱼adropin cDNA3’端和5’端片段，对所得到的PCR产物的回收纯化、连接T载体、转化大肠杆菌、阳性克隆鉴定及DNA测序。
特异引物序列如SEQ ID NO：6-7所示。
实施例2：
罗非鱼adropin的组织表达分布。
采用Real-time PCR检测罗非鱼adropin在各种组织中的表达，结果显示adropin在下丘脑、视顶盖-丘脑及精巢中表达最高，在端脑、小脑、延脑、肾脏、肝脏、垂体和心脏等组织也有表达，而在卵巢和脂肪组织中没有表达，如图2所示。
实施例3：
罗非鱼adropin在饥饿过程的表达变化。
将体重30±5g的罗非鱼随机分配到四个玻璃缸中(分别为对照组和投喂组)，平均每个缸8条罗非鱼。罗非鱼每天定时(上午10点；下午4点左右)投喂两次，食物投喂量约为体重的3％。驯养两周后，实验正式开始。对照组的罗非鱼停止投喂，实验组的罗非鱼在实验开始后正常投喂。在进食1，2和3小时后，分别将实验组和对照组的罗非鱼用MS-222麻醉，经处死收集下丘脑样品，液氮速冻处理，存放于-80℃超低温冰箱备用。通过荧光定量PCR检测罗非鱼下丘脑中adropin基因在对照组和进食组的表达量变化，结果表明：下丘脑adropinmRNA表达水平在进食1小时后被显著抑制，如图3所示。
实施例4：
采用腹腔注射方式研究化学合成的罗非鱼adropin对罗非鱼摄食的影响。
将体重30±5g的罗非鱼随机分配到六个玻璃缸中，平均每个缸8条罗非鱼。罗非鱼每天定时(上午10点；下午4点左右)投喂两次，食物投喂量约为体重的3％。驯养两周后开始正式实验。罗非鱼用MS-222麻醉后，称量每组中个体体重(body weight，BW)，根据体重分别由腹腔注射0.7％鱼类生理盐水(对照组)、50ng/gBW、100ng/gBW、250ng/gBW和500ng/gBWadropin，另外单独注射orexin(100ng/gBW)、共同注射adropin(500ng/gBW)与Orexin(100ng/gBW)。注射15分钟后，分别向每组罗非鱼投喂固定重量的食物，让每组罗非鱼自由摄食2小时。同时将等量的食物放入水中，以计算食物在水中2小时中的重量变化。将各组剩余的食物分别收集并烘干，根据剩余的食物的重量计算各组中单位体重罗非鱼的摄食量，如图4所示。
以上的仅是本发明的优选实施方式，应当指出，对于本领域的普通技术人员来说，在不脱离本发明创造构思的前提下，还可以做出若干变形和改进，这些都属于本发明的保护范围。
Claims (1)
1.一种鱼类促摄食成熟肽及其应用，其特征在于：所述鱼类促摄食成熟肽及其应用包括如下步骤：
(1)通过分析人、鸡及鱼类基因组和EST数据库，在鱼类中发现adropin基因的存在，根据这些序列设计引物，结合分子克隆技术，在罗非鱼中克隆得到adropin cDNA序列480bp，其中开放阅读框(ORF)序列333bp，编码的adropin蛋白前体为110个氨基酸，蛋白前体等电点为7.67，分子量为12.72千道尔顿，其中N-端信号肽由22个氨基酸组成；
(2)采用实时荧光定量PCR检测罗非鱼adropin基因在各种组织中的表达，结果显示在脑、肝脏、脾脏、卵巢、垂体和肌肉中都有较高的表达；另外在心脏和肠的表达量较弱；在肾脏和鳃中检测不到表达信号，从中显示出adropin具有明显的组织表达特异性，Adropin基因在脑和垂体中的高表达预示着adropin在上游神经中枢系统中的调控作用；
(3)用实时荧光定量PCR检测罗非鱼基因在进食状态下的变化，结果表明：在进食1小时后，下丘脑中adropin基因的表达量显著高于对照组(P＜0.05)，表明adropin与摄食调控相关；
(4)鱼类adropin神经肽基因可以用于鱼类摄食调控，采用腹腔注射化学合成的adropin成熟肽，实际应用表明，adropin能够显著性增加罗非鱼的摄食量，并且可以和orexin一起叠加刺激摄食，鱼类adropin神经肽基因编码的成熟肽adropin可以用于诱导鱼类摄食。
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