CN103724415A - 斜带石斑鱼性别调控基因Rspo1及其制备方法和应用 - Google Patents
斜带石斑鱼性别调控基因Rspo1及其制备方法和应用 Download PDFInfo
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Abstract
本发明公开了斜带石斑鱼性别调控基因Rspo1及其应用。基因Rspo1的cDNA序列如SEQIDNO:1所示，全长756bp，其编码蛋白共251个氨基酸，序列如SEQIDNO:2所示。本发明方法利用反转录PCR技术从斜带石斑鱼中克隆到了一个斜带石斑鱼性别调控基因Rspo1，并根据实验结果说明了Rspo1可能参与调控斜带石斑鱼其他性别调控基因的表达，为相关鱼类性别调控的研究提供一定的理论依据和重要线索。基因Rspo1编码的蛋白有望应用于鱼类催产、催熟、人工调控雌雄转换、大规模工业化养殖等方面。
Description
技术领域
本发明属于水生动物基因工程技术领域，涉及一种斜带石斑鱼性别调控基因及其应用，特别涉及斜带石斑鱼性别调控基因Rspo1及其应用。
背景技术
Rspo1～4超家族，作为经典Wnt信号的配体，它们之间有着40%～60%的蛋白一致性和结构同源性。Rspo蛋白包含一个N端信号肽，两段临近的富含半胱氨酸的区域，一段高同源的TSP-1模块，以及一段包含双向核定位信号的碳端尾巴。 他们在各种发育和癌症发生通路中其重要作用。Rspo1的结构具有5`端信号肽，具有转移分泌蛋白的作用；两段含有半胱氨酸的弗林样片段，这是它与其受体结合的重要部位；还有一段TSP-1（凝血酶敏感蛋白-1 ）。在斜带石斑鱼的Rspo1中，未发现C端核定位信号。Rspo1是该超家族中的一种，它的最初发现是在人类性别调控方面发挥的重要作用。传统观点认为雌性性别决定和分化是被动的，但目前对RSPO1的研究却对上述提出了质疑。
RSPO1 是一种小分泌因子，能够刺激经典的Wnt/b-连环素信号通路，提高细胞膜上的Wnt与Rspo1共受体LRP6的水平。重要的是，Wnt/b-连环素通路在卵巢和睾丸早期发育中起到重要作用。雌性小鼠敲除Rspo1很明显发生性逆转，有些生育能力降低。与Wnt4敲除不同，Rspo1敲除的小鼠部分是可育的，卵泡在一定程度上发育，但是大多数的雌性是不育的。敲除XX中RPSO1的小鼠发生明显的性别转化，局部上皮的Rspo1信号对于乳腺的发育是必须的。RSPO1是卵巢性别调控基因，它积极地掌管卵巢的分化，而WNT4是RSPO1的即时的或者是协同的下游基因，它和RSPO1重叠表达，直接或者间接地受RSPO1调节。我们推测，Rspo1在鱼类中也可能存在相同性别调控的功能。
与高等的脊椎动物相比，大多数低等鱼类没有性染色体，其性别决定主要由多种性别调控基因所控制，至今依然没有找到鱼类的性别决定基因。鱼类性别调控的研究，能够使得人为控制鱼类性别的分化和成熟，提早或者延迟鱼类的性成熟，甚至可以人为调控鱼类的性别转化，得到转化后的、具有功能性的雌鱼或雄鱼。此类研究的成果，将对人工养殖的工业化、现代化产生深远影响。
石斑鱼是一种深海性的名贵经济鱼种，营养丰富，肉质细嫩洁白，类似鸡肉，素有“海鸡肉”之称，成为港澳台等地区的美味佳肴。在国际自然保护联盟的“濒危物种红色名录”上，163种石斑鱼类，有20种面临灭绝，另有5种属濒危水平。斜带石斑鱼属鲈形目，属于雌雄同体、雌性先熟的物种，群体生活，出生的时候都是雌性，成年后才会转为雄性。斜带石斑鱼的这些特征使其成为研究性别调控的重要材料。因此，研究斜带石斑鱼及其性别调控机制，对于斜带石斑鱼功能基因组学及斜带石斑鱼育种、人工调控雌雄转换、大规模工业化养殖的实现等等都具有重要的应用价值。
发明内容
本发明的目的在于提供一种斜带石斑鱼性别调控基因Rspo1及其应用。
本发明所采取的技术方案是：
斜带石斑鱼性别调控基因Rspo1的编码蛋白，其氨基酸序列如SEQ ID NO:1所示。
编码斜带石斑鱼性别调控基因Rspo1蛋白的核酸序列。
进一步的，上述核酸序列为基因Rspo1的cDNA，全长756bp，其序列如SEQ ID NO:2所示。
斜带石斑鱼性别调控基因Rspo1的重组蛋白含有如SEQ ID NO:3所示的氨基酸序列。
编码斜带石斑鱼性别调控基因Rspo1的重组蛋白的核酸序列。
进一步的，上述核酸序列含有如SEQ ID NO:4所示的核酸序列。
Rspo1的编码蛋白或重组蛋白在制备鱼类性别催熟剂中的应用。
Rspo1的编码蛋白或重组蛋白在制备鱼类性别转换剂中的应用。
本发明的有益效果是：
本发明方法利用反转录PCR 技术从斜带石斑鱼中克隆到了性别调控基因Rspo1，并构建了Rspo1的原核表达载体，可大量表达获得斜带石斑鱼性别调控基因Rspo1所编码的蛋白。
在本发明中，斜带石斑鱼Rspo1蛋白能够在卵巢组织的大量表达，并说明了Rspo1参与调控斜带石斑鱼中其他性别调控基因的表达，丰富了鱼类性别调控的系统信号通路理论，能为相关鱼类性别调控的研究提供一定的理论依据。
在本发明中，斜带石斑鱼Rspo1蛋白在鱼类催产、催熟、人工调控雌雄转换、大规模工业化养殖等方面具有重要的应用价值。
附图说明
图1为基因Rspo1的PCR扩增图；
图2为重组质粒pQE30-Rspo1转化大肠杆菌M15的菌液PCR验证图；
图3为Rspo1重组蛋白的Western检测图，其中1表示诱导后总菌体，2表示诱导前总菌体，3表示诱导后上清，4表示诱导前上清，5表示诱导后沉淀，6表示诱导前沉淀；
图4为Rspo1重组蛋白纯化后的SDS-PAGE检测图，从左到右箭头所示的泳道分别为50mM、100mM 150mM、200mM、250mM、300mM咪唑洗脱液的洗脱情况。黑色框内的条带为目的重组蛋白，分子量约27kDa；
图5 为Rspo1重组蛋白的SDS-PAGE检测，箭头所示为目的重组蛋白条带；
图6为 Rspo1重组蛋白对相关性别调控基因在mRNA转录水平上的表达调节；A表示Rspo1重组蛋白对Cyp19a表达的影响；B表示Rspo1重组蛋白对Sox9表达的影响；图中T表示用Rspo1重组蛋白处理的实验组，Treat的缩写；C表示不加Rspo1重组蛋白的对照组，Control的缩写。
具体实施方式
下面结合附图和具体实施例进一步详细说明本发明。除非特别说明，本发明采用的试剂、设备和方法为本技术领域常规市购的试剂、设备和常规使用的方法。
实施例1
一、Rspo1重组蛋白的制备
1、斜带石斑鱼RNA 的提取
取健康斜带石斑鱼全鱼，以冰浴麻醉约2 min 后，杀鱼取样，分离卵巢组织，采用Trizol 试剂法抽提获得斜带石斑鱼卵巢组织总RNA。
2、Rspo1基因的克隆
第一链cDNA 的合成：
1) 取出保存的斜带石斑鱼RNA 1μL，加入1μL 的Oligo (dT) 16 (10 mM)，混匀后置于70 ℃中水浴5 min；
2) 冰上放置5 min 后，于冰上向EP 管中先后加入dNTP Mixture (10 mM) 2μL 、5XRT Buffer 4μL、Rnase 抑制剂1μL (10 U/μL)、Rnase-free ddH2O 8μL 、ReverTra Ace 1μL；
3) 将EP 管置于PCR 仪中，按30℃ 10 min，42℃ 60 min，99℃ 5 min，4℃ 5 min 程序后瞬时离心，反转录得到第一链的单链cDNA ，于-20℃冰箱保存备用。
以cDNA 为模板的Rspo1基因克隆：
根据生物信息学中相关的序列比对和结构分析结果，在基因Rspo1的cDNA序列（如SEQ ID NO:2所示，其编码的蛋白质序列如SEQ ID NO:1所示）中去除编码19个氨基酸的信号肽的序列，以剩下的序列在两端设计特异性引物，如下：
Rspo1-RT-F :5' - CGCGGATCCGATGTTCTCAAGCTCTC -3' (SEQ ID NO:5)（下划线标记部分为内切酶Bam H1的识别序列）；
Rspo1-RT-R :5' - CGGGGTACCTTAGCTGACAGAGCTGG -3' (SEQ ID NO:6)（下划线标记部分为内切酶Kpn 1的识别序列）。
以第一链cDNA为模板，采用上游引物Rspo1-RT-F和Rspo1-RT-R进行PCR 扩增，获得不含信息肽的Rspo1基因，扩增片段大小为699bp，如图1所示，扩增序列为如SEQ ID NO:4所示，其编码的蛋白质序列如SEQ ID NO:3所示。电泳分离DNA 片段，切胶回收目的产物。将纯化后的目的产物连接至pTZ57R/T 载体，转化大肠杆菌DH5α，挑选阳性克隆测序，测序结果经BLAST 比对分析，比对结果表明目的产物确为基因Rspo1。
3、基因Rspo1原核表达载体的构建
提取含有基因Rspo1的pTZ57R/T 质粒，分别经过内切酶BamH1和Kpn1的酶切，获得目的片段，将目的片段连接到经BamH1和Kpn1双酶切过的pQE30质粒载体，pQE30为带有His标签的原核表达载体。连接过程中采有连接酶T4 DNA Ligase，温度37℃，连接反应12～16h。
将上述连接产物转化进入扩增宿主-大肠杆菌DH5α的感受态细胞，筛选培养，将获得的阳性单克隆菌落进行菌液PCR验证，PCR扩增的片段大小与目的片段大小相似，约699bp， 并将该阳性单菌落进行测序验证，测序结果与目片段的序列一致，说明成功构建了Rspo1的原核重组表达载体：pQE30-Rspo1。
4、基因Rspo1的原核表达及纯化
转化表达宿主：
提取3中构建好的重组表达载体质粒（pQE30-Rspo1），将质粒转化进入表达宿主-大肠杆菌M15的感受态细胞，用含氨苄霉素（Amp）和卡那霉素（Kana）的培养基，进行筛选培养，挑取阳性单克隆菌落，进行菌液PCR验证（如图2所示），PCR扩增的片段大小与目的片段大小相似，约699bp。验证正确后，以含Amp的培养基过夜扩大培养该阳性菌落，取菌液加入甘油，按8%甘油终浓度保存在-80度冰箱中，进行菌种保存。
重组蛋白的诱导表达：
将含有重组表达载体（pQE30-Rspo1）的大肠杆菌M15先经过LB培养基的扩大培养，再经过2xYT培养基的二级扩大培养，当菌液OD600值介于0.4～0.6之间时，取少量诱导前的菌液，作为后续实验的对照。剩下的菌液加入IPTG进行诱导培养，诱导培养后收集菌液，离心获得菌体，加入1xNi-NTA结合缓冲液重悬菌体，此时取20μL菌体重悬液，作为后续实验对照。将剩下的菌体重悬液超声波破碎，离心，将上清和沉淀分开。
分别将诱导前和诱导后的总菌体、上清和沉淀加入蛋白Loading buffer，混匀，沸水浴中煮5分钟，分别取10μL进行Western blotting检测，检测结果如图3所示。从图3的western blotting检测结果中，可看出Rspo1重组蛋白在菌体的上清和沉淀中都存在。
上述2xYT培养基配方为：胰蛋白胨（Tryptone）16g，酵母提取物（Yeast Extract）10g，氯化钠5g，加水至1L，120℃高压灭菌20分钟。
重组蛋白的纯化、脱盐及冻干：
将诱导表达后的菌体超声破碎，离心，弃沉淀，将上清经孔径0.45微米的滤膜过滤备用。
镍柱准备，往柱子内加入2mL的Ni-NTA His-Bind（Novagen，货号：70666），再加10mL水过柱；再加入10mL的1xNi-NTA结合缓冲液平衡柱子。
挂柱，往50mL离心管中加入结合缓冲液，再加入菌体上清液，盖上盖子，封口膜封口，置于冰上，缓慢摇匀、亲和吸附1小时。将摇匀的融合液过镍柱，收集穿过液，再重复过镍柱两次。
漂洗和洗脱，重组蛋白挂柱后，先用1xNi-NTA漂洗液漂洗，再分别以50mM、100mM 150mM、200mM、250mM浓度的咪唑洗脱液洗脱，对不同浓度咪唑洗脱液洗脱下来的物质进行SDS-PAGE检测，检测结果显示重组蛋白能被浓度为150～200mM的咪唑洗脱下来，其中咪唑浓度为200mM时，洗脱的条带单一（如图4所示），故选用200mM的咪唑洗脱液用于洗脱获得纯化重组蛋白。
基于上述探索出的最优的诱导培养条件、最适浓度的咪唑洗脱液等，再进行大量表达、纯后获得大量重组蛋白，并结合SDS-PAGE 进行检测，检测结果如图5所示。Rspo1重组蛋白的分子量约为27kDa，如图5中的箭头所示。
将纯化后的重组蛋白进行脱盐，先用0.02M的PB缓冲液进行清洗和平衡柱子。加入样品，收集穿过液。待样品完全进入下端柱子后，再加入PB缓冲液洗脱，收集洗脱液，即为脱盐后的重组蛋白。
将脱盐的重组蛋白进行冻存：将含有目的蛋白的收集液置于合适大小的干净管子中，用封口膜封好，在膜上扎几个针眼，以便冻存时水分的散失。管子竖直放于-20度冰箱约2小时以上，待全部冻成冰状，迅速放于-80度冰箱。第二天，将-80度保存的管子置于调试完好的冻存机挂瓶内，开始冻存。冻存时间视水分散失快慢而定，一般8小时以上或者过夜。将冻存完成的、含有目的蛋白的管子取下迅速放于-80度冰箱保存，以便下一步的实验。
二、Rspo1重组蛋白调节性别调控基因的表达
以斜带石斑鱼为研究对象，以高表达基因Rspo1的卵巢组织为材料，用Rspo1重组蛋白离体孵育卵巢碎片，研究Rspo1重组蛋白对相关性别调控基因Cyp19a、Foxl2、Sox9、Wnt4a在mRNA转录水平表达的调节情况。
分别以浓度100、1000、10000μg/μL的Rspo1重组蛋白孵育卵巢碎片8h，然后分别检测性别调控基因Cyp19a 、Foxl2、Sox9、Wnt4a在mRNA转录水平上的表达情况。检测结果如图6所示。
从图6的检测结果可获知，Rspo1重组蛋白对其它几种性别调控因子起不同作用。其中，三种不同浓度（100、1000、10000ng/ml）Rspo1对Cyp19a都有着上调作用；对Foxl2在1000ng/ml浓度时起着明显下调作用；对Sox9在1000和10000ng/ml时有着下调作用，在1000ng/ml时作用最明显；而Rspo1对Wnt4a的作用却不明显。可见，Rspo1重组蛋白能够促进雌性调节基因（Cyp19a），而抑制雄性调节基因（Sox9）的表达，从而可能促进雌性的发生、发育和成熟。说明，Rspo1蛋白可能具有上调雌性激素合成的作用，从而引起雌性性别发生和卵巢发育，同时抑制雄性特征，进而可能引发鱼类从雄性到雌性的性逆转。
<110> 中山大学
<120> 斜带石斑鱼性别调控基因Rspo1及其制备方法和应用
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Claims (8)
1.斜带石斑鱼性别调控基因Rspo1的编码蛋白，其氨基酸序列如SEQ ID NO:1所示。
2.编码权利要求1所述的斜带石斑鱼性别调控基因Rspo1蛋白的核酸序列。
3.根据权利2所述的核酸序列，其特征在于：该核酸序列为基因Rspo1的cDNA，全长756bp，其序列如SEQ ID NO:2所示。
4.斜带石斑鱼性别调控基因Rspo1的重组蛋白，其特征在于：该重组蛋白含有如SEQ ID NO:3所示的氨基酸序列。
5.编码权利要求4所述的斜带石斑鱼性别调控基因Rspo1的重组蛋白的核酸序列。
6.根据权利5所述的核酸序列，其特征在于：其含有如SEQ ID NO:4所示的核酸序列。
7.Rspo1的编码蛋白或重组蛋白在制备鱼类性别催熟剂中的应用。
8.Rspo1的编码蛋白或重组蛋白在制备鱼类性别转换剂中的应用。
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| CN108676801A (zh) * | 2018-04-28 | 2018-10-19 | 中山大学 | 一种斜带石斑鱼的卵母细胞标记基因slbp2的cDNA及其应用 |
| CN108997491A (zh) * | 2018-06-26 | 2018-12-14 | 中山大学 | 斜带石斑鱼天然免疫受体tlr5s基因及其编码蛋白的新用途 |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108676801A (zh) * | 2018-04-28 | 2018-10-19 | 中山大学 | 一种斜带石斑鱼的卵母细胞标记基因slbp2的cDNA及其应用 |
| CN108676801B (zh) * | 2018-04-28 | 2023-06-13 | 中山大学 | 一种斜带石斑鱼的卵母细胞标记基因slbp2的cDNA及其应用 |
| CN108997491A (zh) * | 2018-06-26 | 2018-12-14 | 中山大学 | 斜带石斑鱼天然免疫受体tlr5s基因及其编码蛋白的新用途 |
| CN111172189A (zh) * | 2018-11-13 | 2020-05-19 | 上海海洋大学 | 一种模拟人精卵巢综合征的Sissy青鳉模型的建立方法 |
| CN111172189B (zh) * | 2018-11-13 | 2023-06-13 | 上海海洋大学 | 一种模拟人精卵巢综合征的Sissy青鳉模型的建立方法 |
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