CN112251443B - 一种中华绒螯蟹雄性特异Etse基因及其应用 - Google Patents

一种中华绒螯蟹雄性特异Etse基因及其应用 Download PDF

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CN112251443B
CN112251443B CN202011171258.XA CN202011171258A CN112251443B CN 112251443 B CN112251443 B CN 112251443B CN 202011171258 A CN202011171258 A CN 202011171258A CN 112251443 B CN112251443 B CN 112251443B
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陈晓雯
王军
王成辉
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Abstract

本发明公开了一种中华绒螯蟹雄性特异Etse基因及其应用，其核苷酸序列如SEQ ID NO:1所示，编码蛋白的氨基酸序列如SEQ ID NO:2所示，雄性特异分子标记引物如SEQ ID NO:5和SEQ ID NO:6所示，上述中华绒螯蟹雄性特异Etse基因、其编码蛋白或雄性特异分子标记引物用于中华绒螯蟹性别鉴定，鉴定方法包括：采用雄性特异分子标记引物进行PCR扩增，反应结束后进行电泳检测，结果显示一条特异性条带的中华绒螯蟹为雄性，电泳结果显示没有特异性条带的中华绒螯蟹为雌性，方法简单高效，可在分子水平上快速实现中华绒螯蟹的性别鉴定。

Description

一种中华绒螯蟹雄性特异Etse基因及其应用
技术领域
本发明涉及中华绒螯蟹雄性特异Etse基因及其在性别鉴定中的应用，属于中华绒螯蟹性别鉴定技术领域。
背景技术
中华绒螯蟹(Eriocheir sinensis)，俗称河蟹，隶属于节肢动物门(Arthropoda)、甲壳亚门(Crustacean)、软甲纲(Malacostraca)、十足目(Decapoda)、方蟹科(Grapsidae)，绒螯蟹属(Eriocheir)，广泛分布于我国自北向南的各大水系中，主要包括辽河、黄河、长江、瓯江、闽江、珠江和南流江水系，是我国重要的水产养殖对象和经济蟹类之一。2019年我国中华绒螯蟹养殖的产量达77.9万吨，产值约690亿元。一般而言，中华绒螯蟹的雄性个体较雌蟹大，精巢发育成熟后的副性腺富含人类所需的必需氨基酸、不饱和脂肪酸，维生素等，为人们喜吃的白膏，雌性个体卵巢发育成熟后的红膏味道鲜美，口感与雄蟹性腺(白膏)不一致。白膏和红膏口味不同，深受广大消费者的喜爱。因此开展中华绒螯蟹性别调控研究，实现中华绒螯蟹性别控制育种及养殖具有较大的市场前景。
性别特异基因的获得是实现中华绒螯蟹性别控制育种及阐述其性别决定分子机制的重要前提。然而目前有关中华绒螯蟹的性别决定基因仍未可知，至今仍然没有明确的中华绒螯蟹性别决定机制报道，因此需要获得一种中华绒螯蟹性别特异基因的分子标记。
发明内容
本发明的第一目的是，提供中华绒螯蟹雄性特异Etse基因的核苷酸序列。
本发明的第二目的是，提供上述中华绒螯蟹雄性特异Etse基因编码蛋白的氨基酸序列。
本发明的第三目的是，提供上述中华绒螯蟹雄性特异Etse基因的扩增方法。
本发明的第四目的是，提供一种基于上述中华绒螯蟹雄性特异Etse基因设计的雄性特异分子标记引物。
本发明的第五目的是，提供上述中华绒螯蟹雄性特异Etse基因、上述中华绒螯蟹雄性特异Etse基因编码蛋白或上述雄性特异分子标记引物在中华绒螯蟹性别鉴定中的应用。
本发明的第六目的是，提供一种中华绒螯蟹性别鉴定的方法。
为实现上述第一目的，本发明采用的技术方案是：
中华绒螯蟹雄性特异Etse基因的核苷酸序列如SEQ ID NO:1所示。
为实现上述第二目的，本发明采用的技术方案是：
上述中华绒螯蟹雄性特异Etse基因编码蛋白的氨基酸序列如SEQ ID NO:2所示。
为实现上述第三目的，本发明采用的技术方案是：
上述中华绒螯蟹雄性特异Etse基因的扩增方法，包括以下步骤：
(1)提取中华绒螯蟹雄性性腺的总RNA；
(2)以步骤(1)中的总RNA为模板，以oligo dT为反转录引物，进行精巢cDNA的合成；
(3)以上述cDNA为模板，以如SEQ ID NO:3所示的序列为上游引物、如SEQ ID NO:4所示的序列为下游引物，进行PCR扩增，得到中华绒螯蟹雄性特异Etse基因。
为实现上述第四目的，本发明采用的技术方案是：
一种基于上述中华绒螯蟹雄性特异Etse基因设计的雄性特异分子标记引物，上游引物的序列如SEQ ID NO:5所示，下游引物的序列如SEQ ID NO:6所示。
为实现上述第五目的，本发明采用的技术方案是：
上述中华绒螯蟹雄性特异Etse基因、上述中华绒螯蟹雄性特异Etse基因编码蛋白或上述雄性特异分子标记引物在中华绒螯蟹性别鉴定中的应用。
为实现上述第六目的，本发明采用的技术方案是：
一种中华绒螯蟹性别鉴定的方法，包括以下步骤：
(a)设计并合成如SEQ ID NO:5和SEQ ID NO:6所示的雄性特异分子标记引物；
(b)提取中华绒螯蟹性腺的RNA，并将其反转为cDNA；
(c)以步骤(b)中的cDNA为模板，采用步骤(a)中所述雄性特异分子标记引物进行PCR扩增，反应结束后进行电泳检测；
若结果显示一条特异性条带，分子大小为545bp时，所检测的中华绒螯蟹为雄性，若电泳结果显示没有特异性条带时，所检测的中华绒螯蟹为雌性。
本发明采用上述技术方案，其有益效果如下：
本发明首次克隆获得中华绒螯蟹雄性特异Etse基因的全长编码区序列，该基因只在雄蟹中表达，在雄性性别决定中发挥作用，且利用一对雄性特异分子标记引物进行PCR扩增就能快捷高效地在分子水平上鉴别中华绒螯蟹的性别，根据琼脂糖凝胶电泳扩增产物有无条带就可以鉴别中华绒螯蟹的雌雄，无需测序，快速准确，对中华绒螯蟹在分子水平上鉴定及生产养殖具有积极意义。
附图说明
图1为中华绒螯蟹6只雄性和6只雌性个体Etse基因扩增的凝胶电泳图；其中，第1泳道为分子Marker，第2-7泳道均为雌性模板扩增，8-13泳道为雄性模板扩增的条带。
具体实施方式
下面通过实施例和对比例进一步说明本发明，这些实施例只是用于说明本发明，本发明不限于以下实施例。凡是对本发明技术方案进行修改或者等同替换，而不脱离本发明技术方案的范围，均应涵盖在本发明的保护范围中。
以下实施例中，中华绒螯蟹雄性特异Etse基因的核苷酸序列如SEQ ID NO:1所示，基因没有内含子，cDNA序列跟DNA序列一致，具体如下：
ATGGTCCGTGTTCAATGGCCGCTGTCGGTTGTGATCCTATTGACTGGGGTCAGCACGGCATTATTATTCACCAACAAGAAGTCAGAGGAGGATCCCGGCTATAGCGTCCGGCGCCATGTGGTGAGAGGCTATGAGAGTGACGGGACGCTGACGGGCAGAGGCAGCAATAGATACAATGCGCCTCCCAAGGAGAGTGATAAAGACGACAAAAAAACCGACGGAGACTCTGAAGGCAAAGACGCTGAGGAGAAGGACGGCGGTGCTACAGAGGAAGACAAAAACGCTGCTGAAGGGGACGAAGTTACTGACAAGGAAGACGAAAAAGATAAAAAATCTGCCAAGGACAAAGAAAACACAAACGAAGACAAAAACGCCAAGGAAGACAAAGACGCCAAGGAAGACAAAAACGCCAAGGAAGACAAAGACGCCAAGGAAGACAAAAACGCCAAGGAAGACAAAGACGCCAAGGAAGACAAAAACGCCAAGGAAGACAAAGACGCCAAGGAAGACAAAAACGCCAAGGAAGACAAAAACGCCAAGGAAGACAAAAACGCCAAGGAAGACAAAAACGCCAAGGAAGACAAAGACGCCAAGGAAGACAACAACTCCAAGGAAGACAAAGTCGCCGAGGAAGACAAAGACTCCAAGGAAGACAAAGACGCCGAGGAAGACAAAGGTGCCAAGGAAGACAAAGGTGTTAAGGAAGACAAAGAAATCAAGAAAGACCAAGGTTCTGGAAAGGAGAAAAACTCTAACGGGACCAAATTCACTAACAACGATAAAATTAT
GAATGGTAAAAGGAAGCCTAGGAAAAGGAACTTTGGTAAGCATAGAAAGGGGAAAGGCGGTGCAAC
TAAAACTAAAATATTATTCACTTTCTCCGATGCTGCAAAAGGCAAGAAATCACGAAAAATAGTGCC
ATGCAATTGTTCCGAAACAGTGGAAGGAGGAATAATAACAGGAAGCAGGTCATTAGGCTCTTCCTT
TGGATCAGGAGGAACCGGATCAGGCACTTTGATTGGGGTAGGAGGAACCGAATCAGGCACTTTGAT
TGAAGCAGGAGGAAACGGATCACCAGACTACATCAAGACGGGAGGACGCGGATCATCAGGCTACAT
CGAGATGGGAGGAACCGTATCAGGCACTTTGATTGAGACCGGAGGAACCAGATCAGATGCTGTGAC
TGAGACCGGAGGAACCGGATCAGGCACTCTGATTGAGACCGGAGGAACCGGATCAGGCACTCTGAT
TGAGACCGGAGGAACCGGATCAGGTGCTGTGATTGAGACCGGAGGAACCGGATCAGGCACTCTGAT
TGAGACGGGAGGAGACGGATCAAGCACTATGATTGAGACGGGAGGAGACGGAGCAGGCACTATGAT
TGAGACGGGAGGAGACGGATCAAGCACTATGATTGAGACGGGAGGAGACGGAGCAGGCACTATGAT
TGAGACGGGAGGAGACGGATCAAGCTCTGTGACTGAGACGGGAGGAGACGGATCAGGCACTTTCAT
CGAGACGGGAGGAGACAGATCAAGCTCTGTGATTGAGACGGGAGGAGACGGATCAGGCACTTTCAT
CGAGACGGGAGGAGACGGAGCAGGCACCATGATTGAGACGGGAGGAGACGGATCAGGTACCTTCAT
CGAGACGGGAGGAGACGGAGCAGGGACCATGATTGAGACGGGAGGAGACGGATCAGGCACTTTCAT
CGAGACGGGAGGAGACGGAGCAGGCACCATGATTGAGACGGGAGGAGACGGATCAGGCACTTTCAT
CGAGACGGGAGGAGACGGAGCAGGCACCATGATTGAGACGGGAGGAGACGGATCAGGTGCCGTGAT
TGAGACGGGAGCAGACGGATCAGGCACTTTCATCGAGACGGGAGGAGACGGATCAGGCACTTCCAT
CGAGACGGGAGGAGACGGAGCAGGCACCATGATTGAGACGGGAGGAGACGGATCAGGCACTTTCAT
CGAGACGGGAGGAGACGGATCAAGCACTTTCATCGAGACGGGAGGAGACGGATCAGGCACTTCCAT
CGAGACGGGAGGAGACGGAGCAGGCACCATGATTGAGACGGGAGGAGACGGATCAGGCACTTTCAT
CGAGACGGGAGGAGACGGATCAAGCACTTTCATCGAGACGGGAGGAGACGGAGCAGGCACTATGAT
TGAGACGGGAGGAGACGGATCAGGTGCTGTGGTTGAAACGGGAGGATATGGATCAGGTGCTGTGAT
TGAAACGGGAGGAGACGGATCAGGGACTTTCATCGAGACGGGAGGAGACGGAGCAGGCACCATGAT
TGAGACGGGAGAAGACGGATCAGGTGCTGTGATTGAGACGGGAGGAGACGGATCAGGCACTTTCAT
CGAGACGGGAGGAGACGGAGCAGGCACTATGATTGAGACGGGAGGAGATGGATCAGGTGCTGTGAT
TGAAACGGGAGGAGATGGATCAGGTGCTGTGATTGAAACGGGAGGAGACGGATCAGGTGCTGTGAT
TGAAACGGGAGGAGACGGATCAGGTGCTGTGATTGAAACGGGAGGAGACGGATCAGGTGCTGTGAT
TGAAACGGGAGGAGATGGATCAGGTTATGTGATTGAAACGGGAGGAGATGGATCAGGTGCTGTGAT
TGAAACGGGAGGAGACGGATCAGGTACTGTGATTGAGACGGAAGGAGACGGATCATCAAGCTATTT
GATTGAGAGGGAAGGAGGCGGATCATCAGGCCACATCGAGGCCGGAGGAACCGGATCAGGCCCAGT
GATTGAGACAGGAGGAAAAGGATCGCAAGGCTCTTGGATACAAGGGATAGGGAAAGGATTACAAGG
CTCTTGGATTAAAGGGGGAGGAAAAGGATTACAAGGCTCTTGGATTAAAGGGGGAGGACAAGGATC
ACATGGCTCTTGGATTAAAGGGATAGGAAAAGGATTACAAGGCTCTTGGATTAAAGGGGGAGGAAA
AGGATTACAAGGCTCTTGGATTAAAGGGGGAGGAAAAGAATTACAAGGCTCTTGGATTAAAGGAGA
AGGAAAAGGATCACAAGGCTCTTGGATTAAAGGGGGAGGAAAAGGATCACAAGGCTCTTGGATTAA
AGGGATAGGAAAAGGATTACAAGGCTCTTGGATTAAAGGGGGAGGAAAAGGACTACAAGGCTCTTG
GATTAAAGGAGGAGGAAAAGGATCACAAGGCTCTTGGATTAAAGAGGGAGGAAAAGGATTACAAGG
CTCTTGGATTAAAGGTGAAGGAAAAGGATTACAAGACTCTTGGGTTAAAGAGGGAGGAAAAGGATC
GCAAGGTTCTTGGATTAAAGGGGGAGGAAAAGGATCACAAGGCTCTTGGATTAAAGTAAAAAGAAG
CGGATCACAAGATTCTTGGATCGGATCAGGAGGGAGCAGACCATCAGGCTCATGGACTGGAGCGAG
AGGAAGGAGATCACCAGGCTTTTCCGTCCCATCAGGAGGAAGCGGATTATTAACCTATTACAATAC
AGCGAGAGAGAAAGGACCATTAACCTCTGCCGGTGAAGTGGGAAGGAGGGTACCACTATGCTTGTCCAACAAAGAAGGCGCGACGTCAGACTGCGCCGCTTGA。
上述中华绒螯蟹雄性特异Etse基因编码蛋白的氨基酸序列如SEQ ID NO:2所示，具体如下：
MVRVQWPLSVVILLTGVSTALLFTNKKSEEDPGYSVRRHVVRGYESDGTLTGRGSNRYNAPPKESDKDDKKTDGDSEGKDAEEKDGGATEEDKNAAEGDEVTDKEDEKDKKSAKDKENTNEDKNAKEDKDAKEDKNAKEDKDAKEDKNAKEDKDAKEDKNAKEDKDAKEDKNAKEDKNAKEDKNAKEDKNAKEDKDAKEDNNSKEDKVAEEDKDSKEDKDAEEDKGAKEDKGVKEDKEIKKDQGSGKEKNSNGTKFTNNDKIMNGKRKPRKRNFGKHRKGKGGATKTKILFTFSDAAKGKKSRKIVPCNCSETVEGGIITGSRSLGSSFGSGGTGSGTLIGVGGTESGTLIEAGGNGSPDYIKTGGRGSSGYIEMGGTVSGTLIETGGTRSDAVTETGGTGSGTLIETGGTGSGTLIETGGTGSGAVIETGGTGSGTLIETGGDGSSTMIETGGDGAGTMIETGGDGSSTMIETGGDGAGTMIETGGDGSSSVTETGGDGSGTFIETGGDRSSSVIETGGDGSGTFIETGGDGAGTMIETGGDGSGTFIETGGDGAGTMIETGGDGSGTFIETGGDGAGTMIETGGDGSGTFIETGGDGAGTMIETGGDGSGAVIETGADGSGTFIETGGDGSGTSIETGGDGAGTMIETGGDGSGTFIETGGDGSSTFIETGGDGSGTSIETGGDGAGTMIETGGDGSGTFIETGGDGSSTFIETGGDGAGTMIETGGDGSGAVVETGGYGSGAVIETGGDGSGTFIETGGDGAGTMIETGEDGSGAVIETGGDGSGTFIETGGDGAGTMIETGGDGSGAVIETGGDGSGAVIETGGDGSGAVIETGGDGSGAVIETGGDGSGAVIETGGDGSGYVIETGGDGSGAVIETGGDGSGTVIETEGDGSSSYLIEREGGGSSGHIEAGGTGSGPVIETGGKGSQGSWIQGIGKGLQGSWIKGGGKGLQGSWIKGGGQGSHGSWIKGIGKGLQGSWIKGGGKGLQGSWIKGGGKELQGSWIKGEGKGSQGSWIKGGGKGSQGSWIKGIGKGLQGSWIKGGGKGLQGSWIKGGGKGSQGSWIKEGGKGLQGSWIKGEGKGLQDSWVKEGGKGSQGSWIKGGGKGSQGSWIKVKRSGSQDSWIGSGGSRPSGSWTGARGRRSPGFSVPSGGSGLLTYYNTAREKGPLTSAGEVGRRVPLCLSNKEGATSDCAA。
上述中华绒螯蟹雄性特异Etse基因的扩增方法，包括以下步骤：
(1)提取中华绒螯蟹雄性性腺的总RNA；
(2)以步骤(1)中的总RNA为模板，以oligo dT为反转录引物，进行精巢cDNA的合成；
(3)以上述cDNA为模板，以如SEQ ID NO:3所示的(ATGGTCCGTGTTCAATGGCC)序列为上游引物、如SEQ ID NO:4所示的(TCAAGCGGCGCAGTCTGA)序列为下游引物，进行PCR扩增，得到中华绒螯蟹雄性特异Etse基因。
一种基于上述中华绒螯蟹雄性特异Etse基因设计的雄性特异分子标记引物，上游引物的序列如SEQ ID NO:5所示(ACGCCGAGGAAGACAAAGGTG)，下游引物的序列如SEQ ID NO:6所示(TCCGGTTCCTCCGGTCTCAG)，该引物只能在中华绒螯蟹雄性个体的性腺组织中进行特异性扩增，可在分子水平上快速实现中华绒螯蟹的性别鉴定。
上述中华绒螯蟹雄性特异Etse基因、上述中华绒螯蟹雄性特异Etse基因编码蛋白或上述雄性特异分子标记引物在中华绒螯蟹性别鉴定中的应用。
一种中华绒螯蟹性别鉴定的方法，包括以下步骤：
(a)设计并合成如SEQ ID NO:5和SEQ ID NO:6所示的雄性特异分子标记引物；
(b)提取中华绒螯蟹性腺的RNA，并将其反转为cDNA；
(c)以步骤(b)中的cDNA为模板，采用步骤(a)中所述雄性特异分子标记引物进行PCR扩增，反应结束后进行电泳检测；
若结果显示一条特异性条带，分子大小为545bp时，所检测的中华绒螯蟹为雄性，若电泳结果显示没有特异性条带时，所检测的中华绒螯蟹为雌性。
以下通过具体实施例进一步解释说明。
实施例1
本实施例提供中华绒螯蟹雄性特异Etse基因的扩增方法，包括下述步骤：
由生物公司合成上游引物和下游引物，上下游引物序列分别为ATGGTCCGTGTTCAATGGCC(如SEQ ID NO：3所示)和TCAAGCGGCGCAGTCTGA(如SEQ ID NO：4所示)。
解剖取健康成熟的中华绒螯蟹雄蟹精巢，采用Trizol试剂法抽提获得精巢总RNA，并进行DNA酶处理去除基因组DNA。
取1μg上述中华绒螯蟹精巢总RNA样品与Oligo dT及相关反转录试剂混合，进行反转录，获得cDNA,并将其置于-20℃保存备用。
利用以上述cDNA为模板，以步骤1合成的引物为扩增引物，用高保真Taq酶进行PCR扩增。
PCR的反应体系为：10μl PCR mix,上下游引物各1μl(10uM),cDNA模板100ng,用超纯水补足至20μl。
PCR的反应程序为：首先94℃预变性5分钟，其次进行35个循环扩增(95℃变性30秒，58℃退火30秒，72℃延伸4分钟)，最后72℃延伸5分钟。
扩增获得的片段大小为3597bp，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PMD-19T载体，转化DH5α大肠杆菌，挑选阳性克隆送至生物公司进行测序，获得的核苷酸序列如SEQ ID NO：1所示，利用软件将其核酸序列翻译成氨基酸序列，获得的氨基酸序列如SEQ ID NO：2所示。
实施例2
本实施例提供基于中华绒螯蟹雄性特异Etse基因进行性别鉴定的方法，包括下述步骤：
根据上述Etse基因的核苷酸序列设计引物，上游引物序列为ACGCCGAGGAAGACAAAGGTG(如SEQ ID NO：5所示)，下游引物序列为TCCGGTTCCTCCGGTCTCAG(如SEQ ID NO：6所示)。
中华绒螯蟹cDNA样品制备：
取成熟的中华绒螯蟹雌雄各6只，分别解剖获取其性腺，采用常规Trizol法提取RNA，并将其反转录为cDNA。
PCR扩增验证：
采用上述步骤1的引物对上述雌、雄各6只中华绒螯蟹性腺的cDNA样品进行PCR扩增，PCR产物由1％的琼脂糖凝胶电泳进行检测。
PCR的反应体系为：10μl PCR mix,上下游引物各1μl(10uM),cDNA 100ng,用超纯水补足至20μl。
PCR的反应程序为：首先94℃预变性5分钟，其次进行35个循环扩增(95℃变性30秒，60℃退火30秒，72℃延伸40秒)，最后72℃延伸5分钟。
结果：在6只雌蟹的cDNA样品中没有扩增出特异性条带，在6只雄蟹的cDNA样品中均扩增出特异性条带(图1)。
上述对实施例的描述是为了便于该技术领域的普通技术人员能理解和使用本发明。熟悉本领域技术人员显然可以容易的对这些实施例做出各种修改，并把在此说明的一般原理应用到其他实施例中，而不必经过创造性的劳动。因此，本发明不限于上述实施例。本领域技术人员根据本发明的原理，不脱离本发明的范畴所做出的改进和修改都应该在本发明的保护范围之内。
序列表
<110> 上海海洋大学
<120> 一种中华绒螯蟹雄性特异Etse基因及其应用
<141> 2020-10-28
<160> 6
<170> SIPOSequenceListing 1.0
<210> 1
<211> 3597
<212> DNA
<213> 中华绒螯蟹
<400> 1
atggtccgtg ttcaatggcc gctgtcggtt gtgatcctat tgactggggt cagcacggca 60
ttattattca ccaacaagaa gtcagaggag gatcccggct atagcgtccg gcgccatgtg 120
gtgagaggct atgagagtga cgggacgctg acgggcagag gcagcaatag atacaatgcg 180
cctcccaagg agagtgataa agacgacaaa aaaaccgacg gagactctga aggcaaagac 240
gctgaggaga aggacggcgg tgctacagag gaagacaaaa acgctgctga aggggacgaa 300
gttactgaca aggaagacga aaaagataaa aaatctgcca aggacaaaga aaacacaaac 360
gaagacaaaa acgccaagga agacaaagac gccaaggaag acaaaaacgc caaggaagac 420
aaagacgcca aggaagacaa aaacgccaag gaagacaaag acgccaagga agacaaaaac 480
gccaaggaag acaaagacgc caaggaagac aaaaacgcca aggaagacaa aaacgccaag 540
gaagacaaaa acgccaagga agacaaaaac gccaaggaag acaaagacgc caaggaagac 600
aacaactcca aggaagacaa agtcgccgag gaagacaaag actccaagga agacaaagac 660
gccgaggaag acaaaggtgc caaggaagac aaaggtgtta aggaagacaa agaaatcaag 720
aaagaccaag gttctggaaa ggagaaaaac tctaacggga ccaaattcac taacaacgat 780
aaaattatga atggtaaaag gaagcctagg aaaaggaact ttggtaagca tagaaagggg 840
aaaggcggtg caactaaaac taaaatatta ttcactttct ccgatgctgc aaaaggcaag 900
aaatcacgaa aaatagtgcc atgcaattgt tccgaaacag tggaaggagg aataataaca 960
ggaagcaggt cattaggctc ttcctttgga tcaggaggaa ccggatcagg cactttgatt 1020
ggggtaggag gaaccgaatc aggcactttg attgaagcag gaggaaacgg atcaccagac 1080
tacatcaaga cgggaggacg cggatcatca ggctacatcg agatgggagg aaccgtatca 1140
ggcactttga ttgagaccgg aggaaccaga tcagatgctg tgactgagac cggaggaacc 1200
ggatcaggca ctctgattga gaccggagga accggatcag gcactctgat tgagaccgga 1260
ggaaccggat caggtgctgt gattgagacc ggaggaaccg gatcaggcac tctgattgag 1320
acgggaggag acggatcaag cactatgatt gagacgggag gagacggagc aggcactatg 1380
attgagacgg gaggagacgg atcaagcact atgattgaga cgggaggaga cggagcaggc 1440
actatgattg agacgggagg agacggatca agctctgtga ctgagacggg aggagacgga 1500
tcaggcactt tcatcgagac gggaggagac agatcaagct ctgtgattga gacgggagga 1560
gacggatcag gcactttcat cgagacggga ggagacggag caggcaccat gattgagacg 1620
ggaggagacg gatcaggtac cttcatcgag acgggaggag acggagcagg gaccatgatt 1680
gagacgggag gagacggatc aggcactttc atcgagacgg gaggagacgg agcaggcacc 1740
atgattgaga cgggaggaga cggatcaggc actttcatcg agacgggagg agacggagca 1800
ggcaccatga ttgagacggg aggagacgga tcaggtgccg tgattgagac gggagcagac 1860
ggatcaggca ctttcatcga gacgggagga gacggatcag gcacttccat cgagacggga 1920
ggagacggag caggcaccat gattgagacg ggaggagacg gatcaggcac tttcatcgag 1980
acgggaggag acggatcaag cactttcatc gagacgggag gagacggatc aggcacttcc 2040
atcgagacgg gaggagacgg agcaggcacc atgattgaga cgggaggaga cggatcaggc 2100
actttcatcg agacgggagg agacggatca agcactttca tcgagacggg aggagacgga 2160
gcaggcacta tgattgagac gggaggagac ggatcaggtg ctgtggttga aacgggagga 2220
tatggatcag gtgctgtgat tgaaacggga ggagacggat cagggacttt catcgagacg 2280
ggaggagacg gagcaggcac catgattgag acgggagaag acggatcagg tgctgtgatt 2340
gagacgggag gagacggatc aggcactttc atcgagacgg gaggagacgg agcaggcact 2400
atgattgaga cgggaggaga tggatcaggt gctgtgattg aaacgggagg agatggatca 2460
ggtgctgtga ttgaaacggg aggagacgga tcaggtgctg tgattgaaac gggaggagac 2520
ggatcaggtg ctgtgattga aacgggagga gacggatcag gtgctgtgat tgaaacggga 2580
ggagatggat caggttatgt gattgaaacg ggaggagatg gatcaggtgc tgtgattgaa 2640
acgggaggag acggatcagg tactgtgatt gagacggaag gagacggatc atcaagctat 2700
ttgattgaga gggaaggagg cggatcatca ggccacatcg aggccggagg aaccggatca 2760
ggcccagtga ttgagacagg aggaaaagga tcgcaaggct cttggataca agggataggg 2820
aaaggattac aaggctcttg gattaaaggg ggaggaaaag gattacaagg ctcttggatt 2880
aaagggggag gacaaggatc acatggctct tggattaaag ggataggaaa aggattacaa 2940
ggctcttgga ttaaaggggg aggaaaagga ttacaaggct cttggattaa agggggagga 3000
aaagaattac aaggctcttg gattaaagga gaaggaaaag gatcacaagg ctcttggatt 3060
aaagggggag gaaaaggatc acaaggctct tggattaaag ggataggaaa aggattacaa 3120
ggctcttgga ttaaaggggg aggaaaagga ctacaaggct cttggattaa aggaggagga 3180
aaaggatcac aaggctcttg gattaaagag ggaggaaaag gattacaagg ctcttggatt 3240
aaaggtgaag gaaaaggatt acaagactct tgggttaaag agggaggaaa aggatcgcaa 3300
ggttcttgga ttaaaggggg aggaaaagga tcacaaggct cttggattaa agtaaaaaga 3360
agcggatcac aagattcttg gatcggatca ggagggagca gaccatcagg ctcatggact 3420
ggagcgagag gaaggagatc accaggcttt tccgtcccat caggaggaag cggattatta 3480
acctattaca atacagcgag agagaaagga ccattaacct ctgccggtga agtgggaagg 3540
agggtaccac tatgcttgtc caacaaagaa ggcgcgacgt cagactgcgc cgcttga 3597
<210> 2
<211> 1198
<212> PRT
<213> 中华绒螯蟹
<400> 2
Met Val Arg Val Gln Trp Pro Leu Ser Val Val Ile Leu Leu Thr Gly
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Lys Asp Ala Lys Glu Asp Lys Asn Ala Lys Glu Asp Lys Asp Ala Lys
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Glu Asp Lys Asn Ala Lys Glu Asp Lys Asp Ala Lys Glu Asp Lys Asn
145 150 155 160
Ala Lys Glu Asp Lys Asp Ala Lys Glu Asp Lys Asn Ala Lys Glu Asp
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Lys Asn Ala Lys Glu Asp Lys Asn Ala Lys Glu Asp Lys Asn Ala Lys
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Glu Asp Lys Asp Ala Lys Glu Asp Asn Asn Ser Lys Glu Asp Lys Val
195 200 205
Ala Glu Glu Asp Lys Asp Ser Lys Glu Asp Lys Asp Ala Glu Glu Asp
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Lys Asp Gln Gly Ser Gly Lys Glu Lys Asn Ser Asn Gly Thr Lys Phe
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Thr Asn Asn Asp Lys Ile Met Asn Gly Lys Arg Lys Pro Arg Lys Arg
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Ile Leu Phe Thr Phe Ser Asp Ala Ala Lys Gly Lys Lys Ser Arg Lys
290 295 300
Ile Val Pro Cys Asn Cys Ser Glu Thr Val Glu Gly Gly Ile Ile Thr
305 310 315 320
Gly Ser Arg Ser Leu Gly Ser Ser Phe Gly Ser Gly Gly Thr Gly Ser
325 330 335
Gly Thr Leu Ile Gly Val Gly Gly Thr Glu Ser Gly Thr Leu Ile Glu
340 345 350
Ala Gly Gly Asn Gly Ser Pro Asp Tyr Ile Lys Thr Gly Gly Arg Gly
355 360 365
Ser Ser Gly Tyr Ile Glu Met Gly Gly Thr Val Ser Gly Thr Leu Ile
370 375 380
Glu Thr Gly Gly Thr Arg Ser Asp Ala Val Thr Glu Thr Gly Gly Thr
385 390 395 400
Gly Ser Gly Thr Leu Ile Glu Thr Gly Gly Thr Gly Ser Gly Thr Leu
405 410 415
Ile Glu Thr Gly Gly Thr Gly Ser Gly Ala Val Ile Glu Thr Gly Gly
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Thr Gly Ser Gly Thr Leu Ile Glu Thr Gly Gly Asp Gly Ser Ser Thr
435 440 445
Met Ile Glu Thr Gly Gly Asp Gly Ala Gly Thr Met Ile Glu Thr Gly
450 455 460
Gly Asp Gly Ser Ser Thr Met Ile Glu Thr Gly Gly Asp Gly Ala Gly
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485 490 495
Gly Gly Asp Gly Ser Gly Thr Phe Ile Glu Thr Gly Gly Asp Arg Ser
500 505 510
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515 520 525
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530 535 540
Ser Gly Thr Phe Ile Glu Thr Gly Gly Asp Gly Ala Gly Thr Met Ile
545 550 555 560
Glu Thr Gly Gly Asp Gly Ser Gly Thr Phe Ile Glu Thr Gly Gly Asp
565 570 575
Gly Ala Gly Thr Met Ile Glu Thr Gly Gly Asp Gly Ser Gly Thr Phe
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Ile Glu Thr Gly Gly Asp Gly Ala Gly Thr Met Ile Glu Thr Gly Gly
595 600 605
Asp Gly Ser Gly Ala Val Ile Glu Thr Gly Ala Asp Gly Ser Gly Thr
610 615 620
Phe Ile Glu Thr Gly Gly Asp Gly Ser Gly Thr Ser Ile Glu Thr Gly
625 630 635 640
Gly Asp Gly Ala Gly Thr Met Ile Glu Thr Gly Gly Asp Gly Ser Gly
645 650 655
Thr Phe Ile Glu Thr Gly Gly Asp Gly Ser Ser Thr Phe Ile Glu Thr
660 665 670
Gly Gly Asp Gly Ser Gly Thr Ser Ile Glu Thr Gly Gly Asp Gly Ala
675 680 685
Gly Thr Met Ile Glu Thr Gly Gly Asp Gly Ser Gly Thr Phe Ile Glu
690 695 700
Thr Gly Gly Asp Gly Ser Ser Thr Phe Ile Glu Thr Gly Gly Asp Gly
705 710 715 720
Ala Gly Thr Met Ile Glu Thr Gly Gly Asp Gly Ser Gly Ala Val Val
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Glu Thr Gly Gly Tyr Gly Ser Gly Ala Val Ile Glu Thr Gly Gly Asp
740 745 750
Gly Ser Gly Thr Phe Ile Glu Thr Gly Gly Asp Gly Ala Gly Thr Met
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Ile Glu Thr Gly Glu Asp Gly Ser Gly Ala Val Ile Glu Thr Gly Gly
770 775 780
Asp Gly Ser Gly Thr Phe Ile Glu Thr Gly Gly Asp Gly Ala Gly Thr
785 790 795 800
Met Ile Glu Thr Gly Gly Asp Gly Ser Gly Ala Val Ile Glu Thr Gly
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Gly Asp Gly Ser Gly Ala Val Ile Glu Thr Gly Gly Asp Gly Ser Gly
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Ala Val Ile Glu Thr Gly Gly Asp Gly Ser Gly Ala Val Ile Glu Thr
835 840 845
Gly Gly Asp Gly Ser Gly Ala Val Ile Glu Thr Gly Gly Asp Gly Ser
850 855 860
Gly Tyr Val Ile Glu Thr Gly Gly Asp Gly Ser Gly Ala Val Ile Glu
865 870 875 880
Thr Gly Gly Asp Gly Ser Gly Thr Val Ile Glu Thr Glu Gly Asp Gly
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900 905 910
Ile Glu Ala Gly Gly Thr Gly Ser Gly Pro Val Ile Glu Thr Gly Gly
915 920 925
Lys Gly Ser Gln Gly Ser Trp Ile Gln Gly Ile Gly Lys Gly Leu Gln
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945 950 955 960
Lys Gly Gly Gly Gln Gly Ser His Gly Ser Trp Ile Lys Gly Ile Gly
965 970 975
Lys Gly Leu Gln Gly Ser Trp Ile Lys Gly Gly Gly Lys Gly Leu Gln
980 985 990
Gly Ser Trp Ile Lys Gly Gly Gly Lys Glu Leu Gln Gly Ser Trp Ile
995 1000 1005
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1010 1015 1020
Lys Gly Ser Gln Gly Ser Trp Ile Lys Gly Ile Gly Lys Gly Leu Gln
1025 1030 1035 1040
Gly Ser Trp Ile Lys Gly Gly Gly Lys Gly Leu Gln Gly Ser Trp Ile
1045 1050 1055
Lys Gly Gly Gly Lys Gly Ser Gln Gly Ser Trp Ile Lys Glu Gly Gly
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Lys Gly Leu Gln Gly Ser Trp Ile Lys Gly Glu Gly Lys Gly Leu Gln
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1105 1110 1115 1120
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1125 1130 1135
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<210> 3
<211> 20
<212> DNA
<213> 人工序列
<400> 3
atggtccgtg ttcaatggcc 20
<210> 4
<211> 18
<212> DNA
<213> 人工序列
<400> 4
tcaagcggcg cagtctga 18
<210> 5
<211> 21
<212> DNA
<213> 中华绒螯蟹
<400> 5
acgccgagga agacaaaggt g 21
<210> 6
<211> 20
<212> DNA
<213> 中华绒螯蟹
<400> 6
tccggttcct ccggtctcag 20

Claims (4)

1.一种基于中华绒螯蟹雄性特异Etse基因设计的雄性特异分子标记引物，其特征是：
上游引物的序列如SEQ ID NO:5所示，下游引物的序列如SEQ ID NO:6所示；
其中所述中华绒螯蟹雄性特异Etse基因的核苷酸序列如SEQ ID NO:1所示。
2.权利要求1所述基于中华绒螯蟹雄性特异Etse基因设计的雄性特异分子标记引物在中华绒螯蟹性别鉴定中的应用。
3.一种中华绒螯蟹性别鉴定的方法，其特征是：
所述方法包括以下步骤：
(a)设计并合成权利要求1所述基于中华绒螯蟹雄性特异Etse基因设计的雄性特异分子标记引物；
(b)提取中华绒螯蟹性腺的RNA，并将其反转为cDNA；
(c)以步骤(b)中的cDNA为模板，采用步骤(a)中所述基于中华绒螯蟹雄性特异Etse基因设计的雄性特异分子标记引物进行PCR扩增，反应结束后进行电泳检测；
若结果显示一条特异性条带时，所检测的中华绒螯蟹为雄性，若电泳结果显示没有特异性条带时，所检测的中华绒螯蟹为雌性。
4.根据权利要求3所述中华绒螯蟹性别鉴定的方法，其特征是：
步骤(c)中，所述特异性条带的大小为545bp。
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