CN109251931A - 青虾Rev3基因及其编码蛋白和应用 - Google Patents
青虾Rev3基因及其编码蛋白和应用 Download PDFInfo
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Abstract
本发明公开了青虾Rev3基因及其编码蛋白和应用，Rev3基因部分cDNA序列如SEQ ID NO:1所示，其中3’端为完整序列。与现有技术相比，本发明具有以下优点：(1)本发明所述青虾Rev3基因在青虾雄性分化及发育过程中的重要作用，为青虾雄性分化及其发育机制的研究提供理论依据和思路；(2)本发明所述青虾Rev3基因能够调控青虾免疫应答机制；(3)本发明所述青虾Rev3基因能够用于青虾全雄群体选育中，在青虾养殖过程中具有显著的经济价值和应用前景。
Description
技术领域
本发明属于生物技术领域，涉及青虾的发育调控，具体为青虾Rev3基因及其编码蛋白和应用。
背景技术
青虾，学名日本沼虾(Macrobrachium nipponense)，俗称河虾，隶属十足目(Decapoda)、长臂虾科(Palaemonidae)、沼虾属(Macrobrachium)，广泛分布于我国各地水域，生长快、适应性强、养殖经济效益高，是我国重要的淡水养殖虾类。据2012年《中国渔业年鉴》记载，全国养殖青虾年产量超过23万吨，年产值超过150亿元，是目前淡水养殖业中最有发展前途的品种之一。在青虾养殖过程中存在着显著的雌雄生长差异现象，即雄性青虾的生长速度显著快于雌性青虾，在收获季节，雄性青虾的个体大小为雌性青虾的2-2.5倍，青虾全雄群体选育具有显著的经济价值以及应用前景。因此，全面了解青虾雄性分化以及发育机制，建立青虾性别选育技术刻不容缓。
促雄腺是甲壳类动物特有的内分泌腺，其分泌的激素对甲壳类动物雄性分化及雄性特征维持起重要的作用。先前研究结果表明，在雄性罗氏沼虾中拔除促雄腺，可诱使雄性罗氏沼虾分化出雌性特征；在雌性罗氏沼虾中植入促雄腺，可诱使雌性罗氏沼虾分化出雄性特征。基于促雄腺在甲壳类动物雄性分化及发育过程中的重要作用，对促雄腺中表达基因的研究成为近些年来的热点。本课题组从青虾转录组文库及青虾蛋白质文库中筛选得到编码Rev3基因的mRNA序列以及蛋白质序列。Rev3基因在人类中证明参与免疫应答系统。Rev3基因可促进DNA合成，降低UV照射对培养的人类纤维原细胞突变。但Rev3基因在甲壳类动物中研究尚未见报道，其功能未知。
原位杂交(In situ hybridization)技术是一种广泛存在于真核生物中的高效的序列特异性基因定位技术。原位杂交技术是透过靶基因序列设计带有荧光信号的特异性序列探针，通过与组织学切片杂交，从而对基因在组织中进行准确定位以及功能研究。近年开始在甲壳动物基因功能研究中使用。目前，关于Rev3基因的原位杂交研究尚未见报道。
发明内容
解决的技术问题：为了克服现有技术的不足，阐明Rev3基因在青虾雄性分化及发育过程中的重要作用，为青虾雄性分化及其发育机制的研究提供理论依据和思路，本发明提供了青虾Rev3基因及其编码蛋白和应用。
技术方案：青虾Rev3(DNA polymerase zeta catalytic subunit，Rev3)基因，Rev3基因部分cDNA序列如SEQ ID NO:1所示，其中3’端为完整序列。SEQ ID NO:1序列如下：
GGAATTGTACCCTGTATAACAGGAATTAAAGTTTGTAATGAAAAGATTCCTGATTCCAAAAAAATTGATAAAAAAAGAACAGACTTAAAAGTCAATAAGGGAAATACGGAAAAACTAATTTGTAGTACAAGTAAAGATAAAACAAAAGGAACCAACAGTGAATCTTCAGAGAAATTAAGTCAGTCAGAAAATAAAAAACACAACAAAGAGATGCTTGACAAACAAAAACCACAGTTAGGATCCAAGATGAAAGACCATGGAAAAAGTTTTGATAAAAAGGAGAAAGACAGCAAAGCCAGAGATTTTGTGACACCTTTAGGAAAGGCAGATCAGAATGTATGTGGTAATTCTAGCTCAGAAGGGAATCCTATGAAAATTCACAATACAGAGAATAAAAATGATAGTGAAGAGGTCTCAGTCCCAGACAAAAAAGATTTCTTGTCTGTAGCTGACAAGAACAAAAACAAAACAAGCACAAAAAAGACACACAGACAAGAAAGTTTGAAGGAGCCCCTTGCTCAGAAAAAAAATAAAAATCCTTCAGAAAATTCTTGCAGAAATCCTAGTGTGAATTCGTGTAGCAAAGCTAAAGTATTTACAACATCCAAAGAAAAACACCATTCATGTATGCAGGAGCCCAGCAGAGATAAGATTGATTTGGATAAAAGTGGCGTCACGAGTGTACATCCAGCAAAAATTGAAAGCCATGATACTTTGCCATTGACTCAGAAGAATGAAATGCCCTTGGAACCTTGCAAGGGGCCATTAGGGGAATTGTCTTCCTTGGCTGCAAAGAGTGATTGCACTTCAACACATGACCCTAAAGTCACAGATTATGAAAACGTGAGTATTGAAAACTTGCGCAAATCCTCAGATAAGATTAATCCACCAGAAAAGCTCAGTAGTGTTCAAGGATGTTTTGAAAAACGTGGTGATGTTACTGGTGGTGCCAACTTGCCACCTAAGTTTGATTCTAGCCAAAATGGGGATGGAAAAATACTCTTAGTAACCACAGAGAATAGTTCTGTTAGCAAAGGCAAAAAGCGAATTAGTCGTTGGGATGTTAAGGATAAAAATTCAAGTATAACAGCAGTGACAAAGAAAAGTCGTTGGGATCAAAGTGACAGCAACGCTAGTGGCCAAGAGGCAGCAAAACAAGAGGCAGCAAGTGAAAAAAGAAAAAGATTAAAGCAGATGGTTGCCAACTTGTTTGAAGATTGTGAAAATGAAAATCCCTCGTGTAGACAACAAATGCGTGGCTCAAGTTTGAGGAAGCAGCTGACAGAGTTGTTTGGCCATTCATCCCTTGCTGAAAAGAATGAAGTTTCTGAGGAATACAAACCTCAACCTTCAACATCTAAAGATAAAGTAGGATCGAAGACCATGTTCAAGGAATCCTTAGAGTCTTTATTTGAACCTAGCTCCACTTTTGGTGTAAAGGAAACCAACATGAGATCTAATTGGAAAGAGGATACAGAAAATGAATATCAGCCAACAACAATAGTTAAATCATGTACAAAGGCTGAATACAACCCTTCTTCAATATCTAAGGAAAGGTTAGAAGTAGAGTATAAACCAACATCCTCTACTAAAGTAAGAGTTAAACCTGATGATAAAGGGAGACCACATGTCCTGAATGATGACACAAAGGGAAAATATAAATCTAGCATGAAGCCTAACTCCAAAACTACTTCAGACACACTTGATAAAAAGTCATTGAAATATAGTAAAACAAAGTGTGATTCTATGCGTAAAAAATGCCTGGGACTGGGAGATGTCTCCAGCATAAAAGATGCCAAGACTGTGTGTAGTGCTACCGATGATGAATATTGCCCTCTTCCATTTGGTCTTGGTAAACCTTGTCCTGAATACATTCCAAGTGATATATCTGATATTAAATCCAACAAGCCTTTAGATGACTTGGAAAGTCATGAAGAATATGTTCCTCTACCAGCAAAGGCAATTTCATATGGAATTAGTTACAATGCATCTGGTGAAGAAAAACCATCAAAAATTTCTGTCCCTGAGGCAAACACTTTAGTACCCATTTCTTATGGTGCGGGAAGTACTGGCTTGGGTTTCTACCGTGAGCAGGGTAACAGTGAGAGTACTCAGAGTAAAGAAGGGGAAAGTTCTGTCAAAGATAAAGGAAGATCAAGTAAAAAGAGACAAGAACCATACAAGAACCTTGGAAATAACAAACCTAAGTTGAAAACTTCATTTTCTTCTACAAACGCTTCAGATTCAGTTGTTCTGGTTTACTGTCCACCCCCACCAAGTTGTGATGATATCATTAATAGCCTTGCAGATTATAATATTCCAGATGTTATAAATCCAGGTGCATTTTGTAGTAACAGACTTGATATTCCTGCCAAACCAATGGAAGTTGGAGGGCAAGTTCTGAAGTTAAAATCCAAAGAAGTTTCAGATTTTGAAAGTTTTGAGGGTCCTCATCAAAATAATGGGCTTTACAACTGGCGTTTATTGCTGAGCAGCATGAATTCTGATTGTGCTGGACCATCTACTCAGGAATTAAGTGTGAATAAAAAGGACAAAAATACTTCAAAGTCTATCATGACTCCTGTAAAAGTTCCACCTTCGCCATCAGAGGTGATGAAGTGGGCGGAAGCTCGCAAGATACTAAAGTCTATGAAAAAGAAGGATGAAGATGGAGATGATGGTGGCTCATCCTCAAAATCTGACAAAAAGGTGGATTCAAATAAAAAATCATCCACTTCACTGCCTCCTGGTTCCAATGGTGTTGGTAATGGAAGTATGCAGAAAAATTCAAGTGGACAAAATAACCAACAGTCTAGAGATGGTAATGTCTCTCTGACTCAAAGTTTATCAAGCAGTGCTTTATCCCAGCAGCGTCACCACACAAGTACACCACAGCCACAGAAATTCTCAAAGTTAACACCTCTTCATTTAACACCCATTGGTGGAGACTCCTCTGGTAATGAGAAGACCCCATCTCCTGCATTTCTTAGCAAGAGAAAACCAAAGCGCAGAGTCTCATGGGAAGATGGTAGCCCATCTGTAAAGCCTTCTTTGTTGAAGAAGAGGAGGATCTCTTATGAGGGCTTGCCTGTTTTACATGAAGAAACTCCAGAAGTCAGTGAACCAAAGAAAAGTTTGGTGAGGCGTGTGTCTTTTTCTGGCATAACTCAACCAGCAGGTGGTTTCAAAGTTACAGAAGTTCAAGATCACAGTAAAGCTGAAGAACTGAAGCCCATGCCAGAGAGTCCTGAATCTGCTAAAGAGTGCACAAGTGAGAAAGGTGAAGATTTGGATTCAACTTTAGTGACCCAAGTTGCTGACCCAGACATAGAATCAAGCCCTGAAACTATAATAAGTTCATCACGCTCATTAGATTTAACACTCTCATTGTCTACTAAGGCAGCTTCACAACCATCTAGTCAGATGTCTGACTCTTCTTTAAGACGTCAACTGATGGACACTCAGTTCCGACAACAGTTCCTTAGCTTCTCACAAGTTCCTTCAGGCTCTTCGCAGGGATTAGATGGCCCAACCTTGAACAACACTTATGGCTTTAAATTCTCAAATCATAACCTGCGTGAGGCTAAAGCTTTGCATGAGATCCCCTCATAGAGAAAAGTGGCAATGAAGGTGCATGCGTGAGAAGATTACCTGGGGAAGGGAAATCGAGAGGTCCAGAACGACCTTGACCTGTGTGTCCAAATGGAAAATAGGCAGCTGCAGAATCTTGACCGCCACTTCACTTTTTCCTCCATTTTCATGCATGAATATCTAACCACCATGTGCATCGAGTGTCATGTTACAACACGACCTTCTTTGTTGCCTGACCCATTGTATGATCCCATCTCGGCTGTTATTTTCACCATCATTCAAGATGTACCAGATGATCACCCCTCTCCTCAGAAACTCAAAGGTGCAATAGTGGTTAGTAGTAGCAGCGGAGATCTGATATCTCATAGTGGCATTTGTGGCCTTTACGTGCTTCATGTTGATGATGAGAAGGCTCTCCTAAATGAAGTAGTCACTATTGTCACTACTTGGGATCCAGATATGCTTGTTGGATATGAGGTTCAGATGTCATCTTGGGGATACCTGCTGGAACGAGGATCAGCACTGAACTTAGAAATTATAGCCAGGCTCTCACGAGTGCCAGAAGGAACAGGCAGTCATTATGAAGAAGAGTATGATGAATATGGAGCTGCTCACACTTCTCAGATTCATATTACTGGCCGAATTGTTCTCAATGTTTGGAGAATAATGAGAGCAGAGGTAGCACTGAGGAGTTACACACTTGACAATGTTGCTTATAATGTCTTGCACACTCGGATACCGTCCTTTTCCCATGACAGCCTCAGAGATTGGTGGATTCATGATGCAACAAGGTGGAGGACCTTGGAGCATTATTCAACTCGCTGTGACTTGACTATTGACCTCCTACACAGATTAGATCTTATCCACCGCACAGCTGAGCTGGCACGTCTCTTTGGCATCCAGTTCTATGATGTTCTGGCTCGTGGCTCACAGTATCGTGTGGAATCCATGATGTTGCGTCTTTCTCGTGCTGCTAACATGGTGGCAGTTTCCCCAAGCATACAGCAGCGTGGAGCCATGAAGTCACCTGAATGGTTTGCCCTAACTTTGGAACCAAAGTCAAGATTCTACAGTGATCCTGTGGTGGTGTTAGATTTCCAGTCCTTATACCCATCAATGATGATTGCTTACAATTACTGCTATTCCACTTGTTTGGGACGAATTGATATGATAAATGGATCTGCACCCATGGAATTTGGAGCTACCCATCTACGAGTTCTTCCAGAGGATCTCTTGAAATTTGCTGATGAGATCAATATCTCTCCTTGTGGAGTGGCTTTTGTACCCCATAAAATAAGGCGAGGAGTTTTACCACAGATGCTTGAAGAAATACTCAATACAAGGATTATGGTGAAGCGAATGATGAAGGAGTACAAAGACGATTCATCCCTTCAGCGTGTGCTACATTCACGACAGCTTGGTCTGAAACTTATTGCGAATGTCACATATGGGTACACCAGTGCAAACTTCTCTGGTCGAATGCCTTGTTCTGAGATTGGTGACAGTGTTGTAAGCAAAGGAAGAGAGACTTTAGAACGTTGTATTCAACATGTCAATAAGCACCCTACTTGGAAAGCAGAGGTGGTGTATGGAGATACAGATTCACTCTTCGTACACTTACCTGGACGGTCTAAGGATCAAGCATTTAAAATTGGAAATGAGATTGCTGATGAAGTAACTCAGATGTATCCTAAACCGGTGAAACTTAAATTTGAAAAAGTTTACTTACCCTGCATTTTACAGACCAAGAAACGTTACGTTGGATATATGTATGAGTCCACAACCCAGGAGACCCCAGAGTTTGATGCTAAAGGCATTGAGACTGTTCGTAGAGATGGATGCCCAGCAATGGCAAAGGTTCTTGAAAAATCCTTGCGTATTCTCTTTGAAACAAAGGATATTTCAAAAGTAAAAGACTATATTCAGCGTCAGTTTGTGAAACTTCTACAAGGACAGGTGTCTCTCCAGGATTTGATATTTGCAAAAGAGTATAGAGGAGCCTCTGGCTATAAGCCAGGTGCATGCGTCCCTGCTCTAGAATTAGCAAGAAAATGGACTCGCAAAGATCCTAGAAAAGAACCAAGAGTTGGTGAGCGTGTACCATATGTAATAGTCTATGGTGCTCCAGGCTTACCTTTAATCCAATTAGTAAGATCACCAGATGCTGTTCTAGCAAATCCAGCACTTCGTGTAAATGCAACATACTATATCACAAGAGCAATAATGCCACCTTTAGAAAGATGTTTCTCTCTTCTTGGTGTAGATATTGCTGCTTGGTATAATGACTTGCCAAGATCATCAAGTACAAATGCTTCCCTGGTGAGAGAAATAGCAAATAATCAGAAAGGATCAAGCAACTGGGAAAAACCATCTACTTCCCATGGTATGTCCATTGTTCGTTACTTCACTCCATCAGCATGCATTGTTTGTGGAGCTGCAACTGTTCTAACTGGTGCAGGGGGTGGAGTTGCGTTATGTTCAGACTGCAAGACTACCCCACAAGACACTGTCCTTGCTCTTAATGAGAAAATTCGCCATTATGATAGAGCACTTGACAATATACAAAAGGTTTGCCAGGCATGCCTTGGATGTAGACTGGAAAAGGTGGAGTGCATATCCATTGACTGCCCTATCATTTACCAGCGTAACCAAGCAAACATTGACCAACTTTTAACTGGACAGCTGCAAGAGCTTATTCAAAGATTTACTAGTGTGATATAAAATTTATAAATTCCAGCAGGATAATGGAGAGCTTTTGTGTAACAATATTAAGATTTAAGAGTAAATTATGACTGCAGATCCAGACTGACTTAGTGATGCTGTTAAGTGATTGTTGAATAAGAAAACAAATAAGTTCAGTACCTTCCTTTGGCACTGATATAAGATGGCTTAAGTGCATTAATGTTATAAGATAACTGAATGCAGAGTATTATTAGTGAAATTATTAAATCAAATTTTGTATATATTGAGCTATATAATAGTTCAAGAAATTTATTTTGAAAACACAGAATTATATATGTACATTTAGTGAAAAACCCTTATGTGAATATATAAATAGTACGTATAAAACAAAGCTTTCTGTGGTAAAAGTTTGCATCTATGCATAAAAAAAAACGGTTTTTTATGAAGACTTCAGATTTAGTTTAAATGGCAAGGTAGATGTGAAGATGGAAGGACGACTATTTTAAAGGTGGTTTGGGTCTTTCAAGAGTAAAAAAAAAAAAAAAAAAAAAAAAAAAAA
优选的，Rev3基因cDNA序列克隆的3’端特异性正向引物为：
3Rev3-F1(SEQ ID NO:3)：CCATCTACTTCCCATGGTATGT；和
3Rev3-F2(SEQ ID NO:4)：ATCCATTGACTGCCCTATCATT。
优选的，Rev3基因的荧光定量PCR引物为：
Rev3-RTF(SEQ ID NO:5)：AGTGACAGCAACGCTAGTGG；
Rev3-RTR(SEQ ID NO:6)：GGCCAAACAACTCTGTCAGC。
优选的，Rev3基因的原位杂交试验中，正义原位杂交探针为(SEQ ID NO:7)：CATCTAATCCCTGCGAAGAGCCTGAAGGAACTTGTGAG，反义原位杂交探针为(SEQ ID NO:8)：CTCACAAGTTCCTTCAGGCTCTTCGCAGGGATTAGATG。
由青虾Rev3基因编码的蛋白，该编码蛋白的氨基酸序列如SEQ ID NO:2所示。SEQID NO:2序列如下：
GIVPCITGIKVCNEKIPDSKKIDKKRTDLKVNKGNTEKLICSTSKDKTKGTNSESSEKLSQSENKKHNKEMLDKQKPQLGSKMKDHGKSFDKKEKDSKARDFVTPLGKADQNVCGNSSSEGNPMKIHNTENKNDSEEVSVPDKKDFLSVADKNKNKTSTKKTHRQESLKEPLAQKKNKNPSENSCRNPSVNSCSKAKVFTTSKEKHHSCMQEPSRDKIDLDKSGVTSVHPAKIESHDTLPLTQKNEMPLEPCKGPLGELSSLAAKSDCTSTHDPKVTDYENVSIENLRKSSDKINPPEKLSSVQGCFEKRGDVTGGANLPPKFDSSQNGDGKILLVTTENSSVSKGKKRISRWDVKDKNSSITAVTKKSRWDQSDSNASGQEAAKQEAASEKRKRLKQMVANLFEDCENENPSCRQQMRGSSLRKQLTELFGHSSLAEKNEVSEEYKPQPSTSKDKVGSKTMFKESLESLFEPSSTFGVKETNMRSNWKEDTENEYQPTTIVKSCTKAEYNPSSISKERLEVEYKPTSSTKVRVKPDDKGRPHVLNDDTKGKYKSSMKPNSKTTSDTLDKKSLKYSKTKCDSMRKKCLGLGDVSSIKDAKTVCSATDDEYCPLPFGLGKPCPEYIPSDISDIKSNKPLDDLESHEEYVPLPAKAISYGISYNASGEEKPSKISVPEANTLVPISYGAGSTGLGFYREQGNSESTQSKEGESSVKDKGRSSKKRQEPYKNLGNNKPKLKTSFSSTNASDSVVLVYCPPPPSCDDIINSLADYNIPDVINPGAFCSNRLDIPAKPMEVGGQVLKLKSKEVSDFESFEGPHQNNGLYNWRLLLSSMNSDCAGPSTQELSVNKKDKNTSKSIMTPVKVPPSPSEVMKWAEARKILKSMKKKDEDGDDGGSSSKSDKKVDSNKKSSTSLPPGSNGVGNGSMQKNSSGQNNQQSRDGNVSLTQSLSSSALSQQRHHTSTPQPQKFSKLTPLHLTPIGGDSSGNEKTPSPAFLSKRKPKRRVSWEDGSPSVKPSLLKKRRISYEGLPVLHEETPEVSEPKKSLVRRVSFSGITQPAGGFKVTEVQDHSKAEELKPMPESPESAKECTSEKGEDLDSTLVTQVADPDIESSPETIISSSRSLDLTLSLSTKAASQPSSQMSDSSLRRQLMDTQFRQQFLSFSQVPSGSSQGLDGPTLNNTYGFKFSNHNLREAKALHEIPS
以上任一所述青虾Rev3基因在调控青虾雄性分化及其发育过程中的应用。
以上任一所述青虾Rev3基因在调控青虾免疫应答机制中的应用。
以上任一所述青虾Rev3基因在青虾全雄群体选育中的应用。
有益效果：(1)本发明所述青虾Rev3基因在青虾雄性分化及发育过程中的重要作用，为青虾雄性分化及其发育机制的研究提供理论依据和思路；(2)本发明所述青虾Rev3基因能够调控青虾免疫应答机制；(3)本发明所述青虾Rev3基因能够用于青虾全雄群体选育中，在青虾养殖过程中具有显著的经济价值和应用前景。
附图说明
图1是青虾Rev3基因在不同成体组织中的相对表达量结果图；
图2是青虾Rev3基因在精巢、促雄腺和肝胰腺中的原位杂交结果图；其中ST为精原细胞，SC为精母细胞，SP为精子，M为肌肉，C为促雄腺细胞，FS为索状结构，LG为脂肪粒细胞，HC为肝胰腺细胞，该图标尺为50μm。
具体实施方式
以下实施例进一步说明本发明的内容，但不应理解为对本发明的限制。在不背离本发明精神和实质的情况下，对本发明方法、步骤或条件所作的修改和替换，均属于本发明的范围。若未特别指明，实施例中所用的技术手段为本领域技术人员所熟知的常规手段。
实施例1：青虾Rev3基因部分CDNA(3’端完整)的获得
总RNA提取：选取10只健康成熟的雄性青虾促雄腺，迅速放入盛有液氮的预冷研钵中，迅速的研磨。总RNA的提取使用Takara公司的RNAiso Plus提取试剂结合传统的酚仿抽提法进行，提取方法参照使用说明书。经过琼脂糖凝胶电泳检测RNA的质量，分光光度计分析该样品OD260/280比值在1.8～2.0之间，并测定RNA的浓度。
从青虾促雄腺转录组中获得Rev3基因cDNA中间片段序列。根据Rev3基因cDNA中间片段序列，设计特异性正向引物3Rev3-F1(SEQ ID NO:3)、3Rev3-F2(SEQ ID NO:4)；分别进行cDNA 3’端快速扩增，操作步骤按TaKaRa 3’-RACE试剂盒说明书进行。1.5％琼脂糖凝胶电泳检测。利用上海生工生物工程有限公司(Sangon)的柱式DNA胶回收试剂盒进行目的片段的回收，将产物连接到pMD18-T载体(Takara)，并转化入大肠杆菌DH5α，进行蓝白斑筛选，挑取单克隆白斑扩增培养后，将插入目的片段的阳性克隆送至上海铂尚生物公司进行测序分析。将得到的几段产物进行拼接，得到青虾Rev3基因部分cDNA序列(3’端完成)。经过序列比对显示，青虾Rev3基因与其他物种的同源性为41％，但覆盖度达到35％，其原因为青虾Rev3基因cDNA序列不完整。其核苷酸序列如SEQ ID NO:1所示，其编码蛋白的氨基酸序列如SEQ ID NO:2所示。
实施例2：青虾Rev3基因组织表达
以SEQ ID NO:1核苷酸序列为依据，在开放阅读框以内采用NCBI在线引物设计软件(http://www.ncbi.nlm.nih.gov/tools/primer-blast/)设计青虾Rev3基因的荧光定量PCR的引物(Rev3-RTF、SEQ ID NO:5-Rev3-RTR、SEQ ID NO:6)。从健康成熟的青虾中获取精巢、促雄腺、卵巢、肝胰腺、肠以及心脏等组织(N≥3)，并将组织保存在RNA保存液中(Takara)。按实施例1所述提取各组织总RNA并使用iScriptTM cDNA Synthesis Kitperfect Real Time(Bio-Rad)试剂盒将总RNA反转成cDNA，采用Bio-Rad iCycler iQ5荧光定量PCR分析系统检测Rev3基因在青虾各组织中的相对表达量。结果如图1所示，青虾Rev3基因在肝胰腺中的表达量最高，且与其他组织表达量差异极显著，表明Rev3基因参与青虾免疫应答机制。Rev3基因在精巢及促雄腺中的表达量较高，因此推测该基因可能在青虾雄性分化及其发育过程中起重要的作用。
实施例3：青虾Rev3基因的原位杂交研究
以SEQ ID NO:1核苷酸序列为依据，在开放阅读框内采用Primer5引物设计软件设计制备青虾Rev3基因的原位杂交正义探针序列和反义探针序列，并委托采用上海生工生物工程有限公司(Sangon)合成标有地高辛信号的正义原位杂交探针和反义原位杂交探针(SEQ ID NO:7-SEQ ID NO:8)。从健康成熟的雄性青虾中获取精巢、促雄腺和肝胰腺组织，并保存在4％多聚甲醛溶液中固定。制备各组织的冰冻切片。使用Zytofast PLUS CISHimplementation kit(ZytoVision GmBH,Germany)原位杂交试剂盒对各组织进行组织学定位，并在显微镜下观察定位结果。结果如图2所示，青虾Rev3基因在青虾精巢中的精原细胞、精母细胞以及镜子中均观察到了较强的地高辛信号，因此该基因在精巢整个发育过程中均起重要的作用。该基因在青虾促雄腺中的索状结构中观察到了较强的地高辛信号，但并未直接在促雄腺细胞中观察得到地高辛信号。促雄腺索状结构的形成对促雄腺细胞的形成起重要的促进和支撑作用。因此，该基因并不是促雄腺细胞所分泌，但该基因在促雄腺索状结构的形成过程中重要作用，为促雄腺细胞的形成起到促进和职称的作用。该基因还在青虾肝胰腺细胞中观察到较强的地高辛信号，证明该基因还参与青虾免疫应答机制。结果证明，青虾Rev3基因在青虾雄性分化及其发育过程中起重要的作用，为该基因的新功能，未在其他物种中报道，这一研究结果为青虾雄性分化及其发育机制的研究提供了理论依据，也为后续青虾全雄群体选育提供指导意义。
序列表
<110> 中国水产科学研究院淡水渔业研究中心
<120> 青虾Rev3基因及其编码蛋白和应用
<160> 8
<170> SIPOSequenceListing 1.0
<210> 1
<211> 6832
<212> DNA
<213> 青虾(shrimp)
<400> 1
ggaattgtac cctgtataac aggaattaaa gtttgtaatg aaaagattcc tgattccaaa 60
aaaattgata aaaaaagaac agacttaaaa gtcaataagg gaaatacgga aaaactaatt 120
tgtagtacaa gtaaagataa aacaaaagga accaacagtg aatcttcaga gaaattaagt 180
cagtcagaaa ataaaaaaca caacaaagag atgcttgaca aacaaaaacc acagttagga 240
tccaagatga aagaccatgg aaaaagtttt gataaaaagg agaaagacag caaagccaga 300
gattttgtga cacctttagg aaaggcagat cagaatgtat gtggtaattc tagctcagaa 360
gggaatccta tgaaaattca caatacagag aataaaaatg atagtgaaga ggtctcagtc 420
ccagacaaaa aagatttctt gtctgtagct gacaagaaca aaaacaaaac aagcacaaaa 480
aagacacaca gacaagaaag tttgaaggag ccccttgctc agaaaaaaaa taaaaatcct 540
tcagaaaatt cttgcagaaa tcctagtgtg aattcgtgta gcaaagctaa agtatttaca 600
acatccaaag aaaaacacca ttcatgtatg caggagccca gcagagataa gattgatttg 660
gataaaagtg gcgtcacgag tgtacatcca gcaaaaattg aaagccatga tactttgcca 720
ttgactcaga agaatgaaat gcccttggaa ccttgcaagg ggccattagg ggaattgtct 780
tccttggctg caaagagtga ttgcacttca acacatgacc ctaaagtcac agattatgaa 840
aacgtgagta ttgaaaactt gcgcaaatcc tcagataaga ttaatccacc agaaaagctc 900
agtagtgttc aaggatgttt tgaaaaacgt ggtgatgtta ctggtggtgc caacttgcca 960
cctaagtttg attctagcca aaatggggat ggaaaaatac tcttagtaac cacagagaat 1020
agttctgtta gcaaaggcaa aaagcgaatt agtcgttggg atgttaagga taaaaattca 1080
agtataacag cagtgacaaa gaaaagtcgt tgggatcaaa gtgacagcaa cgctagtggc 1140
caagaggcag caaaacaaga ggcagcaagt gaaaaaagaa aaagattaaa gcagatggtt 1200
gccaacttgt ttgaagattg tgaaaatgaa aatccctcgt gtagacaaca aatgcgtggc 1260
tcaagtttga ggaagcagct gacagagttg tttggccatt catcccttgc tgaaaagaat 1320
gaagtttctg aggaatacaa acctcaacct tcaacatcta aagataaagt aggatcgaag 1380
accatgttca aggaatcctt agagtcttta tttgaaccta gctccacttt tggtgtaaag 1440
gaaaccaaca tgagatctaa ttggaaagag gatacagaaa atgaatatca gccaacaaca 1500
atagttaaat catgtacaaa ggctgaatac aacccttctt caatatctaa ggaaaggtta 1560
gaagtagagt ataaaccaac atcctctact aaagtaagag ttaaacctga tgataaaggg 1620
agaccacatg tcctgaatga tgacacaaag ggaaaatata aatctagcat gaagcctaac 1680
tccaaaacta cttcagacac acttgataaa aagtcattga aatatagtaa aacaaagtgt 1740
gattctatgc gtaaaaaatg cctgggactg ggagatgtct ccagcataaa agatgccaag 1800
actgtgtgta gtgctaccga tgatgaatat tgccctcttc catttggtct tggtaaacct 1860
tgtcctgaat acattccaag tgatatatct gatattaaat ccaacaagcc tttagatgac 1920
ttggaaagtc atgaagaata tgttcctcta ccagcaaagg caatttcata tggaattagt 1980
tacaatgcat ctggtgaaga aaaaccatca aaaatttctg tccctgaggc aaacacttta 2040
gtacccattt cttatggtgc gggaagtact ggcttgggtt tctaccgtga gcagggtaac 2100
agtgagagta ctcagagtaa agaaggggaa agttctgtca aagataaagg aagatcaagt 2160
aaaaagagac aagaaccata caagaacctt ggaaataaca aacctaagtt gaaaacttca 2220
ttttcttcta caaacgcttc agattcagtt gttctggttt actgtccacc cccaccaagt 2280
tgtgatgata tcattaatag ccttgcagat tataatattc cagatgttat aaatccaggt 2340
gcattttgta gtaacagact tgatattcct gccaaaccaa tggaagttgg agggcaagtt 2400
ctgaagttaa aatccaaaga agtttcagat tttgaaagtt ttgagggtcc tcatcaaaat 2460
aatgggcttt acaactggcg tttattgctg agcagcatga attctgattg tgctggacca 2520
tctactcagg aattaagtgt gaataaaaag gacaaaaata cttcaaagtc tatcatgact 2580
cctgtaaaag ttccaccttc gccatcagag gtgatgaagt gggcggaagc tcgcaagata 2640
ctaaagtcta tgaaaaagaa ggatgaagat ggagatgatg gtggctcatc ctcaaaatct 2700
gacaaaaagg tggattcaaa taaaaaatca tccacttcac tgcctcctgg ttccaatggt 2760
gttggtaatg gaagtatgca gaaaaattca agtggacaaa ataaccaaca gtctagagat 2820
ggtaatgtct ctctgactca aagtttatca agcagtgctt tatcccagca gcgtcaccac 2880
acaagtacac cacagccaca gaaattctca aagttaacac ctcttcattt aacacccatt 2940
ggtggagact cctctggtaa tgagaagacc ccatctcctg catttcttag caagagaaaa 3000
ccaaagcgca gagtctcatg ggaagatggt agcccatctg taaagccttc tttgttgaag 3060
aagaggagga tctcttatga gggcttgcct gttttacatg aagaaactcc agaagtcagt 3120
gaaccaaaga aaagtttggt gaggcgtgtg tctttttctg gcataactca accagcaggt 3180
ggtttcaaag ttacagaagt tcaagatcac agtaaagctg aagaactgaa gcccatgcca 3240
gagagtcctg aatctgctaa agagtgcaca agtgagaaag gtgaagattt ggattcaact 3300
ttagtgaccc aagttgctga cccagacata gaatcaagcc ctgaaactat aataagttca 3360
tcacgctcat tagatttaac actctcattg tctactaagg cagcttcaca accatctagt 3420
cagatgtctg actcttcttt aagacgtcaa ctgatggaca ctcagttccg acaacagttc 3480
cttagcttct cacaagttcc ttcaggctct tcgcagggat tagatggccc aaccttgaac 3540
aacacttatg gctttaaatt ctcaaatcat aacctgcgtg aggctaaagc tttgcatgag 3600
atcccctcat agagaaaagt ggcaatgaag gtgcatgcgt gagaagatta cctggggaag 3660
ggaaatcgag aggtccagaa cgaccttgac ctgtgtgtcc aaatggaaaa taggcagctg 3720
cagaatcttg accgccactt cactttttcc tccattttca tgcatgaata tctaaccacc 3780
atgtgcatcg agtgtcatgt tacaacacga ccttctttgt tgcctgaccc attgtatgat 3840
cccatctcgg ctgttatttt caccatcatt caagatgtac cagatgatca cccctctcct 3900
cagaaactca aaggtgcaat agtggttagt agtagcagcg gagatctgat atctcatagt 3960
ggcatttgtg gcctttacgt gcttcatgtt gatgatgaga aggctctcct aaatgaagta 4020
gtcactattg tcactacttg ggatccagat atgcttgttg gatatgaggt tcagatgtca 4080
tcttggggat acctgctgga acgaggatca gcactgaact tagaaattat agccaggctc 4140
tcacgagtgc cagaaggaac aggcagtcat tatgaagaag agtatgatga atatggagct 4200
gctcacactt ctcagattca tattactggc cgaattgttc tcaatgtttg gagaataatg 4260
agagcagagg tagcactgag gagttacaca cttgacaatg ttgcttataa tgtcttgcac 4320
actcggatac cgtccttttc ccatgacagc ctcagagatt ggtggattca tgatgcaaca 4380
aggtggagga ccttggagca ttattcaact cgctgtgact tgactattga cctcctacac 4440
agattagatc ttatccaccg cacagctgag ctggcacgtc tctttggcat ccagttctat 4500
gatgttctgg ctcgtggctc acagtatcgt gtggaatcca tgatgttgcg tctttctcgt 4560
gctgctaaca tggtggcagt ttccccaagc atacagcagc gtggagccat gaagtcacct 4620
gaatggtttg ccctaacttt ggaaccaaag tcaagattct acagtgatcc tgtggtggtg 4680
ttagatttcc agtccttata cccatcaatg atgattgctt acaattactg ctattccact 4740
tgtttgggac gaattgatat gataaatgga tctgcaccca tggaatttgg agctacccat 4800
ctacgagttc ttccagagga tctcttgaaa tttgctgatg agatcaatat ctctccttgt 4860
ggagtggctt ttgtacccca taaaataagg cgaggagttt taccacagat gcttgaagaa 4920
atactcaata caaggattat ggtgaagcga atgatgaagg agtacaaaga cgattcatcc 4980
cttcagcgtg tgctacattc acgacagctt ggtctgaaac ttattgcgaa tgtcacatat 5040
gggtacacca gtgcaaactt ctctggtcga atgccttgtt ctgagattgg tgacagtgtt 5100
gtaagcaaag gaagagagac tttagaacgt tgtattcaac atgtcaataa gcaccctact 5160
tggaaagcag aggtggtgta tggagataca gattcactct tcgtacactt acctggacgg 5220
tctaaggatc aagcatttaa aattggaaat gagattgctg atgaagtaac tcagatgtat 5280
cctaaaccgg tgaaacttaa atttgaaaaa gtttacttac cctgcatttt acagaccaag 5340
aaacgttacg ttggatatat gtatgagtcc acaacccagg agaccccaga gtttgatgct 5400
aaaggcattg agactgttcg tagagatgga tgcccagcaa tggcaaaggt tcttgaaaaa 5460
tccttgcgta ttctctttga aacaaaggat atttcaaaag taaaagacta tattcagcgt 5520
cagtttgtga aacttctaca aggacaggtg tctctccagg atttgatatt tgcaaaagag 5580
tatagaggag cctctggcta taagccaggt gcatgcgtcc ctgctctaga attagcaaga 5640
aaatggactc gcaaagatcc tagaaaagaa ccaagagttg gtgagcgtgt accatatgta 5700
atagtctatg gtgctccagg cttaccttta atccaattag taagatcacc agatgctgtt 5760
ctagcaaatc cagcacttcg tgtaaatgca acatactata tcacaagagc aataatgcca 5820
cctttagaaa gatgtttctc tcttcttggt gtagatattg ctgcttggta taatgacttg 5880
ccaagatcat caagtacaaa tgcttccctg gtgagagaaa tagcaaataa tcagaaagga 5940
tcaagcaact gggaaaaacc atctacttcc catggtatgt ccattgttcg ttacttcact 6000
ccatcagcat gcattgtttg tggagctgca actgttctaa ctggtgcagg gggtggagtt 6060
gcgttatgtt cagactgcaa gactacccca caagacactg tccttgctct taatgagaaa 6120
attcgccatt atgatagagc acttgacaat atacaaaagg tttgccaggc atgccttgga 6180
tgtagactgg aaaaggtgga gtgcatatcc attgactgcc ctatcattta ccagcgtaac 6240
caagcaaaca ttgaccaact tttaactgga cagctgcaag agcttattca aagatttact 6300
agtgtgatat aaaatttata aattccagca ggataatgga gagcttttgt gtaacaatat 6360
taagatttaa gagtaaatta tgactgcaga tccagactga cttagtgatg ctgttaagtg 6420
attgttgaat aagaaaacaa ataagttcag taccttcctt tggcactgat ataagatggc 6480
ttaagtgcat taatgttata agataactga atgcagagta ttattagtga aattattaaa 6540
tcaaattttg tatatattga gctatataat agttcaagaa atttattttg aaaacacaga 6600
attatatatg tacatttagt gaaaaaccct tatgtgaata tataaatagt acgtataaaa 6660
caaagctttc tgtggtaaaa gtttgcatct atgcataaaa aaaaacggtt ttttatgaag 6720
acttcagatt tagtttaaat ggcaaggtag atgtgaagat ggaaggacga ctattttaaa 6780
ggtggtttgg gtctttcaag agtaaaaaaa aaaaaaaaaa aaaaaaaaaa aa 6832
<210> 2
<211> 1203
<212> PRT
<213> 青虾(shrimp)
<400> 2
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Lys Gly Asn Thr Glu Lys Leu Ile Cys Ser Thr Ser Lys Asp Lys Thr
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Lys Gly Thr Asn Ser Glu Ser Ser Glu Lys Leu Ser Gln Ser Glu Asn
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Lys Lys His Asn Lys Glu Met Leu Asp Lys Gln Lys Pro Gln Leu Gly
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Ser Lys Met Lys Asp His Gly Lys Ser Phe Asp Lys Lys Glu Lys Asp
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Ser Lys Ala Arg Asp Phe Val Thr Pro Leu Gly Lys Ala Asp Gln Asn
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Val Cys Gly Asn Ser Ser Ser Glu Gly Asn Pro Met Lys Ile His Asn
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Thr Glu Asn Lys Asn Asp Ser Glu Glu Val Ser Val Pro Asp Lys Lys
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Asp Phe Leu Ser Val Ala Asp Lys Asn Lys Asn Lys Thr Ser Thr Lys
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Lys Thr His Arg Gln Glu Ser Leu Lys Glu Pro Leu Ala Gln Lys Lys
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Asn Lys Asn Pro Ser Glu Asn Ser Cys Arg Asn Pro Ser Val Asn Ser
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Cys Ser Lys Ala Lys Val Phe Thr Thr Ser Lys Glu Lys His His Ser
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Cys Met Gln Glu Pro Ser Arg Asp Lys Ile Asp Leu Asp Lys Ser Gly
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Val Thr Ser Val His Pro Ala Lys Ile Glu Ser His Asp Thr Leu Pro
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Leu Thr Gln Lys Asn Glu Met Pro Leu Glu Pro Cys Lys Gly Pro Leu
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Gly Glu Leu Ser Ser Leu Ala Ala Lys Ser Asp Cys Thr Ser Thr His
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Asp Pro Lys Val Thr Asp Tyr Glu Asn Val Ser Ile Glu Asn Leu Arg
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Lys Ser Ser Asp Lys Ile Asn Pro Pro Glu Lys Leu Ser Ser Val Gln
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Gly Cys Phe Glu Lys Arg Gly Asp Val Thr Gly Gly Ala Asn Leu Pro
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Pro Lys Phe Asp Ser Ser Gln Asn Gly Asp Gly Lys Ile Leu Leu Val
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Thr Thr Glu Asn Ser Ser Val Ser Lys Gly Lys Lys Arg Ile Ser Arg
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Trp Asp Val Lys Asp Lys Asn Ser Ser Ile Thr Ala Val Thr Lys Lys
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Ser Arg Trp Asp Gln Ser Asp Ser Asn Ala Ser Gly Gln Glu Ala Ala
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Lys Gln Glu Ala Ala Ser Glu Lys Arg Lys Arg Leu Lys Gln Met Val
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Ala Asn Leu Phe Glu Asp Cys Glu Asn Glu Asn Pro Ser Cys Arg Gln
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Gln Met Arg Gly Ser Ser Leu Arg Lys Gln Leu Thr Glu Leu Phe Gly
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His Ser Ser Leu Ala Glu Lys Asn Glu Val Ser Glu Glu Tyr Lys Pro
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Gln Pro Ser Thr Ser Lys Asp Lys Val Gly Ser Lys Thr Met Phe Lys
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Glu Ser Leu Glu Ser Leu Phe Glu Pro Ser Ser Thr Phe Gly Val Lys
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Glu Thr Asn Met Arg Ser Asn Trp Lys Glu Asp Thr Glu Asn Glu Tyr
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Gln Pro Thr Thr Ile Val Lys Ser Cys Thr Lys Ala Glu Tyr Asn Pro
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Ser Ser Ile Ser Lys Glu Arg Leu Glu Val Glu Tyr Lys Pro Thr Ser
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Ser Thr Lys Val Arg Val Lys Pro Asp Asp Lys Gly Arg Pro His Val
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Leu Asn Asp Asp Thr Lys Gly Lys Tyr Lys Ser Ser Met Lys Pro Asn
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Ser Lys Thr Thr Ser Asp Thr Leu Asp Lys Lys Ser Leu Lys Tyr Ser
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Lys Thr Lys Cys Asp Ser Met Arg Lys Lys Cys Leu Gly Leu Gly Asp
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Val Ser Ser Ile Lys Asp Ala Lys Thr Val Cys Ser Ala Thr Asp Asp
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Glu Tyr Cys Pro Leu Pro Phe Gly Leu Gly Lys Pro Cys Pro Glu Tyr
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Ile Pro Ser Asp Ile Ser Asp Ile Lys Ser Asn Lys Pro Leu Asp Asp
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Leu Glu Ser His Glu Glu Tyr Val Pro Leu Pro Ala Lys Ala Ile Ser
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Tyr Gly Ile Ser Tyr Asn Ala Ser Gly Glu Glu Lys Pro Ser Lys Ile
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Ser Val Pro Glu Ala Asn Thr Leu Val Pro Ile Ser Tyr Gly Ala Gly
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Ser Thr Gly Leu Gly Phe Tyr Arg Glu Gln Gly Asn Ser Glu Ser Thr
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Gln Ser Lys Glu Gly Glu Ser Ser Val Lys Asp Lys Gly Arg Ser Ser
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Lys Lys Arg Gln Glu Pro Tyr Lys Asn Leu Gly Asn Asn Lys Pro Lys
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Leu Lys Thr Ser Phe Ser Ser Thr Asn Ala Ser Asp Ser Val Val Leu
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Val Tyr Cys Pro Pro Pro Pro Ser Cys Asp Asp Ile Ile Asn Ser Leu
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Ala Asp Tyr Asn Ile Pro Asp Val Ile Asn Pro Gly Ala Phe Cys Ser
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Asn Arg Leu Asp Ile Pro Ala Lys Pro Met Glu Val Gly Gly Gln Val
785 790 795 800
Leu Lys Leu Lys Ser Lys Glu Val Ser Asp Phe Glu Ser Phe Glu Gly
805 810 815
Pro His Gln Asn Asn Gly Leu Tyr Asn Trp Arg Leu Leu Leu Ser Ser
820 825 830
Met Asn Ser Asp Cys Ala Gly Pro Ser Thr Gln Glu Leu Ser Val Asn
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Lys Lys Asp Lys Asn Thr Ser Lys Ser Ile Met Thr Pro Val Lys Val
850 855 860
Pro Pro Ser Pro Ser Glu Val Met Lys Trp Ala Glu Ala Arg Lys Ile
865 870 875 880
Leu Lys Ser Met Lys Lys Lys Asp Glu Asp Gly Asp Asp Gly Gly Ser
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Ser Ser Lys Ser Asp Lys Lys Val Asp Ser Asn Lys Lys Ser Ser Thr
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Ser Leu Pro Pro Gly Ser Asn Gly Val Gly Asn Gly Ser Met Gln Lys
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Asn Ser Ser Gly Gln Asn Asn Gln Gln Ser Arg Asp Gly Asn Val Ser
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Leu Thr Gln Ser Leu Ser Ser Ser Ala Leu Ser Gln Gln Arg His His
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Thr Ser Thr Pro Gln Pro Gln Lys Phe Ser Lys Leu Thr Pro Leu His
965 970 975
Leu Thr Pro Ile Gly Gly Asp Ser Ser Gly Asn Glu Lys Thr Pro Ser
980 985 990
Pro Ala Phe Leu Ser Lys Arg Lys Pro Lys Arg Arg Val Ser Trp Glu
995 1000 1005
Asp Gly Ser Pro Ser Val Lys Pro Ser Leu Leu Lys Lys Arg Arg Ile
1010 1015 1020
Ser Tyr Glu Gly Leu Pro Val Leu His Glu Glu Thr Pro Glu Val Ser
1025 1030 1035 1040
Glu Pro Lys Lys Ser Leu Val Arg Arg Val Ser Phe Ser Gly Ile Thr
1045 1050 1055
Gln Pro Ala Gly Gly Phe Lys Val Thr Glu Val Gln Asp His Ser Lys
1060 1065 1070
Ala Glu Glu Leu Lys Pro Met Pro Glu Ser Pro Glu Ser Ala Lys Glu
1075 1080 1085
Cys Thr Ser Glu Lys Gly Glu Asp Leu Asp Ser Thr Leu Val Thr Gln
1090 1095 1100
Val Ala Asp Pro Asp Ile Glu Ser Ser Pro Glu Thr Ile Ile Ser Ser
1105 1110 1115 1120
Ser Arg Ser Leu Asp Leu Thr Leu Ser Leu Ser Thr Lys Ala Ala Ser
1125 1130 1135
Gln Pro Ser Ser Gln Met Ser Asp Ser Ser Leu Arg Arg Gln Leu Met
1140 1145 1150
Asp Thr Gln Phe Arg Gln Gln Phe Leu Ser Phe Ser Gln Val Pro Ser
1155 1160 1165
Gly Ser Ser Gln Gly Leu Asp Gly Pro Thr Leu Asn Asn Thr Tyr Gly
1170 1175 1180
Phe Lys Phe Ser Asn His Asn Leu Arg Glu Ala Lys Ala Leu His Glu
1185 1190 1195 1200
Ile Pro Ser
<210> 3
<211> 22
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 3
ccatctactt cccatggtat gt 22
<210> 4
<211> 22
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 4
atccattgac tgccctatca tt 22
<210> 5
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 5
agtgacagca acgctagtgg 20
<210> 6
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 6
ggccaaacaa ctctgtcagc 20
<210> 7
<211> 38
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 7
catctaatcc ctgcgaagag cctgaaggaa cttgtgag 38
<210> 8
<211> 38
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 8
ctcacaagtt ccttcaggct cttcgcaggg attagatg 38
Claims (8)
1.青虾Rev3基因，其特征在于，Rev3基因部分cDNA序列如SEQ ID NO:1所示，其中3’端为完整序列。
2.根据权利要求1所述的青虾Rev3基因，其特征在于，Rev3基因cDNA序列克隆的3’端特异性正向引物为：3Rev3-F1、SEQ ID NO:3和3Rev3-F2、SEQ ID NO:4。
3.根据权利要求1所述的青虾Rev3基因，其特征在于，Rev3基因的荧光定量PCR引物为Rev3-RTF、SEQ ID NO:5，Rev3-RTR、SEQ ID NO:6。
4.根据权利要求1所述的青虾Rev3基因，其特征在于，Rev3基因的原位杂交试验中，正义原位杂交探针为SEQ ID NO:7，反义原位杂交探针为SEQ ID NO:8。
5.由青虾Rev3基因编码的蛋白，其特征在于，该编码蛋白的氨基酸序列如SEQ ID NO:2所示。
6.权利要求1-4任一所述青虾Rev3基因在调控青虾雄性分化及其发育过程中的应用。
7.权利要求1-4任一所述青虾Rev3基因在调控青虾免疫应答机制中的应用。
8.权利要求1-4任一所述青虾Rev3基因在青虾全雄群体选育中的应用。
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