CN108998510B - 半滑舌鳎性别标签piR-xtr-979116的应用 - Google Patents
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Abstract
一种半滑舌鳎性别标签piR‑xtr‑979116的应用，所述性别标签piR‑xtr‑979116来源于鱼精浆外泌体，本发明通过smallRNA测序分析，筛选到两种鱼显著差异表达的标签piRNAs作为候选，通过实时定量PCR验证，最终确定了对两种鱼具有指示作用的piRNA生物标志物，标签piRNAs为piR－xtr－979116，序列为CGGGTTCGTTTCCCGGCCAACGCACCA，并以此为基础开发制备了检测试剂盒。本发明方法具有无创，高效的优点，且鉴定结果可靠，是首次通过定量检测来判别半滑舌鳎的遗传性别。
Description
技术领域
本发明属于鱼类生物技术领域，特别是涉及一种半滑舌鳎性别标签piR-xtr-979116的应用。
背景技术
piRNA(Piwi-interactiing RNA)是一类长度约为26-32nt(nucleotides)的非编码RNA，于2006年首次在果蝇、小鼠、大鼠和人的生殖系细胞中被四个独立研究组发现。在所有的非编码RNA中,piRNA数量最多,主要存在于生殖系统。piRNA作为PIWI的向导，调控靶基因的表达以及转录和转录后水平的修饰。piRNA不同于miRNA的序列特征和产生方式,由基因组序列转录加工而来,不需要Dicer的剪切。Piwi结合piRNA后进入核内,参与表观调节,有证据表明可能是转录沉默或者转录激活,具体机制还未知。piRNA在动物发育和生殖调控中起重要作用,在果蝇卵巢生殖细胞中,piRNA簇的转录子转运到胞浆后经过初级加工途径形成初级piRNA,结合到Piwi和Aub上。生殖细胞的初级piRNA还会进入次级加工途径,经PIWI家族蛋白的协同、加工,使细胞中的piRNA大量扩增,称为乒乓循环(“Ping-Pong”cycle),但是目前对乒乓循环的细节和具体机制也所知甚少。
半滑舌鳎(Cynoglossus semilaevis)为我国重要的海水养殖鱼类品种，其经济价值高，适合工厂化养殖，在我国已形成了较大的养殖规模，作为名贵海水鱼具有巨大的市场空间。众所周知，半滑舌鳎雌、雄鱼生长速度和体型差异巨大，养殖中存在雄鱼比例高，经济价值低的问题。现发现雄鱼中有很大比例的伪雄鱼，其遗传上是雌性染色体，但从它生理上表现的是雄性特征。伪雄鱼也可以像雄鱼一样产生精子并繁育后代。实验表明用伪雄鱼作为父本，后代也会遗传父亲的特征成为伪雄鱼，导致了雄鱼比例高的问题。鉴别伪雄鱼的方法通常包括雌性特异微卫星分子标记法和染色体的方法，目前尚未发现半滑舌鳎的雄性相关的分子标记。
发明内容
本发明要解决的技术问题在于提供一种半滑舌鳎性别标签piR-xtr-979116的应用，所述的性别标签piR-xtr-979116来源于半滑舌鳎的精浆外泌体piRNA，以解决半滑舌鳎雄鱼及伪雄鱼遗传性别的区分问题。在海洋动物中外泌体来源的piRNA作为生物标志物的研究几乎没有报道，本发明开发了针对以半滑舌鳎为代表的鱼精浆来源的外泌体提取及初步应用的方法，解决了半滑舌鳎雄鱼及伪雄鱼遗传性别的识别问题。
本发明解决其技术问题是采取以下技术方案实现的：
一种半滑舌鳎性别标签piR-xtr-979116的应用，所述的性别标签piR-xtr-979116来源于半滑舌鳎的精浆外泌体piRNA，序列为CGGGTTCGTTTCCCGGCCAACGCACCA。
本发明还提供一种所述精浆外泌体piRNA的筛选方法，具体步骤如下：
1)精浆外泌体分离鉴定后，对精浆外泌体smallRNA进行测序分析，Small RNA种类繁多，将clean reads依次和Rfam数据库、cDNA序列、物种重复序列库、piRBase数据库进行比对，以对测序结果中的smallRNA进行分类注释；将注释后的序列和piRBase数据库比对，进行已知piRNA的统计；使用未注释上的序列进行新的piRNA预测，piRNA差异表达分析，差异表达piRNA靶基因的通路富集分析和功能富集分析，最后与雄鱼、伪雄鱼样品进行相关性匹配，筛选出一种或者多种对雄鱼和伪雄鱼两种样品具有指示作用的piRNA作为候选的生物标记物；最终筛选到39个候选的piRNA作为下一步验证的备选。
2)提取两组验证样本：经外周血染色体鉴定后的雄鱼及伪雄鱼精浆来源外泌体总RNA，利用MicroRNA定量分析试剂盒定量检测候选piRNA的差异表达情况，筛选出最具标签指示作用的piRNA作为最终的标签piRNA；根据统计学分析原理，P值小于0.05为显著差异；筛选到在雄鱼与伪雄鱼中差异表达的piRNA生物标记物piR-xtr-979116，序列为CGGGTTCGTTTCCCGGCCAACGCACCA。
本发明提供了一种半滑舌鳎雄鱼及伪雄鱼检测试剂盒，其特征在于：所述试剂盒包括piRNA检测引物序列为F：TTAATCGGGTTCGTTTCCC，R:TCGTATCCAGTGCAGGGTC，PCR逆转录引物序列piR-xtr-979116-RT：GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGATGGTGCGT及逆转录试剂和qPCR荧光定量试剂，内参piR为U6。
进一步,U6引物为U6-F：CTCGCTTCGGCAGCACATATACT；U6-R：ACGCTTCACGAATTTGCGTGTC。
进一步，引物探针序列的5’端标记了荧光基团，3’端标记淬灭基团，以适合Taqmanprobe-based qRT-PCR方法检测。
本发明与现有技术相比的有益效果
本发明创新性的运用精液外泌体piRNA进行半滑舌鳎性别鉴定，开发了检测的试剂盒，在水生动物中尚属首次，本鉴定方法具有无创，高效的优点，且鉴定结果可靠，是首次通过定量检测来判别半滑舌鳎的遗传性别。
附图说明
图1本发明提出的标志piRNA经20个验证样本验证后的qRT-PCR表达量；
图2本发明20个验证样本验证后在两组样本中的qRT-PCR表达量均值。
具体实施方式
以下结合具体实施方式对本发明做进一步详述，以下实施例只是描述性的，不是限定性的，不能以此限定本发明的保护范围。
实施例1
一种半滑舌鳎精液来源的外泌体piRNA作为生物标记物的筛选方法
1、精液样本的收集
收集半滑舌鳎性成熟雄鱼精液0.5ml于离心管中用于外泌体分离鉴定。
2、外泌体的提取
(1)取0.5ml精液样本转移1.5mlEP管，放置于4℃，1200g离心15min去除精子细胞，4℃，15000g离心20min去除小细胞杂质和碎片，用PBS稀释1倍后，使用0.45um滤膜进行预过滤，过滤液再经0.22um滤膜过滤。
(2)过滤后的样品用Total Exosome Isolation Kit试剂盒提取外泌体。
(3)收集溶解样并完全转移至一个无RNA酶的2ml管中。
3、外泌体的鉴定：日立H600IV型透射电镜观察，透射电镜分析鉴定后，剩余样品用于RNA提取和测序分析。
4、外泌体的piRNA测序分析
TRizol法提取精浆外泌体的RNA，对精浆外泌体的smallRNA进行测序分析。SmallRNA种类繁多，包括miRNA、tRNA(tiRNA、tRFs)、rRNA、piRNA、snoRNA等，质检后构建小RNA文库并基于illumina平台测序，完成测序后进行数据过滤分析，将clean reads依次和Rfam数据库、cDNA序列、物种重复序列库、piRBase数据库进行比对注释。将过滤后的序列和piRBase数据库比对，进行已知piRNA的统计。使用未注释上的序列进行新的piRNA预测，piRNA差异表达分析，差异表达piRNA靶基因的通路富集分析和功能富集分析，最后与两个样品进行相关性匹配，筛选出一种或者多种，对雄鱼和伪雄鱼两种样品具有指示作用的piRNA作为候选的生物标记物。
5、实时荧光定量PCR分析验证
提取雄鱼及伪雄鱼精浆来源外泌体总RNA，利用MicroRNA定量分析试剂盒定量检测待检测两个样品中piRNA的表达情况，内参选择在两组样品中都具有稳定高表达的U6选取2-ΔΔCt进行计算分析。筛选到在雄鱼与伪雄鱼中差异表达的piRNA生物标记物piR-xtr-979116，序列为CGGGTTCGTTTCCCGGCCAACGCACCA。
实施例2
半滑舌鳎精液外泌体miRNA标志物鉴定雄鱼和伪雄鱼的试剂盒
基于精液外泌体piRNA标志物piR-xtr-979116的鉴定试剂盒包括piR-xtr-979116定量检测引物，引物序列为，F：TTAATCGGGTTCGTTTCCC，R:TCGTATCCAGTGCAGGGTC，PCR逆转录引物piR-xtr-979116-RT：GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGATGGTGCGT及试剂和qPCR荧光定量试剂，内参piR为U6，引物为U6-F：CTCGCTTCGGCAGCACATATACT；U6-R：ACGCTTCACGAATTTGCGTGTC；试剂盒引物包含PCR逆转录引物，定量PCR正向引物和反向引物，此外试剂盒包含定量PCR反应的其它常规试剂：逆转录酶，Taq酶，dNTP，缓冲液，Mgcl2，DEPC水及对照品。该试剂盒应用的反应体系为10ul体系，即0.5ul 10*miRNA引物探针、5ul 2*Master mix、2.5ul ddH2O、2ulcDNA模版。使用Thermo fisher的Q6实时荧光定量PCR检测，反应的程序为：95℃2min；95℃10s,59℃60s,循环40次。样品检测设3个平行。利用筛选到的piR-xtr-979116的定量检测引物和逆转录引物检测经染色体确认的10尾半滑舌鳎伪雄鱼和10尾半滑舌鳎雄鱼的标签piRNA，即piR-xtr-979116的表达情况，结果如表1、图1、图2所示。该标签在两组样本中的表达差异极为显著(p＜0.01)，验证标签piRNA的检测准确率达90％以上。
表1 20尾半滑舌鳎验证样本标签piR-xtr-979116的表达情况
对于本领域的技术人员，对本发明针对的构思及技术要点进行变形，相应的改变应属于本发明所请求的权利要求。
序列表
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gtcgtatcca gtgcagggtc cgaggtattc gcactggata cgatggtgcg t 51
Claims (2)
1.一种半滑舌鳎性别标签piR-xtr-979116的应用，所述的性别标签piR-xtr-979116来源于半滑舌鳎的精浆外泌体piRNA，序列为CGGGTTCGTTTCCCGGCCAACGCACCA。
2.根据权利要求1所述的一种半滑舌鳎性别标签piR-xtr-979116的应用，其特征在于所述精浆外泌体piRNA的筛选方法，具体步骤如下：
1）精浆外泌体分离鉴定后，对精浆外泌体smallRNA进行测序分析，Small RNA 种类繁多，将 clean reads 依次和 Rfam 数据库、cDNA 序列、物种重复序列库、piRBase 数据库进行比对，以对测序结果中的smallRNA 进行分类注释；将注释后的序列和 piRBase 数据库比对，进行已知 piRNA 的统计；使用未注释上的序列进行新的 piRNA 预测，piRNA差异表达分析，差异表达piRNA靶基因的通路富集分析和功能富集分析，最后与雄鱼、伪雄鱼样品进行相关性匹配，筛选出一种或者多种对雄鱼和伪雄鱼两种样品具有指示作用的piRNA作为候选的生物标记物；
2）提取随机两组验证样本：经外周血染色体鉴定后的雄鱼及伪雄鱼精浆来源外泌体总RNA，利用MicroRNA定量分析试剂盒定量检测候选piRNA的差异表达情况，筛选出最具标签指示作用的piRNA作为最终的标签piRNA；根据统计学分析原理，P值小于0.05为显著差异；筛选到在雄鱼与伪雄鱼中差异表达的piRNA生物标记物piR-xtr-979116，序列为CGGGTTCGTTTCCCGGCCAACGCACCA。
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