CN107828893A - 一种臂尾轮属轮虫核基因Hsp82的部分DNA测序及鉴定臂尾轮属轮虫的方法 - Google Patents
一种臂尾轮属轮虫核基因Hsp82的部分DNA测序及鉴定臂尾轮属轮虫的方法 Download PDFInfo
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Abstract
本发明涉及一种臂尾轮属轮虫核基因Hsp82的部分DNA测序及鉴定臂尾轮属轮虫的方法，测序方法步骤包括臂尾轮属轮虫总DNA提取、PCR引物扩增及序列测定、系统发生分析；鉴定方法使用臂尾轮属轮虫核基因Hsp82的DNA进行鉴定。本测序方法中所设计的PCR引物可以扩增出红臂尾轮虫等核基因Hsp82的DNA，测序结果可用于对红臂尾轮虫等进行精确鉴定，解决了利用形态学和超微结构存在的鉴定困难，使得在大规模培养轮虫的过程中，减少种群混杂带来的种群竞争而造成轮虫减产甚至大量死亡的可能性。
Description
技术领域
本发明属于生物技术领域，具体涉及一种臂尾轮属轮虫核基因Hsp82的部分DNA测序及鉴定臂尾轮属轮虫的方法。
背景技术
早期的轮虫分类学和系统学研究主要是依据形态学数据。如Wallace(1989)利用八种形态学特征进行聚类分析研究发现轮虫门(Rotifera)、真轮虫总纲(Eutatoria)和单巢纲(Monogonota)为单系，利用九种形态学特征进行聚类分析研究发现叶轮属(Notholca)分为六个分支。Melone(1998)通过对1种棘头动物、6种轮虫和2个外群(涡虫和颚胃动物)的60个形态特征进行矩阵分析，获得一个简约聚类树，发现双巢纲(Digononta)为并系类群，但不能确定轮虫门的单系性。该系统树不同于依据分子水平数据或演化分类原理而产生的系统树。以形态学数据所研究的轮虫系统学存在缺陷，因为轮虫的形态受遗传和环境因子两方面的影响，尽管遗传因素占主导地位。随着轮虫研究的深入，已有研究者发现形态学数据已经不能满足轮虫部分种类鉴别和系统进化研究(Guiset，1977；Koste，1978；Shiel，1993；Fontaneto，2007)。Sergi(2005)等研究了褶皱臂尾轮虫(B.plicatilis)L-型的两个种B. plicatilis sensu stricto和B.manjavacas之间的形态度量差异，发现B.plicatilis s.s.比B. manjavacas平均长6％，两个种的个体之间表现出较大的形态度量交迭，形态学分类不能用于鉴别这两个种，目前它们的分类只能依据分子指标。2003年，为了了解形态可塑性和基因变异在轮虫的形态变化中的作用，Derry等研究了不同形态的龟甲轮虫(Keratella)线粒体CO Ⅰ基因序列，发现有棘刺和无棘刺的螺形龟甲轮虫(Keratella cochlearis)遗传变异较大，核酸序列的差异达到4.4％，暗示它们是不同的种。而单棘刺和双棘刺的曲腿龟甲轮虫(K.hiemalis) 核酸序列的差异为0.21％，并且氨基酸序列没有变异，说明它们的形态变异归因于环境的诱导。
传统的轮虫分类主要依据头冠、棘刺、足和口器等外部形态和内部结构等形态学特征。但由于轮虫种类众多、体型微小及表型可塑性，轮虫外部形态有些物种非常相似，并且同一物种还有季节性的周期变形，且许多种类缺乏有效地形态学分类特征，仅通过传统的方法进行鉴定和分类存在一定的困难，如壶状臂尾轮虫和红臂尾轮虫均为臂尾轮属常见种类，因它们具有相似的形态，早期的研究曾将这两种轮虫归为一种，称为Brachionusurceus，也译为壶状臂尾轮虫(王家楫)；后来Koste、章宗涉和黄祥飞等根据虫体前端棘刺的两侧是否对称和眼点的形状把B.urceus分为B.urceolaris和B.rubens两种，因此种的界定标准仍存在模糊不清之处，因此传统鉴定方法满足不了鉴定要求，并且内部咀嚼器的超微结构需要电子显微镜的辅助，鉴定困难。
发明内容
针对现有技术的不足，本发明提供一种臂尾轮属轮虫的核基因Hsp82的部分DNA测序及鉴定臂尾轮属轮虫的方法，本鉴定方法使用实验所设计的引物扩增轮虫核基因组上Hsp82DNA序列，并对PCR产物进行纯化、连接、克隆、转化、测序，将序列进行比对分析来鉴别轮虫的种类。对轮虫进行分子生物学的种类鉴定，解决了利用形态学和超微结构存在的鉴定困难，使得在大规模培养轮虫的过程中，减少种群混杂带来的种群竞争而造成轮虫减产甚至大量死亡的可能性。
为解决上述技术问题，本发明的技术方案是：
一种臂尾轮属轮虫的核基因Hsp82的部分DNA测序方法，包括以下步骤：
A、臂尾轮属轮虫总DNA提取；
B、PCR引物扩增及序列测定；
C、系统发生分析。
所述步骤B中PCR引物为：Hsp82F:5’-TGCTCTAGAGCACARTTYGGTGTNGGTTTYTA-3’和Hsp82R:5’-ACGTAAGCTTRTGRTCYTCCCARTCRTTNGT-3’；
扩增反应程序为：95℃预变性5min；95℃变性30s，50℃退火50s，68℃延伸1min，共35个循环；最后72℃延伸10min，4℃保存。
一种鉴定臂尾轮属轮虫的方法，使用臂尾轮属轮虫的核基因Hsp82的部分DNA序列进行鉴定。
基于核基因Hsp82的部分DNA序列鉴定臂尾轮属轮虫，结果准确可靠；本方法中所设计的PCR引物可以扩增出萼花臂尾轮虫、角突臂尾轮虫、红臂尾轮虫、方形臂尾轮虫、镰形臂尾轮虫、十指臂尾轮虫、尾突臂尾轮虫和壶状臂尾轮虫的核基因Hsp82部分DNA，利用对Hsp82DNA比对结果(包括A、C、G、T碱基的含量、变异位点、简约信息位点等)进一步分析了有争议种类的分类地位以及对轮虫种群之间的亲缘关系及轮虫的系统进化做出分子生物学的解释，可对萼花臂尾轮虫、角突臂尾轮虫、红臂尾轮虫、方形臂尾轮虫、镰形臂尾轮虫、十指臂尾轮虫、尾突臂尾轮虫和壶状臂尾轮虫进行精确鉴定。解决了利用形态学和超微结构存在的鉴定困难，使得在大规模培养轮虫的过程中，减少种群混杂带来的种群竞争而造成轮虫减产甚至大量死亡的可能性。
具体实施方式
本鉴定臂尾轮属轮虫的方法所用生物材料为萼花臂尾轮虫、角突臂尾轮虫、红臂尾轮虫、方形臂尾轮虫、镰形臂尾轮虫、十指臂尾轮虫、尾突臂尾轮虫和壶状臂尾轮虫，挑取每种轮虫单个个体，单克隆培养，其中红臂尾轮虫、方形臂尾轮虫采用Gilbert(1963)配方，其余几种采用原地方水样过滤后作培养液，温度与采集时环境温度基本保持一致，所用食物为以HB-4培养液培养的，处于指数生长期的斜生栅藻，当单个克隆轮虫个体数目达到一定多的量的时候，用25μm孔径滤网滤出轮虫，并以无菌蒸馏水冲洗，Eppendof管收集，置于-20℃冰箱保存。
总DNA提取：利用董云伟等(2002)的方法提取轮虫DNA。采用WizardTM基因组 DNA纯化试剂盒(pormega，USA)提取轮虫DNA。将冻存的轮虫从-20℃冰箱中取出，离心数秒，吸去多余水分。加入60μl核裂解液(nuclear lysis solution)，轻弹离心管以使轮虫悬浮均匀，在4℃放置10min，取出。65℃水浴40min。取出冷却至室温，加入20μl蛋白沉降液(protein precipitation solution)，剧烈震荡10s，冰浴5min，13000g离心4min。小心吸取上清，加入 60μl异丙醇，2.5μl糖原(20mg/ml,Sigma，USA)，小心混匀。-70℃放置30min,13000g离心20min，然后弃去上清，用70％酒精洗涤两次。在无菌室将其吹干，然后加入50μl无菌双蒸水，4℃放置过夜以溶解DNA。
PCR扩增及序列测定：扩增反应在PCR扩增仪上进行。PCR引物设计如下：Hsp82F:5’-TGCTCTAGAGCACARTTYGGTGTNGGTTTYTA-3’和Hsp82R：5’-ACGTAAGCTTRTGRTCYTCCCARTCRTTNGT-3’引物于上海Invitrogen公司合成；PCR反应体系的总体积为25μl，包括1×LAPCR BufferⅡ(Mg2+Plus)，0.4mmol/L dNTP，1.5μmol/L引物，DNA模板2μl，1U的TaKaRa LaTaq酶,超纯水补至25μl；扩增反应程序如下:95℃预变性5min；95℃变性30s,48℃退火50s,68℃延伸3min，共15个循环；95℃预变性5min；95℃变性30s，48℃退火50s,68℃延伸3min，延伸时间每个循环加5s，共15个循环；最后72℃延伸10min,4℃保存；扩增完成后，用BBI 试剂盒纯化PCR产物，取2μl于1％的琼脂糖凝胶电泳上检查，其余-20℃保存备用；将纯化产物用TaKaRa Pmd 18-T Vector克隆；最后送上海生工测序。
系统发生分析：对测序结果进行处理并分析，使用CLUSTAL X(1.81)软件(Thompson，1999)进行DNA序列比对。
通过引物测得轮虫Hsp82序列如下：
萼花臂尾轮虫：
ttgctctagaaacacagtttggtgttggtttttactcagcttatttagtcgctgataaagtcgtagtaactactaaacacaatgatgatgaacaatatacttgggaatcatcggctggtggtagttttacagtgagattagacaattcggaaccattaggtagaggaacaaaaattgttttgcacatgaaag aagatcaagccgagtacaatgacgagaaaaaaattaaagaaattgtcaaaaaacactcgcaatttatcggctatccgattaaattagttgttgagaaagaaagagaaaaagaaatagatgaagatgaaggtgaggaagaggtgaaagaagagaaaaaagaggaagaaaaagccaaaatagaagagattgaagaagaggaagagggtgaaaaagaaggtaaaaagaagaaaaaaattaaagaaaagtataaagaagaggaagaattgaataaaactaagccattgtggactagaaatccagaagatattcacaaaatgaatacgctgaattttataaatcgttaaccaacgactgggaagac cataaggcttacgta
角突臂尾轮虫
ttgctctagggacacagtttggtgttggtttttactcagcttacttagtagctgataaagtcacagtcactactaagcacaatgacgatgagcaatacacttgggaatcgtctgctggtggtagtttcacaatcaaattggacaactctgaaccattgggtcgtggtagtaagattgtattgcacatgaaagaggatcaagctgaatttaatgatgagaaaaaaattcgcgaaattgtcaaaaaacattcgcaattcattggctatccaattaaattggtcgttgagaaagaacgtgaaaaggaaataagtgaagacgaggctgaagaagaagagaaaatggaggaaaaaaaagaggaagaaaagccgaaaatagaagaaatcgaagaagaagaagaggataaagaaaaaaagaagaagaaaaaaattaaagaaaaatatttagaagaggaagaattgaacaagacaaagccattgtggacaagaaatccagaggacatatctcaagcagagtatggtgaattttacaaatcgttaacaaacgactgggaagaccacaagcttacgta
红臂尾轮虫
ttttttagagcacaatttggtgttggtttttactcagcttacttggttgctgatagagtaacagtcacaacaaaacacaatgatgatgaacaatacgtatgggaatcatcagctggtggtagtttcactgttaaactcgacaactctgaaccattaggtcgtggtaccaagattgtattgcacatgaaagaagatcaagctgagtacaacgatgagaaaaaagtcaaagaaatcgtcaagaaacactctcaattcattggctacccaatcaaattgttagtcgagaaggaaagagaaaaagaaatcagtgacgatgaagctgaagaagaaaagaaagaagagaaaaaagaaggtgaagaagaaaaaccaaaagtagaagaaattgaagatggttcagacaaagaagatgaaggtgacaaagacaaaaagaagaagaagaaaatcaaagaaaagtacaccgatgaagaagaattgaacaaaacaaaaccattgtggacaagaaatccagaagacatttcacaagctgaatatggtgaattctacaagtcattgaccaacgattgggaagaccacaaagcttacgtaa
方形臂尾轮虫
tttggtgtgggtttatactcaggttacttggttgtcgatagagtaacagtcagttcaaatcacaatgacgatgaacaatacatatgggaatcatcacatggtggcatctttgctgttcagtttgacaactcagaaacaatcggctgtggtactaaactcgttttacacatgaaagaagaccaagcggaatacaatgatgagaagaaaatcaaagaaatggtcaagaaacacttgcaattcattggctatccaatcaaattattagtagaaaaagagagagagaaagaaatcagtgacgatgaaattgaagaggagaagaaggaagaggagaaaaaagagggggaagaagacaagccaaaagtagaagaaattgaagatggatcagacaaagaagatgaaggggaaaaggacaagaagaagaagaagaaaatcaaggagaagtacaatgacgaagaagaattgaacaaaaccaaaccattgtggacaagaaatccaga
镰形臂尾轮虫
ttgctctaggaacacaatttggtgttggtttttactctgcttacttagttgctgacagagtcacagtcaccacaaaaaacaacgatgatgaacaatacgtttgggaatcatcagctggtggtagtttcactgttaaactagacaattcagagccattaggtcgtggtacaaaaattgttttacacatgaaagaagatcaagctgaatacaacgatgagaagaaagtcaaagaaattgtcaagaaacattcacaattcattggctatccaattaaattgatggttgagaaggaaagagaaaaagaaattagcgatgacgaagctgaagaagagaaaaaagaagaaaaaaaagagggtgaagaagataaaccaaaagtagaagaaatagaagatgagtcagataaagaagacgaaggcaataaagacaaaaagaagaagaagaaaatcaaagagaaatatattgatgaagaagagttaaacaaaactaaaccattgtggactcgtaatccagaagatatttcacaagctgaatatggtgaattttacaaatcattg acaaacgactgggaagaccacaagcttacgta
十指臂尾轮虫
accaatttgttttggggttattctcagcttacttggttgtcgatagagtaacagtcagttcaaatcacaatgacgatgaacaatacatatgggaatcatcacatggtggcagctttgctgttcaacttgacaactcagaaccaatcggccgtggtaccaaactcgttttacacatgaaagaagaccaagccgaatacaacgatgagaagaaaatcaaagaaatggtcaagaaacactcgcaattcattggctatccaatcaaattattagtagaaaaagagagagagaaagaaatcagtgacgaggaagttgaagaggagaagaaggaagaggagaaaaaagaggaggaagaagacaagccaaaagtagaagaaatcgaagatggatcagacaaagaagatgaaggcgaaaaggacaagaagaagaagaagaaaatcaaggagaagtacaatgacgaagaagaattgaacaagaccaaaccattgtggacaagaaatccagaagac
尾突臂尾轮虫
gacaatttggtgttggtttttactctgcttacttagttgctgacagagtcacagtcaccacaaaaaacaacgatgatgaacaatacgtttgggaatcatcagctggtggtagtttcactgttaaactagacaattcagagccattaggtcgtggtacaaaaattgttttacacatgaaagaagatcaagctgaatacaacgatgagaagaaagtcaaagaaattgtcaagaaacattcacaattcattggctatccaattaaattgatggttgagaaggaaagagaaaaagaaattagcgatgacgaagctgaagaagagaaaaaagaagaaaaaaaagagggcgaagaagataaaccaaaagtagaagaaatagaagatgagtcagataaagaagacgaaggcaataaagacaaaaagaagaagaagaaaatcaaagagaaatatattgatgaagaagagttaaacaaaactaaaccattgtggactcgtaatccagaagatattcacaa
壶状臂尾轮虫
tttctttagagcccaatttggtgttggtttttattcagcttatttggttgccgatagagttacagtcactacaaagcacaatgatgacgaacaatacacatgggaatcatccgctggtggtagtttcactgttaaacttgacaattctgagccattgggacgtggtactaagattgtattacacatgaaagaagatcaagctgaatataatgatgaaaagaagattaaggaaatcgtcaaaaaacactcgcaatttattggctatccaatcaaattgctcgttgaaaaagaaagagacaaggaaattagcgatgatgaagctgaagaagagaaaaaagaagaaacaaaagaagctgaggaaaagccaaaagtagaagaaattgaagaggaatttgacaaagaggacgagagtggtaaagaaaaaaaaaagaagaagaaaattaaagaaaaattcaaggaagaagaagaattgaataaaacgaaaccgttgtggactagaaatccagaagatatatctcaagctgaatatggggaattttataaatcattgacaaacgattgggaaaaccacaagcttacgta。
Claims (2)
1.一种臂尾轮属轮虫的核基因Hsp82的部分DNA测序的系统发生分析方法，其特征在于：包括以下步骤：
A、臂尾轮属轮虫总DNA提取；
B、PCR引物扩增及序列测定；
C、系统发生分析；
所述步骤B中PCR引物为：Hsp82F:5’-TGCTCTAGAGCACARTTYGGTGTNGGTTTYTA-3’和Hsp82R:5’-ACGTAAGCTTRTGRTCYTCCCARTCRTTNGT-3’，其中R为G或A，Y为T或C，N为A，G，C或T。
2.如权利要求1所述的系统发生分析方法，其特征在于：所述步骤B中扩增反应程序为：95℃预变性5min；95℃变性30s，50℃退火50s，68℃延伸1min，共35个循环；最后72℃延伸10min，4℃保存。
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