【作者】 王庆；

【导师】 傅洪拓；

【作者基本信息】 南京农业大学 ， 水生生物学， 2007， 硕士

【摘要】 日本沼虾(Macrobrachium nipponense)，俗称青虾、河虾，在动物分类学上属节肢动物门、甲壳纲、十足目、长臂虾科、沼虾属。日本沼虾在我国分布很广，具有生长快、繁殖力强、适应性广等特点，是目前淡水养殖业中重要的养殖对象和最有发展前途的品种之一。但近年来，日本沼虾的性状出现了退化，包括性早熟、规格变小、商品率低、抗病能力下降等，极大的影响了日本沼虾养殖业的效益。要实现日本沼虾养殖业的可持续发展，迫切需要对日本沼虾进行资源保护和遗传改良，而对日本沼虾进行遗传背景分析，研究其遗传分化情况，则是资源保护和遗传改良的基础。核rDNA的内转录间隔区(ITS)是由18S和5.8S rRNA之间的ITS-1片段和5.8S和28S rRNA之间的ITS-2片段组成。由于ITS区不加入成熟核糖体，所以受到的选择压力较小，变异丰富。另一方面，18S，5.8S和28S rDNA的高度保守性，能较方便地设计出上下游引物，而使PCR扩增得以实现。利用ITS序列的丰富变异性，可从DNA水平来分析个体间和种群间的遗传变异，为遗传育种提供背景知识。本文将ITS分析技术应用到淡水虾类的遗传研究中，对日本沼虾进行DNA水平的遗传分析。在筛选合适的引物的基础上，采用聚合酶链式反应(PCR)技术对日本沼虾的核rDNA的内转录间隔区的ITS-1进行了扩增，电泳检测得到了一条清晰的扩增产物条带。PCR产物纯化后经T载体连接、克隆和测序，用DNAStar软件拼接处理后得到长度为1763bp的日本沼虾ITS-1序列，是迄今已报道的最长的ITS-1序列，其中A，T，G，C平均含量分别为29.92％、27.71％、28.36％、14.26％，G+C的含量42.44％。日本沼虾ITS-1全序列中存在多个重复序列，其中TTTATTAAAGAAGAAA重复6次，3处存在(GA)_n重复序列，分别重复5次，6次，5次；序列比对发现共有13处碱基转换，9处碱基颠换；共发现4处存在碱基缺失，分别缺失T、GAGA、ATAAAGAGAGAAATTTXTAAAGAGAGAATTT和CAGCAGCAG；发现碱基插入2处，分别插入A和GAGAAATTTTGAAAGAGAGA。本文工作填补了日本沼虾ITS序列研究方面的空白，丰富了日本沼虾遗传背景资料，可为日本沼虾遗传育种研究提供参考。更多还原

【Abstract】 Macrobrachium nipponense, also named river prawn, belongs to Arthopoda,Crustacean, Decapoda, Palaemonide, Macrobrachium in taxonomy. Macrobrachiumnipponense widely distributed in China and characterized by rapid growth, strongreproduction and extensive adaptability was an important breed in freshwater aquacultureand one of the most productive species. Macrobrachium nipponense is one of importantfreshwater species for aquaculture in China. However, serious retrogression of economictraits appeared recently in Macrobrachium nipponense culture, such as prematuration, smallsize, low marketing rate weak disease-resistance which greatly lowered its profits.In orderto realize the sustainable development of Macrobrachium nipponense culture, it’s necessaryto carry out resources protection and genetic improvement and investigation on geneticbackground and genetic divergence is the basis.Nuclear rDNA Internal Transcribed spacer(ITS) are composed of ITS-1 fragmentbetween 18S and 5.8S rRNA and ITS-2 fragment between 5.8S and 28S rRNA. Lessselective pressure and extensive variation were found on ITS due to its absence in matureribosome rDNA. On the other hand, primers were designed conveniently because of highconservation of 18S, 5.8S and 28S rRNA. Analysis on genetic divergences betweenindividuals and populations and genetic background were carried out by using the extensivevariation of ITS sequences.In this paper, ITS technology was applied to genetic research of Macrobrachiumnipponense. Internal Transcribed Spacer (ITS-1) region of nuclear rDNA fromMacrobrachium nipponense were amplified by PCR with suitable primers and a clearamplification band appeared in electrophoresis. ITS-1 sequence with 1763 bp in size wasproduced with DNAStar software after T-carrier connection, clone and sequencing of thepurified PCR products. ITS-1 sequence above was the longest one among reported ITS-1sequences so far. The contents of A, T, G and C were 29.92%, 27.71%, 28.36% and 14.26%respectively. G+C content was 42.44%, and A+T content was higher than G+C content.Repeat sequences were observed in ITS-1 of Macrobrachium nipponense.TTTATTAAAGAAGAAA repeated six times and (GA)_n(n=5, 6, 5 respectively) appearedin three regions of ITS-1. Sequence comparison revealed there were 13 bases of transitions,9 of transversions, 4 of deletions and 2 of insertions.Our research filled in the gap in ITS technique application and broadended genetic background knowledge of Macrobrachium nipponense. The results were helpful to geneticbreeding of Macrobrachium nipponense.更多还原