环境胁迫对单巢类轮虫不同类别基因差异表达的影响-水产养殖专业论文.docxVIP

南京师范大学硕士学位论文 南京师范大学硕士学位论文 唐晟凯 摘要 为了从基因表达水平上探讨单巢类轮虫有性生殖发生的分子机理，本研究以 萼花臂尾轮虫为材料，通过温度刺激，分别获取非混交雌体、混交雌体和雄体， 进而币0用mRNA荧光差异显示的方法对三种类另Ⅱ进行了研究，取得了下列几个 主要结果和结论： (1)用急剧冷休克4小时的方法，得到的轮虫后代中的混交雌体百分含量 最高。 (2)用惭乃0I试剂盒提取了萼花臂尾轮虫非混交雌体、混交雌体和雄体的 RNA，纯度较高。且发现提取雌体的RNA至少需要雌体300只。 (3)进行了20个荧光差异显示反应，获得了34个差异片段。进行PCR 二次扩增得到稳定的差异条带18条。约有1／2的差异条带回收后， 再次扩增时未能扩出条带，可能是假阳性条带。说明萼花臂尾轮虫不 同类别之间在mRNA水平上确实存在着本质的差别。 (4)对差异片段进行克隆测序，获得了16个DNA片段的序列数据。序 列的编号分别为H7．1、H702、F7．1、F7．2、F7-3、F7-4、F7．6、 H8·1、H8_4、 H8．6、X9-1、X9-2、H10．1、H10·3、X11-1、 F13-1。NCBI BLAST数据库中进行同源性分析结果显示H7．1与萼花 臂尾轮虫的内转录间隔区1、2的部分序列、5．8SrRNA基因、18SrRNA 基因有很高的同源性。而其他的片段没有找到与之匹配的已报道的基 因，推断可能是新基因。 关键词：萼花臂尾轮虫；非混交雌体；混交雌体；雄体；荧光差异显示 南京师范大学硕士学位论文 南京师范大学硕士学位论文 唐晟凯 ABSTRACT In order to study the molecular mechanism of sexual reproduction of the Monogononta in gene expression，We obtained amictic female，mictic female and male by temperature stimulating，and then studied their differences in gene expression by Fluorescent Differential Display-PCR．The following results were obtained： (1)The percentage of mietic female in the offspring is the highest in culture that underwent sudden cold shock(ss)treatment for 4h． (2)We extracted the RNA from amicfic female,mictic female and male ofBrachionus calyeiflorus with Trizol kit In addition,We found that at least 300 females were needed in the RNA extraefion， (3)1_11ir移-four differentially expressed eDNA fragments were obtained from twenty DDoPCR reactions．Eighteen differentially expressed eDNA fragments were amplified in reamplifieation reactions．About 1／2 of the tllir哆一four differentially expressed eDNA fragments could not be reamplified,which might be false positive．The results showed that there were remarkable differences in gene expression among amietie female，mictic female and male． (4)We cloned and sequenced the sixteen differentially expressed DNA fragments． these sequences were H7-1、1-17-2、F7-1、F7-2、F7-3、F74、F7—6， H8．1、H8-4、H8．6、X9-1、X9-2、H10．1、H10．3、X11—1 and F13-1．The alignment ofthese sequences through N