暗黑鳃金龟微卫星引物的筛选 动植物检疫学专业

I暗黑鳃金龟微卫星引物的筛选摘 要暗黑鳃金龟(Holotrichia parallela Motschulsky)是花生的主要害虫，盛期时造成花生产量大幅度降低，严重时甚至使花生绝收。微卫星 DNA（microsatellite），又称 SSR(simple sequence repeat，简单重复序列)是以 1～6 个核苷酸为重复单元组成的短串联重复序列。经过数据库检索，我们发现暗黑鳃金龟的 SSR 引物序列从未被发表过，关于暗黑鳃金龟 SSR 的研究也未见报道，不能满足其种群遗传研究结构的需要。本研究基于前期测序获得的暗黑鳃金龟转录组序列，筛选其 EST(Expressed Sequence Tag，表达序列标签）-SSR 序列。用微卫星搜寻软件 MicroSAtellite 查找暗黑鳃金龟转录组中的 SSR 信息，并用 Primer 5 软件对 SSR位点进行引物设计。结果表明，暗黑鳃金龟转录组中富含大量的 SSR 位点。在 58,763 条序列中共搜寻到 14,433 个 SSR 位点，分布在 10,686 条序列中，平均每 3.58kb 就能发现一个SSR 位点。基于上述数据，我们设计了 32 对 SSR 引物并将之用于梯度 PCR 扩增，其中扩增出特异性条带的引物有 22 对。这说明基于转录组数据开发暗黑鳃金龟的微卫星引物是可行的，本研究不仅丰富了暗黑鳃金龟在 SSR 方面的研究，也为近缘种群的遗传学研究提供了工具。关键词：暗黑鳃金龟；简单重复序列；引物；筛选 IIAbstractHolotrichia parallela is the main pest of peanut. Peanut yield was greatly reduced in peak period, and even no peanut harvest was achieved in severe period. Microsatellite DNA (SSR), also known as simple sequence repeat (SSR), is a short tandem repeat sequence consisting of 1 to 6 nucleotides as repeat units. After searching the database, we found that the SSR primer sequence of the Holotrichia parallela has never been published, and the SSR research of the Holotrichia parallela has not been reported, which can not meet the needs of population genetic research structure. EST-SSR sequences were screened, based on the transcriptome sequences obtained previously. MicroSAtellite (MISA) microsatellite search software was used to find SSR information in the...