中华绒螯蟹微卫星单引物扩增产物的初步分析

Amplification products by singly microsatellite-primed PCR in Eriocheir sinensis

采用人工合成的 6个串联重复寡聚核苷酸单引物 ,以及 2个加锚的二核苷酸引物 ,利用SPAR和ASSR技术 ,对中华绒螯蟹 (Eriocheirsinensis)基因组进行PCR扩增 ,结果发现 ,对于GC含量丰富的三、四核苷酸引物如(CGA) 5、(GACA) 4 ,在不同退火温度的PCR反应中 ,都能扩增出清晰的条带 ,可以作为PCR扩增引物 ;反之 ,GC含量 <5 0 %的 (CATA) 4 和 (GATA) 4 很难检测到扩增产物 ;在基本PCR条件下 ,2个加锚的二核苷酸引物能扩增出可分辨的条带 ,但随着退火温度的升高 ,其中部分条带消失 ;对于不加锚的二核苷酸引物如 (AC) 8、(GA) 8,无论怎样改变退火温度 ,终难形成清晰条带。将不同引物扩增产物分别克隆到T Vector上 ,选取其中 9个阳性克隆进行序列测定 ,发现在 (GACA) 4 引物扩增的 2个片段中 ,序列结构相似 ,除引物序列外 ,都出现了 2～ 3个内部重复序列和高含量的AT ;其余

Six microsatellite primers and two anchored dinucleotid repeat primers were used to amplify the genomic DNA sample from Eriocheir sinensis. At different annealing temperatures the GC rich trinucleoid and tetranucleotid, such as (CGA) 5 and (GACA) 4, can generate distinct amplification bands, so they can be used as the PCR primers.However, when GC content<50%, such as (CATA) 4 and (GATA) 4, the products of primers are difficult to detect. Under normal PCR condition, the two anchored dinucleotide primers can produce distinct bands, but some of them dissapper at higher annealing temperatures.For the unanchored dinucleotid such as (AV) 8 and (GA) 8, no distinct bands are produced at any annealing temperatures. When the different amplified products are cloned to T Vector and nine positive clones are selected to determine their sequences, the sequences of the two fragments derived from (GACA) 4 primer reveal high frequency of internal repeat, while the sequences of the other seven fragments do not reveal internal repeat.