摘要:
以中国对虾血液、眼柄、卵巢、雌虾头胸部、雄虾头胸部和三倍体对虾头胸部组织为材料,采用异硫氰酸胍-酸酚法提取总RNA;用磁珠法纯化mRNA;用Uni ZAP cDNA合成试剂盒合成双链cDNA并进行修饰,将cDNA定向连接在UniZAP载体上;经Gigapack Gold Package包装试剂盒包装成为噬菌体颗粒,转染宿主XL1 Blue MRF’菌株细胞,形成初级文库.初级文库经转染进一步扩增,形成稳定的cDNA文库.6个文库的库容在0.2×106~1.3×106之间,重组率都超过90%.从各文库随机取出6~10个清晰的噬菌斑进行PCR扩增,用琼脂糖凝胶电泳检测,其插入片段长度为500~2500bp.由初步功能基因克隆获得阳性结果.多项指标表明,所构建的对虾cDNA文库质量较高,为进一步筛选目的基因、EST测序和制作基因芯片提供了有效的工具.
Abstract:
Six cDNA libraries of blood,eyestalks,ovary and cephalathorax of female,male,triploid Chinese shrimp(Fenneropenaeus chinensis) were constructed using the cDNA synthesis and ZAP express kit (Stratagene).Total RNA was extracted with guanidine thiocyanate/phenol/chloroform.Synthesized cDNA was ligated with the E coR I adapter and phosphory lated.After EcoR I adapters were digested and eliminated with Sephrose-2Bspun column,the cDNA fragments that had more than 400 bp were collected and ligated with the ZAP express vector.And then the ligated cDNAs were packed and incubated with XL blue MRF.The content of each library is 0.78×106 (blood),0.60×106(eyestalks),0.36×106(ovary),1.3×106(female),0.20×106(male) and 1.2×106(triploid),respectively.In order to detect the quality of these libraries,the inserted fragments were am plified with the T3 and T7 primers.The size of the inserted fragments was 500~2500 bp.Moreover,antimicrobial peptide and-actingenes were cloned from the libraries.All these results indicate that high quality cDN A libraries were obtained.These cDNA libraries provide avery powerful tool to find new functional genes,analyze ESTs,and prepare genechips and so on.
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