中国明对虾“黄海1号”血细胞和肌肉cDNA文库的构建

Construction of cDNA libraries from Chinese shrimp Fenneropenaeus chinensis "Huanghai No. 1"

采用SMART(Switching mechanism at5′end of RNA transcript)技术构建了中国明对虾(Fenneropenaeus chinensis)血细胞和肌肉组织全长cDNA文库。以中国明对虾"黄海1号"血细胞和肌肉组织为材料,Trizol法提取总RNA,磁珠法纯化mRNA后,用SMART cDNA合成试剂盒合成双链cDNA。双链cDNA经SfiⅠ酶切和CHROMA SPINTM-400过柱分级分离后,与λTriplEx2载体两臂进行连接,随后进行λ噬菌体体外包装反应,构建血细胞和肌肉全长cDNA文库。肌肉组织原始文库滴度为0.77×106pfu/mL,血细胞原始文库滴度为2.36×106pfu/mL;扩增后,肌肉组织文库滴度为3.0×109pfu/mL,血细胞文库滴度为5.6×109pfu/mL,重组率均达到98%以上。从各文库随机取出10个清晰的噬菌斑进行PCR扩增,琼脂糖凝胶电泳检测,其插入片段长度为400-2000bp,证明2个文库均具有较高的质量。

cDNA libraries provide useful resources for functional genomic research. In order to discover and understand the molecular mechanism of Chinese shrimp Fenneropenaeus chinensis＂ Huanghai No.1 ＂. Two cDNA libraries from hemacyte and muscle were constructed using the technique of switching mechanism at 5′ end of RNA transcript （SMART）. Total RNA were extracted and mRNA were separated and purified. After digested with restriction enzyme SfiⅠand fractionated with CHROMA SPINTM-400 columns,cDNA fragments longer than 400 bp were collected and ligated into the Sfi Ⅰ -digested ,λ TriplEx2TM vector. The ligation mixture was packaged into lambda capsids using the Packagene Lambda DNA Packaging extract （Promega） to generate full-length hemacyte and muscle libraries of Fenneropenaeus chirtensis. The titers of un-amplified libraries were 0.77 × 10^6 pfu/mL for the muscle library and 2.36 × 10^6 pfu/mL for the hemacyte library, and the titers of amplified libraries were 3.0×10^9 pfu/mL for the muscle library and 5.6 × 10^9 pfu/mL for the hemacyte library. In order to detect quality of these libraries, inserted fragments were amplified with sequencing primers. The sizes of the inserted fragments ranged from 400 bp to 2 000 bp. Quality of these cDNA libraries is good enough for further cloning of hemacyte and muscle specific expression genes in hemacyte and muscle. [Journal of Fishery Sciences of China,2009,16 （5）： 781-785]