中国被毛孢有性发育启动前后cDNA文库的构建

Construction of cDNA libraries from Hirsutella sinensis before and after sexual development

构建了中国被毛孢有性发育启动前后的cDNA文库.采用Total RNA Kit I 法提取总RNA,磁珠法分离mRNA,反转录合成双链cDNA.获得的cDNA经蛋白酶K与Sfi I消化酶切并纯化,连接到载体pUC19上,采用电转化法将其转化到感受态细胞DH-5a 中.之后测定文库的克隆数、重组率、插人片段大小.结果显示,文库1 含1.09×10^6个重组体,蓝白斑实验显示重组率为92.5%;文库2 含1.13×10^6个重组体,蓝白斑实验显示重组率为92% ;两文库重组DNA片段的大小范围均在500-2000bp.说明本试验中所提取的RNA其纯度很好,完全可以达到后续研究的要求.

The cDNA libraries were constructed from Hirsutella sinensis before and after the sexual development,respectively. Total RNA KitⅠ method was used to extract total RNA. The mRNA was separated by magnetic particles,and then double stranded cDNA was synthesized by reverse transcription. The obtained cDNA was purified by proteinase K and SfiⅠdigestive enzymes and connected to the carrier pUC19,then transferred into strain DH-5α with competent cells,by electrotransformation. Afterwards,the clone number,reconstruction ratio and the size of the insert fragment of the library were measured. The results showed that library 1 contained 1. 09 × 10~6 recombinants,the blue and white test showed the reconstruction ratio was 92. 5%,library 2 contained 1. 13 × 10~6 recombinants,and the blue and white test showed the reconstruction ratio was 92%. The size of reconstructed DNA fragments in two libraries ranged from 500 to 2 000 bp. It showed that RNA extracted in this experiment had good purification degree,which was enough to meet the requirements for subsequent research.