中国被毛孢有性发育启动前后cDNA文库的构建

doi:10.3969/j.issn.1004-1524.2016.11.14

浙江农业学报 ›› 2016, Vol. 28 ›› Issue (11): 1895-1900.DOI: 10.3969/j.issn.1004-1524.2016.11.14

中国被毛孢有性发育启动前后cDNA文库的构建

谢放, 常黎明, 李建宏, 张育, 张亚军, 刘帅

兰州交通大学化学与生物工程学院,甘肃兰州 730070

收稿日期:2016-03-16 出版日期:2016-11-15 发布日期:2016-11-16
作者简介:谢放(1962—),男,甘肃兰州人,副教授,研究方向为资源与环境微生物。 E-mail:xfrankf@163.com
基金资助:
国家自然科学基金项目(215069)

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

XIE Fang, CHANG Li-ming, LI Jian-hong, ZHANG Yu, ZHANG Ya-jun, LIU Shuai

School of Chemical and Biological Engineering, Lanzhou Jiaotong University, Lanzhou 730070, China

Received:2016-03-16 Online:2016-11-15 Published:2016-11-16

摘要/Abstract

摘要： 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SfiⅠ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-5α中。之后测定文库的克隆数、重组率、插入片段大小。结果显示,文库1含1.09×10⁶个重组体,蓝白斑实验显示重组率为92.5%;文库2含1.13×10⁶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500~2 000 bp。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。

关键词: 中国被毛孢, 有性发育, cDNA文库

Abstract: 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 1.09×10⁶ recombinants, the blue and white test showed the reconstruction ratio was 92.5%, library 2 contained 1.13×10⁶ 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.

Key words: Hirsutella sinensis, sexual development, cDNA library

中图分类号:

谢放, 常黎明, 李建宏, 张育, 张亚军, 刘帅. 中国被毛孢有性发育启动前后cDNA文库的构建[J]. 浙江农业学报, 2016, 28(11): 1895-1900.

XIE Fang, CHANG Li-ming, LI Jian-hong, ZHANG Yu, ZHANG Ya-jun, LIU Shuai. Construction of cDNA libraries from Hirsutella sinensis before and after sexual development[J]. , 2016, 28(11): 1895-1900.

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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

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