摘要
[目的]研究线性质粒的转化,提高衣藻外源基因的转化效率。[方法]进行p SP108质粒单酶切试验,采用玻璃珠法转化线性质粒并对转化的关键条件进行优化,最后经抗性筛选、PCR及RT-PCR验证转化情况。[结果]经Bam HⅠ、XbaⅠ、EcoRⅠ、SacⅠ单酶切制备的p SP108线性质粒均能成功转化,Bam HⅠ酶切后转化率最高,质粒最佳用量为4μg,最佳震荡时间为25 s,博来霉素最适筛选浓度为15μg/m L。[结论]为构建衣藻生物反应器提供实验基础。
[ Objective ] Researching linear plasmids transformation system and improving the transformation efficiency of C. reinhardtii. [ Methods ] The single enzyme cutting test of pSP108 plasmid was carried out,the linear plasmid was transformed by glassbead method and key transformation conditions were optimized, finally, the transformation result was verified by resistance screening,PCR and RT -PCR detection. [ Results] The linear plasmid single enzymed by BamH I ,Xba I ,EcoR I and Sac I could be successfully transformed, and the transformation efficiency of BamH I was the highest, the best plasmid dosage was 4 μg, the best vortex time was 25 s, and the optimal zeocin concentration was 15 μg/mL. [ Conclusion I This study provided experimental basis for constructing Chlamydomortas reinhardtii bioreactor.
作者
黄亚萍
王川
冯秀芳
张宇
Yaping Huang;Chuan Wang;Xiufang Feng;Yu Zhang(College of Bioengineering,Sichuan University of Science ＆ Engineering,Zigong 643000,China)
出处
《生物技术》
CAS
2018年第5期467-472,共6页
Biotechnology
基金
四川省科技厅应用基础项目("高活力表达人表皮生长因子(EGF)的衣藻生物反应器的研究"
No.2014JY0203)
四川理工学院研究生创新基金项目("莱茵衣藻细胞核稳定转化外源基因条件的优化"
No.y2017046)
自贡市科技局创新苗子工程项目("莱茵衣藻细胞核转化体系的优化及CRP表达的研究"
No.2018CXMZ03)
关键词
莱茵衣藻
玻璃珠法
单酶切
线性质粒
转化
Chlarnydomonas reinhardtii
glass - bead method
single enzyme digestion,linear plasmids
transformation
