CrPP2C蛋白影响衣藻细胞鞭毛过渡区结构

doi:10.13523/j.cb.20160510

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中国生物工程杂志 ›› 2016, Vol. 36 ›› Issue (5) : 68-73. DOI: 10.13523/j.cb.20160510

研究报告

CrPP2C蛋白影响衣藻细胞鞭毛过渡区结构

何炜, 潘俊敏

作者信息 +

Protein Phosphatase 2CInvolved in the Flagellar Transition Zone Structure of Chlamydomonas reinhardtii

HE Wei, PAN Jun-min

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文章历史 +

摘要

目的:在莱茵衣藻细胞中构建并筛选鞭毛组装缺陷突变体,克隆缺陷基因,探索其对鞭毛组装的影响。方法:使用带有巴龙霉素(Paromomycin)抗性的基因片段随机插入衣藻细胞基因组中,通过性状筛选和基因序列分析获得与CrPP2C(Chlamydomonas reinhardtii type 2C protein phosphatase)基因相关的鞭毛异常突变体,根据突变体基本生物学性状和生化分析对CrPP2C基因的功能进行分析。结果:采用电转法成功获得衣藻细胞鞭毛缺陷相关突变体,部分细胞具有短鞭毛,部分细胞则不具有鞭毛;通过RESDA-PCR(restriction enzyme site-directed amplification PCR)对突变体基因序列分析,鞭毛缺陷性状由CrPP2C基因遭到破坏导致;把含有完整CrPP2C基因的重组质粒通过电转法导入突变体后,其鞭毛几乎恢复为野生型长度,并可检测到PP2C-HA融合蛋白的表达;观察鞭毛再生,突变体鞭毛只能再生为原有长度;使用药物处理使鞭毛缩短,突变体鞭毛能正常解聚;电镜检测突变体的鞭毛显微结构,发现过渡区的Y形结构缺陷。结论:CrPP2C基因的破坏导致鞭毛过渡区结构缺失,影响鞭毛组装过程,不组装鞭毛或组装短鞭毛。

Abstract

Objective:Using insertional mutagenesis in Chlamydomonas to identify genes required for flagellar assembly, so as to researching the function of the gene related to flagellar assembly. Methods:By transforming the DNA fragment harboring paromomycinresistance gene into Chlamydomonas randomly, the mutant strainwith flagellar defect is obtained and identified the disrupted gene was CrPP2C(Chlamydomonas reinhardtii type 2C protein phosphatase).Then the CrPP2C gene function is analyzed by biochemistry methods. Results:Theflagella abnormal mutantsthat show short flagella or none flagella are obtained by electroporation transformation.DNA sequence analysis show that the CrPP2C gene is disrupted. Transformation of mutants with CrPP2C-HA could rescue the flagellar phenotype.The mutants could regenerateflagella to the original length and shorte normally. Electron microscopyimages show incomplete Y-link structure in the transition zone of flagella. Conclusion:The CrPP2C gene is associated with the transition zone structure and affects the assembly of flagella.

导出引用

何炜, 潘俊敏. CrPP2C蛋白影响衣藻细胞鞭毛过渡区结构[J]. 中国生物工程杂志, 2016, 36(5): 68-73 https://doi.org/10.13523/j.cb.20160510

HE Wei, PAN Jun-min. Protein Phosphatase 2CInvolved in the Flagellar Transition Zone Structure of Chlamydomonas reinhardtii[J]. China Biotechnology, 2016, 36(5): 68-73 https://doi.org/10.13523/j.cb.20160510

中图分类号： Q819

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