筛选冠突伪尾柱虫有小核细胞系和无小核细胞系饥饿期差异表达的ESTs
IDENTIFICATION OF ESTs EXPRESSED DIFFERENTIALLY BETWEEN MICRONUCLEATES AND AMICRONUCLEATES OF HYPOTRICHOUS CILIATE, PSEUDOUROSTYLA CRISTATA DURING STARVATION
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摘要: 利用显微切割的方法建立大型腹毛目纤毛虫---冠突伪尾柱虫(Pseudourostyla cristata)的无小核细胞系,分别提取有小核和无小核细胞系的总RNA,用SMART-PCR方法直接合成dscDNA,进而运用抑制性消减杂交技术(Sup-pression subtractive hybridization,SSH)对冠突伪尾柱虫去小核前后的差异表达基因进行研究,构建了有与无小核细胞系在饥饿期差异表达基因的消减文库,随机挑取了284个克隆,应用cDNA阵列技术鉴定阳性克隆。将鉴定出的17个阳性克隆进行Blastx分析,结果表明17个EST(Expressed sequence tag)均可能与小核体功能密切相关,此为进一步阐明小核的遗传机理奠定了基础。Abstract: Based on morphologically comparative studies on amicronucleates and micronucleates of a ciliate protozoa,Pseudourostyla cristata, it is supposed that the micronucleus absence may result in abnormal development of the subsurface cytoskeleton and/or other supportive elements in the vicinity of the AZM during depression phase such as starvation.To understand the molecular events governing this mode, we employed suppression subtractive hybridization(SSH)to identify candidate genes which express in micronucleate cells but not in amicronucleate specimens during starvation.A total of 284 transcripts were screened by cDNA array.17 clones were confirmed to be differentially expressed among micronucleate cells.The ESTs(expressed sequence tags)were analyzed by blaster matching in GeneBank at nucleotide and deduced amino acid sequence:11 clones showing no homolgous protein;4 clones showing myoglobin protein;1 clone showing, cytosolic malate dehydrogenase.Interestingly, the last clone, homolog of hsp90.Traditionally, heat shock proteins are known for their function during cellular stress as molecular chaperones responsible for the folding, refolding and transport of newly synthesized protein.As to whether Hsp90 homolog is associated with microtubules in AZM to hold up its structure, and why it is significantly enhanced in micronucleate cells during starvation, further research need to be carried out.
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