日本三角涡虫热休克蛋白90基因克隆及其表达模式  

Cloning and expression pattern of heat shock protein 90 gene from planarian Dugesia japonica

作　　者：马克学[1] 冯程程[1] 豆贺 程方方[1] 陈广文[1] 刘德增[1] 

机构地区：[1]河南师范大学生命科学学院,河南新乡453007

出　　处：《解剖学报》2013年第6期778-783,共6页Acta Anatomica Sinica

基　　金：国家自然科学基金资助项目(31170357;30870368;30670247;30170119);河南省杰出青年科学基金资助项目(0312001100);河南省创新人才基金资助项目(2005126);河南省教育厅自然科学研究计划资助项目(2011B180031)

摘　　要：目的克隆日本三角涡虫热休克蛋白90(Djhsp90)基因,并对其在不同应激条件下表达模式进行分析。方法采用PCR技术克隆日本三角涡虫hsp90 cDNA全长序列和基因组DNA序列,利用生物信息学软件对其序列进行分析,采用实时荧光定量聚合酶链反应(Real-time PCR)技术检测其在再生、饥饿和热激过程中的表达模式。结果 Djhsp90 cDNA全长2354 bp,含有2148 bp的开放阅读框(ORF),编码715个氨基酸,含有真核生物HSP90家族蛋白的5个标签序列和末端高度保守序列MEEVD。基因组DNA测序表明,该基因仅含有1个内含子(48 bp),内含子的5’-和3’-剪切位点符合经典的"GT-AG"法则。涡虫切断后1d Djhsp90的表达量显著增加,2d达到高峰,3d后逐步下降到正常水平。持久饥饿过程中Djhsp90的表达量维持在较高水平。提高培养温度能显著诱导Djhsp90的表达,且涡虫头部对热效应最敏感。结论我们成功克隆了日本三角涡虫hsp90基因,并证实切割、饥饿和热激能诱导其表达上调。Objective To clone heat shock protein 90 gene from planarian Dugesia japonica （Djhsp90）, and to examine its expression pattern in response to different stressors. Methods The full-length eDNA and genomie DNA of Djhsp90 was cloned using PCR methods, and its sequence was analyzed by the bioinformatics software, and its expression pattern in response to different stressors was detected by the fluorescent Real-time RT PCR. Results The full-length cDNA of Djhsp90 was 2354 bp containing an open reading frame （ORF） of 2148 bp, which encoded a polypeptide of 715 amino acids with five signatures motifs and the C-terminal consensus MEEVD of eukaryotic HSP90 family. The ORF sequence of genomie DNA was sequenced, and found that only one intron （48 bp） existed in Djhsp90 gene structure. The 5＇ and 3＇ putative splicing site followed the typical ＂ GT-AG＂ rule. Djhsp90 expression began to rise at 1 day following the amputation of planarians, reached a peak at day 2 and decreased gradually at day 3 to the normal level. During the prolonged starvation, Djhsp90 expression remained a relative higher level in contrast to the controls. We verified that elevating the culture temperature strongly induced Djhsp90 expression, and the most sensitive induction occurred in the head fragment of planarians. Conclusion We have successfully cloned hsp90 gene from planarian Dugesia japonica, and verified that amputation, starvation and thermal stress can induce the up-regulation of Djhsp90.

关 键 词：热休克蛋白90 基因克隆 实时荧光定量聚合酶链反应 涡虫 

分 类 号：Q71[生物学—分子生物学]