杜氏盐藻FKBP cDNA的克隆及其在鞭毛解组装中的功能

Cloning of FKBP cDNA from Dunaliella salina and analysis of its functions in flagellar disassembly

目的:克隆杜氏盐藻FK506结合蛋白(FKBP)的cDNA片段并探讨其与鞭毛微管解组装的联系。方法:提取杜氏盐藻总RNA,根据已知盐藻FKBPcDNA3’端序列,用5’RACE方法扩增该cDNA的5’端序列,拼接得到全长并对其序列进行分析。秋水仙碱处理对数生长期杜氏盐藻,处理0、20、40、60、80、100、120、140和160min后采用实时荧光定量PCR检测FKBPmRNA的表达情况。结果:经5’RACE得到681bp的FKBPcDNA片段,拼接后得到的cDNA全长1075bp,序列分析显示其开放阅读框为762bp,编码253个氨基酸;经BLAST比对,其氨基酸序列具有FKBP家族特有的功能结构域,与莱茵衣藻、团藻、小球藻、大麦及水稻的同源性分别为61%、54%、47%、48%和51%。杜氏盐藻细胞FKBPmRNA表达量在秋水仙碱处理后20～40min明显升高,而在40～100min时逐渐下降,但仍高于对照组(F组间=32.580,P<0.001;F时间=5.543,P=0.001;F交互=237.306,P<0.001)。结论:得到了杜氏盐藻的FKBPcDNA全长序列;FKBP参与了杜氏盐藻微管解组装过程,可能参与了细胞通过鞭毛对外界应激反应的应答。

Aim：To clone the cDNA fragment of FK506 binding protein（FKBP）from Dunaliella salina（D.salina） and to investigate its function in the process of microtubule disassembly in flagella.Methods：The total RNA was extracted from D.salina and the full length of FKBP cDNA was amplified by 5＇RACE method according to the known 3＇terminal sequence.The expression of FKBP mRNA in D.salina treated with colchicine for 0,20,40,60,80,100,120,140 and 160 min,was detected by real-time fluorescence quantitative PCR.Results：A 681 bp DNA fragment was obtained by 5＇RACE.The full length of FKBP cDNA was 1 075 bp long and contained an open reading frame of 762 bp which encodes 253 amino acids.Sequence analysis showed that it had the conserved domain of FKBP family and was homologous with Chlamydomonas reinhardtii（61%）,Volvox carteri f.nagariensis（54%）,Chlorella variabilis（47%）,Hordeum vulgare subsp.Vulgare （48%）and Oryza sativa Indica Group（51%）.The expression of FKBP mRNA in D.salina 20～40 min after colchicine treatment significantly increased,and decreased gradually within 40～100 min after colchicine treatment,but still higher than the control（F group =32.580,P0.001;F time =5.543,P=0.001;F interaction =237.306,P0.001）.Conclusion：The FKBP cDNA sequence of D.salina has been obtained successfully.The expression of FKBP has correlation with the process of microtubule disassembly in D.salina,and it may be involved in the response of the cells to external stress through the flagella.