杜氏盐藻磷脂酶C基因DsPLC的克隆及盐胁迫下的表达分析  

Cloning and Expression Analysis of DsPLC under Salt Stress from Dunaliella salina

作　　者：韩冬梅[1] 柴晓杰[1] 王逸云[1] 刘世才[1] 岳文静[1] 

机构地区：[1]大连海洋大学/农业部北方海水增养殖重点实验室/辽宁省海洋生物资源恢复与生境修复重点实验室,辽宁大连116023

出　　处：《核农学报》2014年第10期1773-1780,共8页Journal of Nuclear Agricultural Sciences

基　　金：国家自然科学基金项目(30972240;31472260);辽宁省教育厅科学技术研究项目(2008T023)

摘　　要：为了研究磷脂酶C基因的结构与功能,采用RT-PCR和RACE技术从盐藻中克隆出了一种磷脂酶C基因(GenBank Accession No.KF573428),命名为DsPLC,并对其进行了生物信息学分析和盐胁迫条件下的表达分析。结果表明,DsPLC的cDNA全长2 628 bp,开放阅读框1 782 bp,编码593个氨基酸。该蛋白质为亲水性稳定蛋白,没有信号肽,也没有跨膜区域,是一种非跨膜蛋白,存在多个潜在的磷酸化位点,二级结构的主要元件为无规卷曲和α-螺旋。进一步的进化分析结果表明,盐藻与衣藻、团藻的亲缘关系最近。实时荧光定量PCR结果显示,正常情况下DsPLC的表达量很低,当受到高盐胁迫时表达量急剧升高,且在胁迫4 h时表达量达到最高,是对照组的10倍左右,差异极显著(P<0.01),表明DsPLC可能在盐藻抵御外界盐胁迫的过程中起重要作用。这些研究结果为进一步阐明DsPLC的功能及其作用机制奠定了分子基础。The aims were to clone Phosphatidase C gene from Dunaliella salina （named DsPLC） and to learn the structure and function of DsPLC. In the present study, DsPLC （GenBank Accession No. KF573428 ） was cloned by RT- PCR and RACE technology, then further analyzed using bioinformatics approaches. The expression of DsPLC gene in Dunaliella salina under salt stress was examined by Real-time fluorescence quantitative PCR （QRT-PCR）. The result showed that full length of cDNA for DsPLC was 2 628 bp, open reading frame （ORF） was 1 782 bp coding 593 amino acids. DsPI,C was a stable hydrophilic protein, without signal peptide and transmembrane region. By analysis we knew that DsPLC belonging to a non-transmembrane protein categoxry, possessing multiple potential phosphorylation sites, with random coil and c~ - helix as main components of its secondary structure. Further phylogenic analysis displayed that Dunaliella salina had the closest genetic relationship with Chlamydomonas reinhardtii and Volvox carteri f. nagariensis. The result of QRT-PCR showed that the expression level of DsPLC was significantly up-regulated and reached the peak level at 4 h under high salt stress, which was 9 times more than that of the control group （P 〈 O. O1 ）. The result demonstrated that in Dunaliella salina, it probably played an important role in the response to salt stress. These results of the current study laid a foundation for further illustrating the function and working mechanism of DsPLC.

关 键 词：杜氏盐藻 磷脂酶C 全长CDNA 生物信息学分析 实时荧光定量PCR 

分 类 号：Q943.2[生物学—植物学]