坛紫菜核糖体蛋白S15a基因的克隆及高温胁迫表达分析

Molecular cloning and expression analysis under high temperature stress of ribosomal protein S15a gene from Porphyra haitanensis

坛紫菜栽培中经常遭遇高温胁迫危害而产生烂菜现象,为研究坛紫菜在高温胁迫条件下的分子应答机制,分离并克隆高温胁迫应答的相关基因,应用引物退火控制（ACP）技术对耐高温型纯系Z-61叶状体在高温胁迫条件下的差异表达基因进行筛选时,获得一条在高温胁迫条件下表达水平显著降低的基因片段,通过5′-RACE技术获得了它的全长,命名为Phrps15a（GenBank登录号：JN991055.1）。该基因序列全长676 bp,包含一个390 bp的开放阅读框编码130个氨基酸的核糖体S15a蛋白（PhRPS15a）,蛋白分子式为C664H1066N188O180S7,由3条螺旋、7个片层及8个环状连接组成,与多个物种的RPS15a蛋白具有较高的序列一致性（78%~80%）。系统进化分析表明,动物、高等植物、绿藻和坛紫菜PhRPS15a蛋白均享有独立的进化分支,但坛紫菜PhRPS15a蛋白与团藻和莱茵衣藻的亲缘关系更近。实时荧光定量PCR分析结果表明,Phrps15a基因的表达水平与高温胁迫密切相关,在高温胁迫条件下,Phrps15a基因的表达水平极显著下调。

Porphyra haitanensis is an economically important marine crop in southern China.A major problem in Porphyra farming is thallus decay in response to high temperature stress.Molecular studies of temperature stress can help to resolve the issue of thallus decay.In this study,to study the molecular mechanism of high temperature tolerance in P.haitanensis,the technology of annealling control primer（ACP） was used to screen the differential expressed genes in gametophytic blades of an F4 high temperature tolerance line Z-61.By the primers combination of dT-RSL and RSL2,one differential expressed gene fragment was cloned.And the full length sequence of this gene fragment was cloned by 5＇RACE technology and named as Phrps15a（acceession number JN991055.1）.The Phrps15a gene with a 676bp sequences contains an open reading frame of 390 bp encoding a PhRPS15a protein of 130 amino acid residues.The PhRPS15a protein which was assembled by 3 helices,7 sheets and 8 cycles shared high amino acid sequence identity with RPS15 from other organisms（78%-80%）,and its molecular formula was C664H1066N188O180S7.Phylogenetic analysis showed that the evolution of PhRPS15a of animal,higher plant,green algae and P.haitanensis has unattached evolutional groups,and the PhRPS15a protein of P.haitanensis has closer relation with RPS15a proteins from green alga than ones from other organisms.The results of real-time quantitative PCR indicated that the expressed level of Phrps15a has close relation with high temperature stress.Under high temperature stress,the expression of Phrps15a gene was down-regulated significantly.