石蒜LrP5CS基因的克隆与表达特性分析  

Cloning and Expression Characteristic Analysis of P5CS Gene fromLycoris radiata

作　　者：徐晟[1] 江宜龙 蒋明敏[1] 卜多[1] 傅江燕 夏冰[1] 汪仁[1] 

机构地区：[1]江苏省中国科学院植物研究所,南京210014

出　　处：《西北植物学报》2015年第10期1925-1933,共9页Acta Botanica Boreali-Occidentalia Sinica

基　　金：国家自然科学基金(31301798;31270339);江苏省产学研联合创新资金-前瞻性联合研究项目(BY2014131)

摘　　要：Δ1-吡咯啉-5-羧酸合成酶(P5CS)是植物渗透胁迫下谷氨酸途径合成脯氨酸的关键酶。该研究以石蒜(Lycoris radiata)为材料,采用同源克隆、RACE方法结合RT-PCR技术克隆获得LrP5CS基因全长cDNA序列。序列分析表明,LrP5CS基因全长2 521bp,其中开放阅读框(ORF)为2 139bp,编码713个氨基酸,预测编码蛋白质的分子量为77.19kD,等电点为6.34;LrP5CS是1个稳定的疏水蛋白,不含信号肽,不具有跨膜结构,具有AAK超基因家族和ALDH-SF超基因家族的保守结构域。氨基酸序列比对和系统进化树分析发现,LrP5CS与植物其他P5CS蛋白具有较高的一致性,且与海枣PdP5CS及油棕EgP5CS聚为一类,亲缘关系最近。实时荧光定量PCR分析结果表明,LrP5CS在根、鳞茎和叶片中均有表达,其中在鳞茎中的表达量最高。LrP5CS在20%聚乙二醇(PEG)处理下的表达模式分析发现,LrP5CS受PEG胁迫处理的诱导表达,其基因相对表达量在处理后6h达到最高;随着处理时间的延长,LrP5CS基因相对表达量水平逐渐下调至对照水平。将LrP5CS连接到表达载体pET-28a上,转化获得LrP5CS编码基因的大肠杆菌BL21(DE3)工程菌,通过IPTG诱导表达,SDS-PAGE分析表达产物发现成功表达目的蛋白。该研究结果为进一步分析LrP5CS基因功能及石蒜抗逆分子育种奠定了基础。Δ1-Pyrroline-5-carboxylate synthetase（P5CS）is the committed-step enzyme of proline biosynthesis under drought stress in many plants.In this study,aP5 CSgene was obtained from Lycoris radiata based on homology cloning,RACE method and RT-PCR technology.The results showed that the fulllength cDNA of LrP5 CSis 2 521 bp,containing a 2 139 bp open reading frame（ORF）which encoded 713 amino acids with a predicted molecular weight of 77.19 kD and pI 6.34.LrP5 CS is a stable hydrophobic protein,and had no signal peptide and transmembrane structure.It had AAK superfamily and ALDH-SF superfamily conserved domains.Multiple sequence alignment and phylogenetic tree analysis showed that the deduced protein LrP5 CS shares higher identity with P5 CS from other plants,and belongs to the same branch with Phoenix dactylifera PdP5 CS and Elaeis guineensis EgP5 CS.Subsequently,quantitative realtime PCR analysis indicated that LrP5 CSwas expressed in leaves,bulbs,and roots with the highest expression level in bulbs.LrP5 CStranscript levels were significantly induced by 20% polyethylene glycol（PEG）treatment,and peaked in 6htreatment.Additionally,the LrP5 CS was ligated into pET-28 avector,and transferred into E.coli strain BL21（DE3）for heterologous expression.The recombinant protein wasinduced by isopropyl-β-D-thiogalactopyranoside（IPTG）and its molecular weight is about 82.58 kD.Above results might lay a foundation for the further function analysis of LrP5 CSand adversity resistance molecular breeding of L.radiata.

关 键 词：石蒜 P5CS 脯氨酸 序列分析 

分 类 号：Q785[生物学—分子生物学] Q786