苦瓜Ⅲ型过氧化物酶Prxs基因及其启动子的克隆与表达分析

Cloning and Expression Pattern Analysis of Class Ⅲ Peroxidases Gene( Prxs) and Its Promoter From Momordica Charantia L.

过氧化还原蛋白(Prxs)是广泛存在于植物体内重要的抗氧化酶。为深入研究Prx基因在苦瓜非生物胁迫中的作用,从苦瓜叶片均一化文库中获得McPrx基因的cDNA全长序列,并命名为McPrx(KJ722768.1),该cDNA序列全长1 068 bp,开放阅读框972 bp,编码324个氨基酸,属于ClassⅢ基因家族成员。采用基因组步移法分离获得McPrx基因5'上游1 237 bp的启动子调控序列,运用Plant CARE对其进行顺式作用元件分析,结果显示该序列除含有CAAT-box、TATA-box等核心启动子元件,还具有激素、抗病与抗逆应答元件,表明McPrx基因的表达可能受多种外界环境条件的调控。qRT-PCR分析表明,McPrx在根、茎、雌花等器官中的表达量存在极显著差异。其中在茎中表达量最大,在雌花中表达量最低,说明该基因的表达具有器官表达特异性;低温胁迫1 h时,McPrx表达量显著上调,胁迫3 h时McPrx表达量达到最大,随后下降,说明McPrx响应了低温胁迫的应答。本研究结果为进一步研究McPrx的生物学功能及其应用奠定了基础。

Peroxiredoxins（ Prxs） are important antioxidant enzymes in nearly all plant tissues. To further characterize their functions under cold stress,the full length of Prx c DNA sequence was cloned from c DNA librarys of the Momordica charantia,which is named McPrx（ KJ722767. 1）. McPrx is 1 068 bp length with 972 bp ORF（ open reading frame）,encodse 324 amino acids and belongs to Class Ⅲ family. Using a genome walking method,1 237 bp promoter sequence of McPrx was obtained. By Plant CARE promoter analysis,it was showed that the McPrx promoter contains not only core promoter elements,but also cis-regulatory elements that response to hormone,bio-stress and abiotic stresses,implying the expression of the McPrx gene was possibly regulated by various environmental factors. qRT-PCR analysis indicated that McPrx is highly expressed in root,stem,female flowers and other tissues,with the highest in stem and the lowest in female flowers,that indicated the expression of McPrx was tissue-specific. Further cold stress experiment showed that the mRNA level of McPrx was significantly increased after 3 hours cold treatment,impling McPrx was responsed to cold stress. The foundation is established by this paper for further research of McPrx biological function and application in the future.