草莓β-半乳糖苷酶基因FaTβgal的克隆与表达分析

Cloning and Expression Analysis of FaTβgal of β-Galactosidase Family from Fragaria ananassa Fruit

利用SSH和RACE技术,从‘丰香’草莓果实中分离了1个草莓β-半乳糖苷酶（β-Gal）基因,命名为FaTβgal。FaTβgal基因cDNA序列全长2 891bp,ORF区2 448bp,编码815个氨基酸,含有保守序列GGPIILSQIENEY和凝集素结构域。FaTβgal推导氨基酸序列与已报道的3个草莓β-Gal基因Faβgal1（CAC44500）、Faβgal2（CAC44501）、Faβgal3（CAC44502）氨基酸序列有47.1%～48.1%的相似性。与其它物种24个β-Gal基因聚类分析表明,FaTβgal聚在一个独立的分枝上。采用实时荧光定量PCR技术,对FaTβgal基因在果实发育成熟过程中的表达分析表明,该基因在果实中特异表达,随着果实成熟表达量升高,粉红期达到峰值,全红期迅速下降;2个软硬不同的品种表达模式趋于一致。研究认为,FaTβgal基因是β-Gal基因家族的一个新基因,该基因可能在果实成熟软化过程中发挥作用。

To identify the function ofβ-galactosidase（β-Gal）gene during ripening and softening,we isolated aβ-Gal gene,named FaTβgal,from strawberry fruit（Fragaria ananassa Duch.cv Toyonoka）by suppression subtraction hybridization（SSH）and RACE techniques.The full length cDNA sequence of the FaTβgal gene is 2 891 bp and the open reading frame（ORF）is 2 448 bp which encoded a deduced protein including 815 amino acid residues.The FaTβgal protein is a member of theβ-Gal gene family of glycoside hydrolase family 35 members,including conservative base sequence ＂GGPIILSQIENEY＂and a galactose binding lectin domain.Blastp analysis revealed 47.1% to 48.1% similarities between FaTβgal and three reported F.ananassagenes of Faβgal1（CAC44500）,Faβgal2（CAC44501）and Faβgal3（CAC44502）.Phylogenetic analysis showed that FaTβgal was clustered on one independent branch with 24β-Gal genes of other species.The relative expression of FaTβgal was determined by quantitative real-time PCR（qRTPCR）during ripening and softening.The results showed that FaTβgal gene was expressed specifically at the mature stage of fruits,and the relative expression levels of FaTβgal genes increased rapidly with fruit ripening and reached a peak at the pink stage,then markedly declined at red fruit stage.The trends ofβ-Gal expression in two cultivars of Toyonoka and Sweet Charlie were similar during fruit development and ripening.These results indicated that the FaTβgal is a novel gene of theβ-Gal gene family,and may playan important role in strawberry fruit ripening and softening.