中华猕猴桃‘红阳’Alase家族基因的克隆及表达分析

Cloning and expression analysis of Alase family genes in kiwifruit (Actinidia chinensis)

在高等植物中醛糖酸内酯酶(Alase)催化L-半乳糖酸(L-Gal A)形成抗坏血酸(Ascorbic acid,As A)的前体物质L-半乳糖-1,4-内酯(L-Ga IL),是As A合成分支途径D-半乳糖醛酸途径中的关键步骤.基于最新的猕猴桃基因组数据库,分离并克隆到3个Alase家族基因,分别命名为Alase1、Alase2、Alase3,并从基因信息、蛋白质理化性质、二级结构及序列特征方面对其进行预测及分析.结果表明,Alase为亲水性蛋白,二级结构主要以α-螺旋和不规则卷曲为主,3个Alase基因具有高度的相似性.定量RT-PCR分析表明3个Alase家族基因在不同组织中均有表达,且在成熟的叶片中表达量最高.同时在猕猴桃果实发育及成熟过程中,Alase家族基因在幼果中的表达水平较低,而在果实发育后期及果实成熟过程中表达量不断升高.本研究可为Alase家族基因后续功能研究及猕猴桃中As A生物合成和积累分子机制解析奠定基础.

In higher plants, aldonolactonase （Alase） is an important enzyme in the D-galacturonate pathway, which converts L-galactonate to L-galactono-1,4-lactone, the precursor of ascorbic acid （AsA）. Based on the recently published kiwifruit genome sequence, specific primers were designed to isolate Alase family genes and the physical and chemical characteristics, secondary structures, and conserved motifs among Alase family members were further predicted and analyzed. Using quantitative RT-PCR, relative expression levels of Alase family genes in different tissues were revealed in kiwifruit. We identified three Alase family genes, named as Alase1, Alase2, and Alase3. Our results indicated that Alase proteins are hydrophilic and the secondary structures mainly consist of α-helices and irregular curls. Motif analysis showed that these Alase members are highly conserved in kiwifruit. Moreover, quantitative RT-PCR analysis revealed that transcripts of Alase genes are detectable in all tissues examined with higher expression observed in mature leaves. Moreover, the expression of all Alase genes increased from 30 days after pollination during fruit development and ripening in kiwifruit. Our results provide a foundation for the functional analysis of Alase family genes in AsA biosynthesis and accumulation in kiwifruit.