红花草莓FamiR156a及其靶基因FaSPL13A的克隆与表达分析  

Cloning and Expression of FamiR156a and Its Target Gene FaSPL13A in Pink-flowered Strawberry

作　　者：岳静宇 刘明 雷家军[1] 薛莉[1] YUE Jing-yu;LIU Ming;LEI Jia-jun;XUE Li(College of Horticulture,Shenyang Agricultural University,Shenyang 110161,China)

机构地区：[1]沈阳农业大学园艺学院,沈阳110161

出　　处：《沈阳农业大学学报》2021年第6期660-667,共8页Journal of Shenyang Agricultural University

基　　金：国家自然科学基金项目(31701964)。

摘　　要：红花草莓是一种可食用的观赏花卉,花色是红花草莓最主要的观赏性状。miRNAs作为重要的转录后调节因子,在植物中发挥着重要的作用,但在红花草莓中尚未见报道。通过对红花草莓品种四季红不同发育进程的花蕾进行miRNA和降解组测序,利用趋势分析,筛选到与花色调控相关的差异FamiR156a及其差异靶基因FaSPL13A,对其进行克隆、过表达载体的构建、生物信息学分析和表达分析。结果表明:FamiR156a前体序列为104 bp,靶基因FaSPL13A编码区序列为1236 bp,并对获得的序列分别进行生物信息学分析和系统进化树构建,发现FamiR156a前体序列与蔷薇科的苹果、桃同为一个分支,FaSPL13A的氨基酸序列与蔷薇科的森林草莓和月季聚类在同一分支上,说明它们亲缘关系较近、功能相似且克隆结果准确。同时,构建了FamiR156a及其靶基因FaSPL13A过表达载体pRI-101-AN-FamiR156a和pRI-101-AN-FaSPL13A,成功转入到农杆菌中。随着花蕾的发育,花瓣的颜色逐渐加深,实时荧光定量结果发现,FaSPL13A基因在红花草莓花瓣中表达逐渐下降,而FamiR156a的表达量逐渐升高,二者表达量呈相反趋势,表明在红花草莓花瓣呈色过程中FamiR156a及其靶基因FaSPL13A是负调控的关系,从而说明FamiR156a可能调控红花草莓花瓣的着色。研究结果为揭示红花草莓花瓣花青苷的生物合成提供了转录后水平调控的分子依据,为进一步深入研究红花草莓花瓣呈色机理及分子育种奠定了基础。Pink-flowered strawberry is a kind of edible ornamental plant and its petal color is the main ornamental character.As an important post-transcriptional regulator,miRNAs play important roles in plants,but it has not been reported in pink-flowered strawberry.In this study,the flower buds of different developmental stages were used for miRNAs and degradome sequencing in pinkflowered strawberry cultivar Sijihong,differentially expressed FamiR156a and target gene FaSPL13A related to flower color regulation were selected and cloned via trend analysis.The results showed that the precursor sequence of FamiR156a was 104 bp,and the coding region sequence of FaSPL13A was 1236 bp and the obtained sequences were used for bioinformatic analysis and phylogenetic tree construction,respectively.It was found that the precursor sequence of FamiR156a was clustered in the same branch as apple and peach in Rosaceae,and the amino acid sequence of FaSPL13A was clustered in the same branch as Fragaria vesca and rose in Rosaceae,which indicated that they had similar genetic relationship and similar functions,and our cloning results were accurate.Furthermore,FamiR156a and its target gene FaSPL13A overexpression vectors pRI-101-AN-FamiR156a and pRI-101-ANFaSPL13A were constructed and successfully transferred into Agrobacterium.With the development of flower buds,the color of petals deepened gradually.qRT-PCR results showed that the expression of FaSPL13A gene decreased gradually,while the expression of FamiR156a increased gradually with the opposite expression trend.These results indicated that FamiR156a and its target gene FaSPL13A were negatively regulated during the petal coloring process,which suggested that FamiR156a might play a role in regulating the petal coloring of pink-flowered strawberry.The results provide a molecular basis for post-transcriptional regulation of anthocyanin biosynthesis in petals of pink-flowered strawberry and lay the foundation for further study on the color mechanism of petals and molecular breeding in pink-

关 键 词：红花草莓 FamiR156a FaSPL13A 克隆 过表达载体 

分 类 号：S668.4[农业科学—果树学]