多叶羽扇豆'鲁冰妮'LpluF3'5'H基因的克隆与表达分析  

Cloning and Expression Analysis of LpluF3′5'H in Lupinus polyphyllus'Lupine'

作　　者：胡钧舒 朱云涛 赵一燃 杨捷 孙婷婷 何恒斌 HU Jun-Shu;ZHU Yun-Tao;ZHAO Yi-Ran;YANG Jie;SUN Ting-Ting;HE Heng-Bin(Beijing Key Laboratory of Flower Germplasm Innovation&Molecular Breeding/National Engineering Research Center for Floriculture/Beijing Laboratory of Urban and Rural Ecological Environment/Key Laboratory of Genetics and Breeding in Forest Trees&Ornamental Plants Ministry of Education,College of Landscape Architecture,Beijing Forestry University,Beijing 100083,China)

机构地区：[1]北京林业大学园林学院花卉种质创新与分子育种北京市重点实验室/国家花卉工程技术研究中心/城乡生态环境北京实验室/林木、花卉遗传育种教育部重点实验室,北京100083

出　　处：《农业生物技术学报》2023年第6期1171-1181,共11页Journal of Agricultural Biotechnology

基　　金：国家自然科学基金(31672191);北京林业大学2018年大学生创新创业训练项目(20181002014);中央高校基本科研业务费专项资金(2015ZCQ-YL-03);中央高校基本科研业务费专项资金(YX2015-16);北京林业大学建设世界一流学科和特色发展引导专项资金(2019XKJS0323)。

摘　　要：类黄酮3’,5’羟化酶(flavonoid-3’,5’-hydroxylase,F3’5’H)基因是自然界绝大多数蓝色花呈色的关键基因。为探究多叶羽扇豆(Lupinus polyphyllus)F3’5’H的生物学功能,本研究以多叶羽扇豆’鲁冰妮’(’Lupine’)的花部组织为实验材料,对其F3’5’H进行克隆、系统进化等生物信息学分析、亚细胞定位和qPCR检测。结果表明,克隆获得多叶羽扇豆LpluF3’5’H(GenBank No.OQ657278)CDS序列长1620 bp,编码539个氨基酸,生物信息学预测发现其含有细胞色素P450超家族结构域。系统进化分析表明,LpluF3’5’H编码的氨基酸序列与双子叶植物狭叶羽扇豆(L.angustifolius)的相似度最高,单子叶植物则聚为另一支。亚细胞定位表达结果表明,LpluF3’5’H蛋白在荧光下呈点状离散分布,可能定位于叶绿体。qPCR结果显示,LpluF3’5’H在花不同的发育阶段都有不同程度的表达,在’鲁冰妮蓝色’花中的表达水平显著高于’鲁冰妮白色’花。在’鲁冰妮蓝色’花不同发育阶段中,LpluF3’5’H的表达水平在第二发育阶段的花瓣中达到峰值,随后降低。在’鲁冰妮蓝色’花的不同瓣型中,LpluF3’5’H在翼瓣的表达水平最高,其次是旗瓣,在龙骨瓣中表达水平最低。本研究说明LpluF3’5’H可能在多叶羽扇豆蓝色花花色形成过程中起到重要作用,可为后续探究多叶羽扇豆蓝色花的形成机理提供参考。The flavonoid-3',5'-hydroxylase(F3′5′H)gene is crucial for the development of blue flowers in nature.To provide more resources and reference for blue flower breeding by cloning and expression analysis of F3′5′H in Lupinus polyphyllus,the petals of L.polyphyllus'Lupine'were used as the material to clone LpluF3′5′H and the sequence was used for bioinformatics analysis by software such as MEGA 10.0 and online websites.The subcellular localization of LpluF3'5'H protein was determined by tobacco injection method.qPCR was used to analyze the expression of LpluF3′5′H in petals with different colors and developmental stages.The LpluF3′5′H cDNA sequence had the encoding region of 1620 bp encoding 539 amino acids,and was named LpluF3′5′H(GenBank No.OQ657278).Bioinformatics predicted that LpluF3′5′H belongs to the CYP75A subfamily,cytochrome P450 superfamily.Phylogenetic analysis revealed that the protein encoded by LpluF3′5′H was the highest similarity to the F3'5'H protein of L.angustifolius,and the monocotyledonous plants F3'5'H protein clustered into another family.Subcellular localization showed that LpluF3'5'H was localized in chloroplasts.qPCR analysis indicated that the expression of LpluF3′5′H could be detected in different development stages and petals.Besides,LpluF3′5′H showed the stronger expression in'Lupine Blue'and lower in'Lupine White'.In the different developmental stages of'Lupine Blue',the expression levels of LpluF3′5′H peaked in the second developmental stage and then decreased.Among the different petals of'Lupine Blue',LpluF3′5′H was the most highly expressed in the wings,followed by the vexils,and lowest expressed in the keels.LpluF3′5′H might play an important role in the flower color formation of'Lupine Blue',and the study provides a basis for further research into the flower color mechanism of blue-flowered L.polyphyllus formation.

关 键 词：多叶羽扇豆’鲁冰妮’ 类黄酮3’ 5’羟化酶(F3’5’H)基因 克隆 基因表达 

分 类 号：S68[农业科学—观赏园艺]