三倍体枇杷花期调控基因EjAGL6的表达特性和功能分析  

Expression Characterization and Function Analysis of the EjAGL6 Gene in Triploid Loquat

作　　者：蒲籽言 胡若倩 徐欣羽 郭启高[1,2] 夏燕 景丹龙 PU Ziyan;HU Ruoqian;XU Xinyu;GUO Qigao;XIA Yan;JING Danlong(Key Laboratory of Horticulture Science for Southern Mountains Regions of Ministry of Education,College of Horticulture and Landscape Architecture,Southwest University,Chongqing 400715,China;State Cultivation Base of Crop Stress Biology for Southern Mountainous Land of Southwest University,Academy of Agricultural Sciences of Southwest University,Chongqing 400715,China;Chongqing Chaoyang Middle School,Chongqing 400700,China)

机构地区：[1]南方山地园艺学教育部重点实验室,西南大学园艺园林学院,重庆400715 [2]西南大学南方山地作物逆境生物学国家级培育基地,西南大学农业科学研究院,重庆400715 [3]重庆市朝阳中学,重庆400700

出　　处：《西北植物学报》2022年第8期1263-1272,共10页Acta Botanica Boreali-Occidentalia Sinica

基　　金：国家自然科学青年基金(32102321);国家级大学生创新创业训练计划(202110635115);重庆市科技局(cstc2021jcyj-sxmX1156,cstc2021jscx-gksbX0010);重庆市高校创新研究群体项目(CXQT19005);重庆市中小学创新人才培养工程项目(CY210203)。

摘　　要：以三倍体枇杷(Eriobotrya japonica)‘华玉无核1号’的花芽为材料,采用基因克隆技术获得EjAGL6基因,分析其序列、亚细胞定位特性以及在二倍体和三倍体枇杷早晚花品种中的表达水平。采用花序浸染转化拟南芥,并利用实时荧光定量PCR分析转基因拟南芥植株的EjAGL6基因表达量,进一步观察野生型与EjAGL6转基因拟南芥的表型差异,分析EjAGL6基因的功能,为解析EjAGL6基因参与三倍体枇杷花期调控机制提供理论依据。结果显示:(1)成功获得MADS-box基因EjAGL6;该基因的编码区序列(CDS)为732 bp,编码243个氨基酸,分子质量为27.88 kD,等电点为9.05,脂溶指数为79.05;系统进化树分析表明,枇杷EjAGL6与苹果MdAGL6蛋白质的相似性较高,聚在同一分支。(2)蛋白序列比对发现,EjAGL6的M区有57个氨基酸,I区有30个氨基酸,K区有82个氨基酸,C区有74个氨基酸,其中C区包含高度保守的AGL6基序Ⅰ和AGL6基序Ⅱ。(3)亚细胞定位分析表明,EjAGL6蛋白定位在细胞核,具有典型的MADS-box转录因子亚细胞定位特性。(4)实时荧光定量PCR分析表明,EjAGL6基因在二倍体和三倍体枇杷早、晚花品种中均有表达,主要集中于小花分化期(S6)、花蕾露白期(S7)和盛花期(S8),且EjAGL6基因在二倍体和三倍体早花品种中的花蕾露白期的表达量均较高。(5)转基因拟南芥株系的EjAGL6基因表达量显著高于野生型拟南芥;转EjAGL6基因植株表型观察显示,EjAGL6基因在拟南芥中过量表达能够使转EjAGL6基因拟南芥的开花时间提前1周左右。研究认为,EjAGL6基因可促使枇杷开花时间提前,推测EjAGL6基因在花蕾露白期发挥调控花期的关键作用。In this study, the EjAGL6 gene was cloned from the flower buds of ‘Huayuwuhe No.1’ in triploid loquat(Eriobotrya japonica) by gene cloning, and its sequence, subcellular localization and expression level in diploid and triploid loquat cultivars were analyzed. Arabidopsis thaliana was infected by flower dipping method. The expression level of EjAGL6 gene in transgenic A. thaliana plants was analyzed by qRT-PCR. The phenotypic differences between wild-type A. thaliana and EjAGL6 transgenic A. thaliana were observed, and the function of EjAGL6 gene was analyzed, providing theoretical basis for analyzing the mechanism of EjAGL6 gene involved in triploid loquat flowering. The results show:(1) Mads-box gene EjAGL6 was obtained successfully. The encoding region(CDS) of EjAGL6 is 732 bp, encoding 243 amino acids. The molecular weight of the putative protein is 27.88 kD, isoelectric point is 9.05, and liposolubility index is 79.05. Phylogenetic tree analysis showed that EjAGL6 and MdAGL6 had high homology and clustered into the same branch.(2) Protein sequence alignment suggested that EjAGL6 includes M domain(57 aa), I domain(30 aa), K domain(82 aa) and the C domain(74 aa), and two highly conserved motifs of AGL6 motifs I and II in the C-terminal domains.(3) Subcellular localization analysis indicated that the functional region of EjAGL6 protein was located in the nucleus, suggesting it has subcellular localization characteristics of MADS-box transcription factor.(4) The qRT-PCR analysis showed that EjAGL6 was expressed in diploid and triploid loquat early and late flowering cultivars. Among them, EjAGL6 expression level was mainly concentrated in the latter three stages of flower development, including the stages of florets(S6), white corollas of floral buds(S7) and full bloom(S8), and the EjAGL6 expression level of diploid and triploid early flowering cultivars were higher in white corollars stage of floral buds.(5) The expression level of EjAGL6 gene in transgenic A. thaliana was significantly higher than that in w

关 键 词：三倍体枇杷 EjAGL6 亚细胞定位 表达分析 功能分析 

分 类 号：Q785[生物学—分子生物学] Q786Q789