三倍体枇杷花期调控基因EjAGL6的表达特性和功能分析
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引用本文:蒲籽言,胡若倩,徐欣羽,郭启高,夏 燕,景丹龙.三倍体枇杷花期调控基因EjAGL6的表达特性和功能分析[J].西北植物学报,2022,42(8):1263~1272
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作者单位
蒲籽言1,2,胡若倩1,2,徐欣羽3,郭启高1,2,夏 燕1,2,景丹龙1,2* (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 4007152 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4007153 重庆市朝阳中学重庆 400700) 
基金项目:国家自然科学青年基金(32102321);
中文摘要: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。
中文关键词:三倍体枇杷  EjAGL6  亚细胞定位  表达分析  功能分析
 
Expression Characterization and Function Analysis of the EjAGL6 Gene in Triploid Loquat
Abstract: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 wild type A. thaliana. Phenotype observation of transgenic plants showed that overexpression of EjAGL6 gene in A. thaliana could advance flowering time of transgenic A. thaliana by one week. The results showed that EjAGL6 gene could promote the flowering time of loquat earlier, suggesting that EjAGL6 gene plays a key role in regulating flowering time during white corollas stage of floral buds.
keywords:triploid loquat  EjAGL6  subcellular localization  expression analysis  functional analysis
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