龙州凤仙花ImLHY和ImC4H基因克隆及表达分析
Cloning and expression analysis of ImLHY and ImC4H genes in Impatiens morsei Hook.f.
赵潞秋 1张晓丽 1李凡 1谭弋 1石万磊 1黄美娟 1黄海泉1
作者信息
- 1. 西南林业大学园林园艺学院/国家林业和草原局西南风景园林工程技术研究中心/云南省功能性花卉资源及产业化技术工程研究中心/西南林业大学园林园艺花卉研发中心,云南昆明 650224
- 折叠
摘要
[目的]对龙州凤仙花晚期下胚轴蛋白基因(ImLHY)和肉桂酸-4-羟基化酶基因(ImC4H)进行克隆及表达分析,探讨龙州凤仙花旗瓣和翼瓣的彩斑区与非斑区颜色差异,克隆LHY和C4H基因,为凤仙花彩斑分子调控机制及花色改良和新品种培育提供理论依据.[方法]以龙州凤仙花为材料,测定其旗瓣和翼瓣的彩斑区与非斑区色度值以及总花色苷和总黄酮含量,对ImLHY和ImC4H基因进行克隆,利用生物学软件对其进行序列特征、蛋白结构和系统发育分析,利用实时荧光定量PCR检测ImLHY和ImC4H基因在不同发育关键时期(花苞期、盛花期和衰败期)及旗瓣和翼瓣不同着色区的表达模式.[结果]旗瓣和翼瓣的彩斑区a*(红度值)和C*(彩度值)均高于非斑区,非斑区的L*(亮度值)大于彩斑区;旗瓣彩斑区b*(黄度值)高于旗瓣非斑区,而翼瓣彩斑区与非斑区的b*(黄度值)差异较小,且旗瓣和翼瓣的彩斑区总花色苷和总黄酮含量均高于非斑区.克隆获得ImLHY和ImC4H基因的cDNA全长分别为1698和1518 bp,分别编码565和505个氨基酸残基,GenBank登录号分别为 OR096417和OR096418.ImLHY和ImC4H蛋白均为亲水性不稳定蛋白,均不具有跨膜结构域.ImLHY蛋白有1个Myb DNA-binding保守结构域,且具有MYB转录因子家族的SANT结构域,定位于细胞核中;ImC4H蛋白含有1个P450超家族结构域(PLN02394),定位于内质网上.ImLHY和ImC4H蛋白分别与喜马拉雅凤仙花LHY和C4H的氨基酸序列相似性达68%和94%,且在系统发育进化树中两者分别均与喜马拉雅凤仙花LHY和C4H聚在一支.2个基因在花发育的3个关键时期(花苞期、盛花期和衰败期)及旗瓣和翼瓣的彩斑区与非斑区均有表达,且整体表现为在盛花期的相对表达量较高;在盛花期时,ImLHY和ImC4H基因在旗瓣的相对表达量均表现为彩斑区显著高于非斑区,在翼瓣的相对表达量均表现为非斑区高于彩斑区.[结论]ImLHY和ImC4H基因在盛花期时主要调控旗瓣彩斑形成,而在翼瓣主要调控非斑区形成,推测旗瓣和翼瓣的彩斑形成存在不一样的调控机制.
Abstract
[Objective]Cloning and expression analysis of ImLHY and ImC4H genes were carried out to investigate the color difference between variegation and non-variegation areas in vexil and wing petals of Impatiens morsei.Cloning of LHY and C4H genes provided theoretical basis for molecular regulation mechanism of flower variegation,flower color improvement and new variety breeding of Impatiens.[Method]The I.morsei as the material,the colorimetric values,con-tents of total anthocyanins and total flavonoids of variegation and non-variegation areas in vexil and wing petals were de-termined.The ImLHY and ImC4H genes were cloned,and their sequence characteristics,protein structure and phylogeny were analyzed by biological software.Real-time quantitative PCR was used to detect the expression patterns of ImLHY and ImC4H genes at different developmental stages(bud stage,blooming stage and decay stage)and in different colored regions of vexil and wing petals.[Result]The a* value(redness value)and C* value(chroma value)of the variegation were higher than the non-variegation area in vexil and wing petals,and L*(brightness value)of the non-variegation area was is greater than the variegation area;and the b* value(yellowness value)of the variegation area was higher than that of the non-variegation area in vexil petal,while the b* value(yellowness value)of the variegation area was less different from that of the non-variegation area in wing petal.The contents of total anthocyanins and flavonoids in vexil and wing petals were higher than those in non-variegation area.The full-length cDNA sequences of ImLHY and ImC4H genes were 1698bp and 1518bp,encoding 565aa and 505aa,respectively.The GenBank login numbers were No.OR096417 and No.OR096417.ImLHY and ImC4H genes were hydrophilic unstable proteins without transmembrane domains.ImLHY pro-tein had a Myb DNA-binding conserved domain and a SANT domain of the MYB transcription factor family,which was localized in the nucleus;ImC4H protein contained a P450 superfamily domain(PLN02394),and located in the endoplas-mic reticulum The amino acid sequences of the ImLHY and ImC4H proteins had 68%and 94%similarity with Impatiens glandulifera.In phylogenetic evolutionary tree,both of them were clustered together with LHY and C4H of I.glandu-lifera respectively.The two genes were expressed in the three key stages of flower development(bud stage,blooming stage and decay stage)and in the variegation and non-variegation areas of vexil and wing petals,and the expression level was the highest at the blooming stage.At blooming stage,the expression levels of ImLHY and ImC4H genes were higher in variegation area of vexil petals than in non-variegation area,and higher in non-variegation area of the wing petals than in variegation area.[Conclusion]ImLHY and ImC4H genes mainly regulate the formation of variegation in vexil petals during the blooming stage,and mainly regulate the formation of variegation in wing petals.It was speculate that there were different regulatory mechanisms for the formation of variegation in vexil and wing petals of I.morsei.
关键词
龙州凤仙花/旗瓣/翼瓣/彩斑/LHY基因/C4H基因/表达分析Key words
Impatiens morsei/variegation/phenotypic assay/LHY gene/C4H gene/expression analysis引用本文复制引用
基金项目
国家自然科学基金项目(32060364)
国家自然科学基金项目(32060366)
云南省农业联合专项重点项目(202301BD070001-011)
云南省重大科技专项(202102AE090052)
云南省园林植物遗传改良与高效繁育博士生导师团队(503210103)
出版年
2023
国家科技期刊平台
NSTL
万方数据