桃PpIDD11调控花发育的功能研究

doi:10.16420/j.issn.0513-353x.2021-0566

摘要/Abstract

摘要：

从‘秋蜜红’桃中克隆到1个IDD转录因子基因PpIDD11。结果显示：PpIDD11编码区全长1 575 bp,编码524个氨基酸,具有保守的ID域。烟草亚细胞定位显示PpIDD11蛋白定位于细胞核,酵母转化试验表明其具有酵母转录自激活活性。荧光定量PCR分析显示PpIDD11主要在花器官中表达,特别在雌蕊中转录水平最高。过表达PpIDD11拟南芥株系出现了柱头高于野生型、荚果变短且结实率下降的表型,同时也出现了莲座叶叶片卷曲、植株变矮的现象。转录组测序分析显示,PpIDD11转基因拟南芥株系共有上调基因3 378个,下调基因3 156个,其中包含LOX4、LOX3、GLC、E6L1等花器官发育调控基因。

关键词: 桃, PpIDD11基因, 雌蕊, 株高, 叶片卷曲

Abstract:

In this study,an IDD transcription factor gene PpIDD11 was cloned from peach‘Qiumihong’. The coding sequence of PpIDD11 is 1 575 bp,it encodes 524 amino acids and has a conserved ID domain. The assay of subcellular localization showed that PpIDD11 was localized in the nucleus. PpIDD11 showed the transcriptional activation activity in yeast. Quantitative real-time PCR analysis indicated that PpIDD11 was mainly expressed in floral organ,with a highest transcript level in pistil. Overexpress PpIDD11 Arabidopsis exhibited a phenotype of higher stigma,shorter siliqua and fewer seeds compared with wild type. In addition,the transgenic lines showed a curled rosette leaves and dwarf phenotype. Transcriptome sequencing analysis showed that the PpIDD11 transgenic Arabidopsis line had 3 378 up-regulated genes and 3 156 down-regulated genes,including the regulatory genes（LOX4,LOX3,GLC,E6L1,etc）of floral organ development.

Key words: peach, PpIDD11, pistil, plant height, leaf curling

中图分类号:

S662.1

蒋亚君, 陈佳佳, 谭彬, 郑先波, 王伟, 张郎郎, 程钧, 冯建灿. 桃PpIDD11调控花发育的功能研究[J]. 园艺学报, 2022, 49(9): 1841-1852.

JIANG Yajun, CHEN Jiajia, TAN Bin, ZHENG Xianbo, WANG Wei, ZHANG Langlang, CHENG Jun, FENG Jiancan. Function Exploration of PpIDD11 in Regulating Peach Flower Development[J]. Acta Horticulturae Sinica, 2022, 49(9): 1841-1852.

图/表 10

表1 本研究中所用的引物序列

Table 1 Primer sequences used in this study

用途 Use	引物名称 Primer name	序列（5′-3′） Sequence
克隆 Clone	PpIDD11-F	TGGGGAAAGAAAACAAAGGAGA
克隆 Clone	PpIDD11-R	TCAATTAAACCTTTGCATTCTTAGG
亚细胞定位 Subcellular localization	PpIDD11-GFP-EcoRⅠ-F	GTGGATCCAAAGAATTCTCCTTTTTATCTGTATCTTCCTCCA
亚细胞定位 Subcellular localization	PpIDD11-GFP-EcoRⅠ-R	CTCCTTTACCCATGAATTCATTAAACCTTTGCATTCTTAGG
自激活鉴定 Self-activation	PpIDD11-BD-EcoRⅠ-F	CCGGAATTCTTTCCTTTTTATCTGTATCTTCCTCCA
自激活鉴定 Self-activation	PpIDD11-BD-PstⅠ-R	GCACTGCAGTCAATTAAACCTTTGCATTCTTAGG
过表达 Primers for overexpression sites	PpIDD11-SAK-EcoRⅠ-F	CCGGAATTCTGGGGAAAGAAAACAAAGGAGA
过表达 Primers for overexpression sites	PpIDD11-SAK-XhoⅠ-R	CCGCTCGAGTCAATTAAACCTTTGCATTCTTAGG
定量 qRT-PCR	PpIDD11-qPCR-F	ATGTATGGCGGGCTTTTATTC
定量 qRT-PCR	PpIDD11-qPCR-R	TGCTGATGCTGGTCATGTTG
定量桃内参 Primers for peach internal control	PpTEF2-qPCR-F	GGTGTGACGATGAAGAGTGATG
定量桃内参 Primers for peach internal control	PpTEF2-qPCR-R	TGAAGGAGAGGGAAGGTGAAAG
定量拟南芥内参 Primers for Arabidopsis internal control	AtUBC-qPCR-F	CTGCGACTCAGGGAATCTTCTAA
定量拟南芥内参 Primers for Arabidopsis internal control	AtUBC-qPCR-R	TTGTGCCATTGAATTGAACCC

图1 序列比对和IDD保守域 At：拟南芥;Os：水稻;Pp：桃;Zm：玉米。下同。

Fig. 1 Sequence alignment and IDD conserved domain At：Arabidopsis;Os：Rice;Pp：Peach;Zm：Maize. The same below.

图2 IDD家族蛋白与桃PpIDD11聚类树

Fig. 1 Phylogenetic tree of IDD family proteins and PpIDD11

图3 PpIDD11蛋白的亚细胞定位

Fig. 3 Subcellular localization of PpIDD11 protein

图4 转录自激活验证

Fig. 4 Transcriptional self-activation verification of PpIDD11 protein

图5 PpIDD11在‘秋蜜红’桃不同组织（A）和不同花发育时期（B）中的相对表达量 1：花芽未分化期;2：花萼分化期;3：花瓣分化期;4：雄蕊分化期;5：雌蕊分化期;6：花蕾;7：花苞;8：盛花。

Fig. 5 The relative expression of PpIDD11 in various tissues（A）and different stages of flower development（B）of‘Qiumihong’peach 1：Undifferentiated flower bud period;2：Calyx differentiation period;3：Petal differentiation period;4：Stamen differentiation period;5：Pistil differentiation period;6：Buds period;7：Budding flower period;8：Full bloom period.

图6 转PpIDD11拟南芥株系的PpIDD11相对表达量（A）及其花和角果特征（B ~ G） * P < 0.05;** P < 0.01.

Fig. 6 Relative expression level（A）,flower and silique characteristics（B-G）of PpIDD11 overexpressed Arabidopsis lines

图7 野生型与过表达PpIDD11拟南芥株系表型

Fig. 7 Phenotype of wild type and PpIDD11 overexpressed Arabidopsis lines

图8 PpIDD11转基因拟南芥叶片横/纵曲率和株高 * P < 0.05;** P < 0.01.

Fig. 8 Identification of phenotypic analysis of PpIDD11 overexpressed Arabidopsis lines

表2 与转基因表型相关的差异基因及其功能

Table 2 Differential genes related to transgenic phenotypes and their functions

基因ID Gene ID	表达差异 log₂（IDD11/WT）	名称 Name	功能 Function	参考文献 Reference
AT1G21910	2.77242	DREB26	植物生长发育 Plant growth and development	Krishnaswamy et al.,2011
AT3G18010	2.40747	WOX1	分生组织发育 Meristematic tissue development	Zhang et al.,2011
AT1G65450	-3.77981	GLC	参与植物受精过程 Involved in the process of plant fertilisation	Leshem et al.,2012
AT3G12500	-3.07184	ATHCHIB	叶片形态发育Leaf blade morphology development	Robles et al.,2001
AT2G33850	-2.78991	E6L1	参与植物早期授粉过程 Involved in the early pollination process of plants	Doucet et al.,2019
AT1G72520	-2.61478	LOX4	花器官发育 Floral organ development	Caldelari et al.,2011
AT1G17420	-2.54068	LOX3	花器官发育 Floral organ development	Caldelari et al.,2011

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