百子莲2个ARF基因与2个Aux/IAA基因的全长克隆与序列分析

doi:10.3969/j.issn.1004-1524.2019.01.12

浙江农业学报 ›› 2019, Vol. 31 ›› Issue (1): 86-97.DOI: 10.3969/j.issn.1004-1524.2019.01.12

百子莲2个ARF基因与2个Aux/IAA基因的全长克隆与序列分析

杨舟, 吕可, 吕珊, 王俊杰, 张荻^*

上海交通大学农业与生物学院园林科学与工程系,上海 200240

收稿日期:2018-03-24 出版日期:2019-01-25 发布日期:2019-04-09
通讯作者: 张荻,E-mail: zhangdi2013@sjtu.edu.cn
作者简介:杨舟(1994—),女,浙江杭州人,硕士研究生,研究方向为观赏植物种质资源保存与创新。E-mail: xinranzhuimeng@sjtu.edu.cn
基金资助:
国家自然科学基金(31670693,31300580)

Cloning and sequence analysis of two ARF genes and two Aux/IAA genes in Agapanthus praecox ssp. orientalis

YANG Zhou, LYU Ke, LYU Shan, WANG Junjie, ZHANG Di^*

Department of Landscape Science and Engineering, School of Agriculture and Biology, Shanghai Jiao Tong University, Shanghai 200240, China

Received:2018-03-24 Online:2019-01-25 Published:2019-04-09

摘要/Abstract

摘要： 采用不同浓度外源生长素类调节物质毒莠定(PIC)对百子莲(Agapanthus praecox ssp. orientalis)进行继代培养,并对其胚性愈伤组织进行比较转录组学研究,获得了百子莲生长素信号转导途径中的2个ARF和2个Aux/IAA家族基因,推测其为体胚发生过程中生长素信号传导途径相关的重要调控因子。采用RACE技术获得ApARF1、ApARF2、ApAux/IAA2与ApAux/IAA3的cDNA全长,分别为2 343、2 888、1 034和821 bp,开放阅读框(ORF)长度分别为1 770、2 349、480和549 bp,分别编码589、782、159和182个氨基酸残基。生物学分析表明,ApARF1与ApARF2蛋白均具有ARF家族的典型结构,ApAux/IAA2蛋白结构域Ⅳ不完整,ApAux/IAA3结构域Ⅱ部分缺失,但仍具有Aux/IAA蛋白的典型功能。转基因实验表明,ApAux/IAA过表达植株表现出生长延缓及发育受阻的表型,ApARF过表达植株表现出生长促进、花期提前的表型,验证了百子莲ApARF和ApAux/IAA家族蛋白对植物生长发育的调控作用。

关键词: 百子莲, 生长素信号, ARF, Aux/IAA, 基因克隆, 胚性愈伤组织

Abstract: Using the Illumina sequencing platform, transcriptome sequencing of Agapanthus praecox ssp. orientalis callus and embryonic callus treated with different concentrations of exogenous auxin picloram (PIC) were performed. Two ARF genes and two Aux/IAA genes that expressed differentially during the auxin signal transduction pathway were obtained and named as ApARF1, ApARF2, ApAux/IAA2 and ApAux/IAA3. To further understand their function in auxin signal transduction during somatic embryogenesis, their full-length cDNA sequences were cloned by RACE technique. The full-length cDNA sequences of ApARF1, ApARF2, ApAux/IAA2 and ApAux/IAA3 were 2 343, 2 888, 1 034 and 821 bp, each contained an open reading frame of 1 770, 2 349, 480 and 549 bp, respectively, which encoded 589, 782, 159 and 182 amino acids, respectively. Through bioinformatic analysis including protein domain analysis, phylogenetic tree analysis, and protein homology analysis, we concluded that ApARF1 and ApARF2 proteins had the typical construction of ARFs, and might be related to plant aging; ApAux/IAA2 lacked a completed domain Ⅳ, ApAux/IAA3 lacked a completed domain Ⅱ; however, they may still have the typical function of Aux/IAA family. We constructed gene over-expression vectors with pHB vector and transformed them into wild-type Arabidopsis thaliana. The phenotypic results showed that compared with the wide-type, the over-expression of ApARF1 and ApARF2 could promote the growth and flowering of transgenic plants, while the over-expression of ApAux/IAA2 and Aux/IAA3 showed a depressing influence on the growth of transgenic plants. The transgenic results combined with the sequence analysis proved that ApARFs and ApAux/IAAs had a regulatory role in auxin signal transduction pathway and plant growth.

Key words: Agapanthus praecox, auxin signal, ARF (auxin response factors), Aux/IAA (auxin/indoleacetic acid proteins), gene cloning, embryonic callus

中图分类号:

杨舟, 吕可, 吕珊, 王俊杰, 张荻. 百子莲2个ARF基因与2个Aux/IAA基因的全长克隆与序列分析[J]. 浙江农业学报, 2019, 31(1): 86-97.

YANG Zhou, LYU Ke, LYU Shan, WANG Junjie, ZHANG Di. Cloning and sequence analysis of two ARF genes and two Aux/IAA genes in Agapanthus praecox ssp. orientalis[J]. , 2019, 31(1): 86-97.

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百子莲2个ARF基因与2个Aux/IAA基因的全长克隆与序列分析

Cloning and sequence analysis of two ARF genes and two Aux/IAA genes in Agapanthus praecox ssp. orientalis

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