油茶3个ARF基因的克隆及表达分析

余姝姝1,2,3,4,5,周俊琴1,3,4,5,卢梦琪1,3,4,5,刘懿瑶1,3,4,5,杨进1,3,4,5,谭晓风1,3,4,5,*
1中南林业科技大学林学院, 长沙410004;2重庆市梁平区湿地保护中心, 重庆405200;3经济林培育与保护教育部重点实验室, 长沙410004;4经济林育种与栽培国家林业局重点实验室, 长沙410004;5经济林培育与利用湖南省2011协同创新中心, 长沙410004

通信作者:谭晓风;E-mail: tanxiaofengcn@126.com

摘 要:

ARF (auxin response factor)又称生长素响应因子, 是生长素信号转导途径中调控生长素响应基因表达的转录因子, 参与植物的生长发育。本文采用PCR技术从油茶中克隆得到3ARF基因并将其分别命名为CoARF5CoARF9CoARF19, 其全长编码序列(CDS)分别为2 8352 0763 234 bp, 分别编码9446911 077个氨基酸。将油茶ARF蛋白与拟南芥ARF家族基因蛋白构建系统进化树, 分析发现CoARF5AtARF5CoARF9AtARF9CoARF19AtARF19亲缘关系最近。多序列比对结果显示, CoARF5CoARF9CoARF19蛋白均具有ARF家族特有的3个结构域: B3 DNA结合结构域、 Auxin_resp结构域和AUX/IAA结构域。实时荧光定量PCR结果显示, 不同组织表达差异明显, CoARF19基因在老茎和芽中表达较多; CoARF5CoARF9基因在芽中特异性表达。自交、异交和未授粉的子房及花柱表达分析发现,CoARF5CoARF9CoARF19在授粉24 h, 自交花柱的表达量均明显高于同期异交及未授粉的表达量;在授粉78~84 h, 异交子房的表达量均明显高于同期自交及未授粉的表达量, 由此推测CoARF5CoARF9CoARF19基因可能参与油茶自交不亲和反应。亚细胞定位结果显示3ARF基因均定位于细胞核, 符合转录因子的特点。以上研究结果表明3CoARF基因可能通过植物激素调控参与油茶的自交不亲和反应。

关键词:油茶; ARF; 基因克隆; 表达; 亚细胞定位

收稿:2020-06-22   修定:2021-03-09

资助:国家自然科学基金(31730016)

Cloning and expression analysis of three ARF genes in Camellia oleifera

YU Shushu1,2,3,4,5, ZHOU Junqin1,3,4,5, LU Mengq1,3,4,5, LIU Yiyao1,3,4,5, YANG Jin1,3,4,5, TAN Xiaofeng1,3,4,5,*
1College of Forestry, Central South University of Forestry and Technology, Changsha 410004, China; 2Chongqing Liangping Wetland Protection Center, Chongqing 405200, China; 3Key Laboratory of Cultivation and Protection for Non-wood Forest Trees, Ministry of Education, Changsha 410004, China; 4Key Lab of Non-wood Forest Products of State Forestry Administration, Changsha 410004, China; 5Cooperative Innovation Center of Cultivation and Utilization for Non-wood Forest Trees of Hunan Province, Changsha 410004, China

Corresponding author: TAN Xiaofeng; E-mail: tanxiaofengcn@126.com

Abstract:

ARF, known as auxin response factor, is a transcription factor that regulates the expression of auxin response gene in auxin signal transduction pathway. ARF also participates in plant growth and development. Three ARF genes were cloned from Camellia oleifera by PCR and named CoARF5, CoARF9 and CoARF19, respectively. Their full-length coding sequences (CDSs) are 2 835, 2 076 and 3 234 bp, respectively, encoding 944, 691 and 1 077 amino acids. The phylogenetic tree was constructed by the ARF proteins of C. oleifera and the ARF family of Arabidopsis thaliana. It was found that CoARF5 and AtARF5, CoARF9 and AtARF9, and CoARF19 and AtARF19 were closely related. The results of multiple sequence alignment show that CoARF5, CoARF9 and CoARF19 proteins all have three characteristic domains of ARF family: B3 DNA binding domain, Auxin_resp domain and AUX/IAA domain. The results of quantitative real-time PCR show that the expressions of different tissues were signifcantly different and the expression of CoARF19 gene was highly expressed in the old stem and bud, and the expressions of CoARF5 and CoARF9 genes were highly expressed in the bud. Expression analysis of self-pollinated, outcrossing and unfertilized ovaries and styles revealed that the expressions of CoARF5, CoARF9 and CoARF19 in self-pollination style were signifcantly higher than those in cross-pollination and non-pollination at 24 h of pollination, and the expressions of these genes in cross-pollination ovary were signifcantly higher than those in self-pollination and non-pollination at 78–84 h of pollination. The expression of CoARF5, CoARF9 and CoARF19 might be involved in the self-incompatibility of C. oleifera. The result of subcellular localization shows that all three ARF genes are located in the nucleus, which is consistent with the characteristics of transcription factors. The results show that the three CoARF genes might be involved in the self-incompatibility of C. oleifera by phytohormone regulation.

Key words: Camellia oleifera; ARF; gene cloning; expression; subcellular localization

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