甘蓝型油菜光敏色素互作因子4 (BnaPIF4)基因克隆和功能分析

第2期
冯韬等: 甘蓝型油菜光敏色素互作因子 4 (B
PIF4 proteins in the closely related species are highly clustered in the phylogenetic tree. PIF4 protein repeats were observed in a large number of plants and the degree of differentiation of PIF4 was lower in lower plants than in higher plants. It indicates that PIF4 protein differentiation is a late evolutionary event and there may be functional redundancy in PIF4 protein. Yeast hybridization experiments showed that there were interactions between BnaPIF4 and BnaBZR proteins, but BnaPIF4 could not interact with the promoter of BnaBZR gene, indicating that BnaPIF4 interacts with BnaBZR at the protein level but not at the transcription level. The genes expression patterns of BnaPIF4_A03 and BnaPIF4_C03 in Xiangyou 15 were consistent. BnaPIF4 gene was mainly expressed in the green tissue of B. napus L., with higher expression levels in stem epidermis, immature pods and leaves, and lower expression levels in flowers and roots, and the gene expression level of BnaPIF4 gradually decreased in the development process of B. napus L. Keywords: Brassica napus L; phytochrome interacting factor 4; gene clone; interaction of gene; gene expression; bionformatic analysis
Abstract: Phytochrome interacting factor 4 (PIF4) is a key transcription factor in light signaling pathway of plants, PIF4 interacts with Brassinazole-resistant (BZR) to mediate the interaction between light signal and brassinosteroid signal and participates in plant photoresponse. In this study, two novel PIF4 gene were isolated from Brassica napus L. cv. Xiangyou 15, they were identified on chromosomes A03 and C03 and encoding 413 and 414 amino acids, respectively, named as BnaPIF4_A03 and BnaPIF4_C03, their coding sequence (CDS), full-length mRNA and full-length gene were 1242 bp and 1245 bp, 1701 bp and 1731 bp, 2527 bp and 2665 bp, respectively. BnaPIF4_A03 and BnaPIF4_C03 had seven and eight exons, six and seven introns, respectively. Compared with the sequenced Zhongshuang 11, BnaPIF4_A03 gene had a single base insertion mutation in the first intron, a deletion mutation in the fourth and sixth introns, and a longer 3'-UTR. Other sequences of the two genes did not differ between Xiangyou 15 and Zhongshuang 11. The BnaPIF4_A03 and BnaPIF4_C03 gene-encoded proteins had a typical plant bHLH domain and were subcellularly localized in the nucleus. They are typical plant PIF4 proteins. Multiple sequence alignment and phylogenetic analysis showed that the BnaPIF4 protein was highly homologous to the PIF4 protein of Brassica oleracea, Arabidopsis thalian, and Eruca sativa. The evolutionary relationship of PIF4 protein was consistent with that of species, and the
Cloning and characterization of phytochrome interacting factor 4 (BnaPIF4) gene from Brassica napus L.
FENG Tao and GUAN Chun-Yun*
College of Agronomy, Hunan Agricultural University / National Oilseed Crops Improvement Center in Hunan, Changsha 410128, Hunan, China
低光寡照影响甘蓝型油菜结实, 是造成油菜减 产的重要原因之一[1], 增强油菜对低光胁迫的抗性 是生产上面临的重要议题。低光如何影响油菜产量 尚无明确的机制研究, 光信号通过何种转化最终影 响油菜产量形成尚不明确。有研究显示施用外源油 菜素内酯可以增强番茄[2]、水稻[3]等多种作物光合效
率、增强弱光抗性、增加产量, 过表达油菜素内酯 合成基因 DWF4 增强甘蓝型油菜非生物胁迫抗性和 产量[4], 表明光信号途径与油菜素内酯信号途径的 互作可能是光最终影响油菜产量的原因之一。在拟 南芥和水稻中均证实光敏色素互作因子 4 (phytochrome interacting factor 4, PIF4)与芸薹素唑抗性因 子(brassinazole-resistant, BZR/BES)互作介导光信号 途径与油菜素内酯信号途径的信号转导[5], 对甘蓝 型油菜 PIF4 基因及其与 BZR/BES 互作的相关研究 是阐明弱光胁迫对油菜产量影响的关键问题之一, 作者前期工作中已克隆了甘蓝型油菜 BZR/BES 基因, 本文主要关注甘蓝型油菜 PIF4 基因相关研究。
作物学报 ACTA AGRONOMICA SINICA 2019, 45(2): 204213 ISSN 0496-3490; CN 11-1809/S; CODEN TSHPA9
/ E-mail: zwxb301@
DOI: 10.3724/SP.J.1006.2019.84085
甘蓝型油菜光敏色素互作因子 4 (BnaPIF4)基因克隆和功能分析
冯 韬 官春云*
湖南农业大学农学院 / 国家油料改良中心湖南分中心, 湖南长沙 410128
摘 要: 光敏色素互作因子 4 (phytochrome interacting factor 4, PIF4)是光信号途径关键转录因子, PIF4 与 BZR 互作介 导光信号与油菜素内酯信号互作, 参与植物光响应。本文在甘蓝型油菜湘油 15 号中克隆到 2 个 PIF4 基因, 分别定位 于 A03 号染色体和 C03 号染色体, 命名为 BnaPIF4_A03 和 BnaPIF4_C03, 全长编码序列(coding sequence, CDS)、全 长 mRNA 和全长基因分别为 1242 bp 和 1245 bp、1701 bp 和 1731 bp、2527 bp 和 2665 bp, 各自编码 413 和 414 个氨 基酸。BnaPIF4_A03 具有 7 个外显子和 6 个内含子, BnaPIF4_C03 具有 8 个外显子和 7 个内含子, 与测序品种中双 11 号相比, BnaPIF4_A03 基因第 1 内含子存在单碱基插入突变, 第 4 和第 6 内含子存在缺失突变, 且具有更长的 3'-UTR, 两基因其他序列在湘油 15 号和中双 11 号之间无差别。BnaPIF4_A03 和 BnaPIF4_C03 基因编码蛋白具有典型的植物 bHLH 结构域, 亚细胞定位于细胞核, 是典型的植物 PIF4 蛋白。多序列比对和进化分析表明, BnaPIF4 蛋白与白菜、 拟南芥、亚麻芥等 PIF4 蛋白高度同源。PIF4 蛋白进化关系与物种进化关系一致, 近缘物种中的 PIF4 蛋白在进化树 中高度聚类, 大量植物中可见 PIF4 蛋白重复且低等植物中分化程度明显低于高等植物中, 表明 PIF4 蛋白是一个晚期 进化事件且可能存在功能冗余。酵母杂交实验表明 BnaPIF4 与 BnaBZR 蛋白存在互作, 但 BnaPIF4 不能与 BnaBZR 基因的启动子互作, 表明 BnaPIF4 与 BnaBZR 在蛋白水平而非转录水平发生互作。湘油 15 号中 BnaPIF4_A03 和 BnaPIF4_C03 基因表达规律一致, BnaPIF4 基因主要表达于油菜茎表皮、未成熟角果和叶中, 在花和根中表达较低, 且其表达随油菜生育进程逐渐降低。 关键词: 甘蓝型油菜; 光敏色素互作因子 4; 基因克隆; 基因互作; 基因表达; 生物信息学分析
本研究由国家重点基础研究发展计划(973计划)项目(2015CB150206)资助。 This study was supported by the National Basic Research Program (973 Program) (2015CB150206). * 通信作者(Corresponding author): 官春云, E-mail: guancy2011@ 第一作者联系方式: E-mail: 812298771@ Received(收稿日期): 2018-06-21; Accepted(接受日期): 2018-08-20; Published online(网络出版日期): 2018-09-25. URL: /kcms/detail/11.1809.S.20180920.1555.002.html