甘蓝型油菜光敏色素互作因子4(BnaPIF4)基因克隆和功能分析

Cloning and characterization of phytochrome interacting factor 4(BnaPIF4) gene from Brassica napus L.

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。

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 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.