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 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.

Key words: Brassica napus L, phytochrome interacting factor 4, gene clone, interaction of gene, gene expression, bionformatic analysis