Welcome to Acta Agronomica Sinica,

Acta Agronomica Sinica ›› 2019, Vol. 45 ›› Issue (2): 204-213.doi: 10.3724/SP.J.1006.2019.84085


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

Tao FENG,Chun-Yun GUAN()   

  1. College of Agronomy, Hunan Agricultural University / National Oilseed Crops Improvement Center in Hunan, Changsha 410128, Hunan, China
  • Received:2018-06-21 Accepted:2018-08-20 Online:2019-02-12 Published:2018-09-25
  • Contact: Chun-Yun GUAN E-mail:guancy2011@aliyun.com
  • Supported by:
    This study was supported by the National Basic Research Program (973 Program)(2015CB150206)


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

Fig. 1

Cloning of BnaPIF4_A03 and BnaPIF4_C03 A: CDS; B: mRNA; C: Full length gene; M1: 2K DNA marker; M2: 2K plus DNA marker; 1: BnaPIF4_A03; 2: BnaPIF4_C03."

Fig. 2

Gene structure of BnaPIF4_A03 and BnaPIF4_C03"

Table 1

Summary of the deduced BnaPIF4_A03 and BnaPIF4_ C03 proteins"

Number of amino acid residues
Molar mass (Da)
Isoelectric point
BnaPIF4-A03 413 46,374.937 5.535
BnaPIF4-C03 414 46,260.633 5.595

Fig. 3

Secondary structure of BnaPIF4_A03 and BnaPIF4_C03"

Table 2

Summary of specific region of BnaPIF4_A03 and BnaPIF4_C03"

Serial number
Domains type
Sequence motif
BnaPIF4_A03 PS00001 4 158-161, 185-188, 234-237, 253-256 NQSQ, NSSS, NKSN, NLSE N-{P}-[ST]-{P}
PS00005 7 43-45, 61-63, 242-244, 243-245, 255-257, 280-282, 400-402 THR, TLR, STR, TRR, SER, TDK, SQR [ST]-x-[RK]
PS00006 9 7-10, 43-46, 66-69, 97-100, 160-163, 162-165, 175-178, 226-229, 284-287 SFEE, THRD, TFLE, STID, SQTD, TDLD, TIDE, SQSD, SILE [ST]-x(2)-[DE]
PS00007 1 174-181 KTIDERLY [RK]-x(2,3)-[DE]-x(2,3)-Y
PS00008 8 156-161, 189-194, 190-195, 193-198, 232-237, 306-311, 308-313, 393-398 GSNQSQ, GGSSGC, GSSGCS, GCSLGK, GNNKSN, GSGMAG, GMAGAA, GSPAGQ G-{EDRKHPFYW}-x(2)-[STAGCN]-{P}
PS00845 2 371-377, 368-377 GRYVGLF, DRFGRYVGLF [GD]-x(1)-[FYWA]-x(1)-G-[LIVM]-x(0)-[LIVMFYD]
BnaPIF4_C03 PS00001 4 13-16, 158-161, 185-188, 253-256 NLSN, NQSQ, NSSS, NLSE N-{P}-[ST]-{P}
PS00005 8 43-45, 61-63, 109-111, 242-244, 243-245, 255-257, 280-282, 400-402 THR, TLR, STR, TRR, SER, TDK, SQR, THR, TLR, TVK, STR, TRR, SER, TDK, SQR [ST]-x-[RK]
PS00006 10 7-10, 43-46, 66-69, 97-100, 160-163, 162-165, 175-178, 226-229, 278-281, 284-287 SFEE, THRD, TFLE, STID, SQTD, TDLD, TIDE, SQSD, TKTD, SILE [ST]-x(2)-[DE]
PS00007 1 174-181 KTIDERLY [RK]-x(2,3)-[DE]-x(2,3)-Y
PS00008 9 156-161, 189-194, 190-195, 193-198, 232-237, 239-244, 306-311, 308-313, 393-398 GSNQSQ, GGSSGC, GSSGCS, GCSLGK, GNNKSN, GSGSTR, GSGMAG, GMAGAA, GSPAGQ G-{EDRKHPFYW}-x(2)-[STAGCN]-{P}
PS00845 2 371-377, 368-377 GRYVGLF, DRFGRYVGLF [GD]-x(1)-[FYWA]-x(1)-G-[LIVM]-x(0)-[LIVMFYD]

Fig. 4

Comparison of the amino acid sequences of BnaPIF4"

Fig. 5

Phylogenetic analysis of PIF4 in different plant species"

Fig. 6

Interaction of BnaPIF4 and BnaBZR/BES"

Fig. 7

Expression levels of BnaPIF4_A03 and BnaPIF4_C03 in root, stem, leaf, flower, and silique of Xiangyou 15 at different developmental stages DAG means days after seed germination."

[1] 卢坤, 申鸽子, 梁颖, 符明联, 贺斌, 铁琳梅, 张烨, 彭柳, 李加纳 . 适合不同产量的环境下油菜高收获指数的产量构成因素分析. 作物学报, 2017,43:82-96.
doi: 10.3724/SP.J.1006.2017.00082
Lu K, Shen G Z, Liang Y, Fu M L, He B, Tie L M, Zhang Y, Peng L, Li J N . Analysis of yield components with high harvest index in Brassica napus under environments fitting different yield levels. Acta Agron Sin, 2017,43:82-96 (in Chinese with English abstract).
doi: 10.3724/SP.J.1006.2017.00082
[2] Irfan M, Alam J, Ahmad I, Ali I, Gul H . Effects of exogenous and foliar applications of brassinosteroid (BRs) and salt stress on the growth, yield and physiological parameters of Lycopersicon esculentum(Mill.). Plant Sci Today, 2017,4:88-101.
[3] Thussagunpanit J, Jutamanee K, Kaveeta L, Chaiarree W, Pankean P, Homvisasevongsa S, Suksamrarn A . Comparative effects of brassinosteroid and brassinosteroid mimic on improving photosynthesis, lipid peroxidation, and rice seed set under heat stress. J Plant Growth Regul, 2015,34:320-331.
doi: 10.1007/s00344-014-9467-4
[4] Sahni S, Prasad B D, Liu Q, Grbic V, Sharpe A, Singh S P, Krishna P . Overexpression of the brassinosteroid biosynthetic gene DWF4 in Brassica napus simultaneously increases seed yield and stress tolerance. Sci Rep, 2016,6:28298.
doi: 10.1038/srep28298 pmid: 4915011
[5] Oh E, Zhu J Y, Wang Z Y . Interaction between BZR1 and PIF4 integrates brassinosteroid and environmental responses. Nat Cell Biol, 2012,14:802-809.
doi: 10.1038/ncb2545 pmid: 22820378
[6] 杨剑飞, 王宇, 杨琳, 李玉花 . 光敏色素互作因子 PIFs 是整合多种信号调控植物生长发育的核心元件. 植物生理学报, 2014,50:1109-1118.
Yang J F, Wang Y, Yang L, Li Y H . Phytochrome-interacting factors integrate multiple signals to control plant growth and development. Plant Physiol J, 2014,50:1109-1118 (in Chinese with English abstract).
[7] Huq E, Quail P H . PIF4, a phytochrome-interacting bHLH factor, functions as a negative regulator of phytochrome B signaling in Arabidopsis. EMBO J, 2002,21:2441-2450.
doi: 10.1093/emboj/21.10.2441 pmid: 12006496
[8] Castillon A, Shen H, Huq E . Phytochrome interacting factors: central players in phytochrome-mediated light signaling networks. Trends Plant Sci, 2007,12:514-521.
doi: 10.1016/j.tplants.2007.10.001
[9] Casson S A, Franklin K A, Gray J E, Grierson C S, Whitelam G C, Hetherington A M . Phytochrome B and PIF4 regulate stomatal development in response to light quantity. Curr Biol, 2009,19:229-234.
doi: 10.1016/j.cub.2008.12.046 pmid: 19185498
[10] Koini M A, Alvey L, Allen T, Tilley C A, Harberd N P, Whitelam G C, Franklin K A . High temperature-mediated adaptations inplant architecture require the bHLH transcription factor PIF4. Curr Biol, 2009,19:408-413.
doi: 10.1016/j.cub.2009.01.046 pmid: 19249207
[11] Franklin K A, Lee S H, Patel D, Kumar S V, Spartz A K, Gu C, Wigge P A . Phytochrome-interacting factor 4 (PIF4) regulates auxin biosynthesis at high temperature. Proc Natl Acad Sci USA, 2011,108:20231-20235.
doi: 10.1073/pnas.1110682108
[12] Xu H, Liu Q, Yao T, Fu X . Shedding light on integrative GA signaling. Curr Opin Plant Biol, 2014,21:89-95.
doi: 10.1016/j.pbi.2014.06.010 pmid: 25061896
[13] Bernardo-García S, Lucas M, Martínez C, Espinosa-Ruiz A, Davière J M, Prat S . BR-dependent phosphorylation modulates PIF4 transcriptional activity and shapes diurnal hypocotyl growth. Genes Dev, 2014,28:1681-1694.
doi: 10.1101/gad.243675.114 pmid: 25085420
[14] 韩霜, 陈素梅, 蒋甲福, 房伟民, 管志勇, 陈发棣 . 弱光下菊花‘清露’的激素水平及相关基因表达. 中国农业科学, 2015,48:324-333.
doi: 10.3864/j.issn.0578-1752.2015.02.12
Han S, Chen S M, Jiang J F, Fang W M, Guan Z Y, Chen F T . Hormone levels and gene expression analysis of chrysanthemum cultivar ‘puma sunny’ under low light intensity. Sci Agric Sin, 2015,48:324-333 (in Chinese with English abstract).
doi: 10.3864/j.issn.0578-1752.2015.02.12
[15] Chalhoub B, Denoeud F, Liu S, Parkin I A, Tang H, Wang X, Corréa M . Early allopolyploid evolution in the post- Neolithic Brassica napus oilseed genome. Science, 2014,345:950-953.
[16] Carretero-Paulet L, Galstyan A, Roig-Villanova I, Martínez Gacía J F, Bilbao-Castro J R, Robertson D L . Genome-wide classification and evolutionary analysis of the bHLH family of transcription factors in Arabidopsis, poplar, rice, moss, and algae. Plant Physiol, 2010,153:1398-1412.
doi: 10.1104/pp.110.153593 pmid: 20472752
[17] Feller A, Machemer K, Braun E L, Grotewold E . Evolutionary and comparative analysis of MYB and bHLH plant transcription factors. Plant J, 2011,66:94-116.
doi: 10.1111/j.1365-313X.2010.04459.x pmid: 21443626
[18] Surhone L M, Timpledon M T, Marseken S F. Rapeseed. Germany: Betascript Publishing, 2010. pp 6-8.
[19] Kumar S V, Lucyshyn D, Jaeger K E, Alós E, Alvey E, Harberd N P, Wigge P A . Transcription factor PIF4 controls the thermosensory activation of flowering. Nature, 2012,484:242-245.
doi: 10.1038/nature10928 pmid: 22437497
[20] Lucas M, Prat S . PIFs get BR right: PHYTOCHROME INTERACTING FACTORs as integrators of light and hormonal signals. New Phytol, 2014,202:1126-1141.
doi: 10.1111/nph.12725 pmid: 24571056
[21] Choi H, Oh E . PIF4 integrates multiple environmental and hormonal signals for plant growth regulation in Arabidopsis. Mol Cell, 2016,39:587-593.
doi: 10.14348/molcells.2016.0126 pmid: 4990750
[22] Wei Z, Yuan T, Tarkowská D, Kim J, Nam H G, Novák O, Li J . Brassinosteroid biosynthesis is modulated via a transcription factor cascade of COG1, PIF4 and PIF5. Plant Physiol, 2017,174:1260-1273.
doi: 10.1104/pp.16.01778 pmid: 28438793
[23] Wang Z Y, Nakano T, Gendron J, He J, Chen M, Vafeados D, Chory J . Nuclear-localized BZR1 mediates brassinosteroid- induced growth and feedback suppression of brassinosteroid biosynthesis. Dev Cell, 2002,2:505-513.
doi: 10.1016/S1534-5807(02)00153-3 pmid: 11970900
[1] LI Hai-Fen, WEI Hao, WEN Shi-Jie, LU Qing, LIU Hao, LI Shao-Xiong, HONG Yan-Bin, CHEN Xiao-Ping, LIANG Xuan-Qiang. Cloning and expression analysis of voltage dependent anion channel (AhVDAC) gene in the geotropism response of the peanut gynophores [J]. Acta Agronomica Sinica, 2022, 48(6): 1558-1565.
[2] JIN Rong, JIANG Wei, LIU Ming, ZHAO Peng, ZHANG Qiang-Qiang, LI Tie-Xin, WANG Dan-Feng, FAN Wen-Jing, ZHANG Ai-Jun, TANG Zhong-Hou. Genome-wide characterization and expression analysis of Dof family genes in sweetpotato [J]. Acta Agronomica Sinica, 2022, 48(3): 608-623.
[3] WANG Rui, CHEN Xue, GUO Qing-Qing, ZHOU Rong, CHEN Lei, LI Jia-Na. Development of linkage InDel markers of the white petal gene based on whole-genome re-sequencing data in Brassica napus L. [J]. Acta Agronomica Sinica, 2022, 48(3): 759-769.
[4] QU Jian-Zhou, FENG Wen-Hao, ZHANG Xing-Hua, XU Shu-Tu, XUE Ji-Quan. Dissecting the genetic architecture of maize kernel size based on genome-wide association study [J]. Acta Agronomica Sinica, 2022, 48(2): 304-319.
[5] CHEN Xin-Yi, SONG Yu-Hang, ZHANG Meng-Han, LI Xiao-Yan, LI Hua, WANG Yue-Xia, QI Xue-Li. Effects of water deficit on physiology and biochemistry of seedlings of different wheat varieties and the alleviation effect of exogenous application of 5-aminolevulinic acid [J]. Acta Agronomica Sinica, 2022, 48(2): 478-487.
[6] WANG Yan-Peng, LING Lei, ZHANG Wen-Rui, WANG Dan, GUO Chang-Hong. Genome-wide identification and expression analysis of B-box gene family in wheat [J]. Acta Agronomica Sinica, 2021, 47(8): 1437-1449.
[7] SONG Tian-Xiao, LIU Yi, RAO Li-Ping, Soviguidi Deka Reine Judesse, ZHU Guo-Peng, YANG Xin-Sun. Identification and expression analysis of cell wall invertase IbCWIN gene family members in sweet potato [J]. Acta Agronomica Sinica, 2021, 47(7): 1297-1308.
[8] ZHOU Xin-Tong, GUO Qing-Qing, CHEN Xue, LI Jia-Na, WANG Rui. Construction of a high-density genetic map using genotyping by sequencing (GBS) for quantitative trait loci (QTL) analysis of pink petal trait in Brassica napus L. [J]. Acta Agronomica Sinica, 2021, 47(4): 587-598.
[9] LI Shu-Yu, HUANG Yang, XIONG Jie, DING Ge, CHEN Lun-Lin, SONG Lai-Qiang. QTL mapping and candidate genes screening of earliness traits in Brassica napus L. [J]. Acta Agronomica Sinica, 2021, 47(4): 626-637.
[10] QIN Tian-Yuan, LIU Yu-Hui, SUN Chao, BI Zhen-Zhen, LI An-Yi, XU De-Rong, WANG Yi-Hao, ZHANG Jun-Lian, BAI Jiang-Ping. Identification of StIgt gene family and expression profile analysis of response to drought stress in potato [J]. Acta Agronomica Sinica, 2021, 47(4): 780-786.
[11] MENG Jiang-Yu, LIANG Guang-Wei, HE Ya-Jun, QIAN Wei. QTL mapping of salt and drought tolerance related traits in Brassica napus L. [J]. Acta Agronomica Sinica, 2021, 47(3): 462-471.
[12] WANG Rui-Li, WANG Liu-Yan, LEI Wei, WU Jia-Yi, SHI Hong-Song, LI Chen-Yang, TANG Zhang-Lin, LI Jia-Na, ZHOU Qing-Yuan, CUI Cui. Screening candidate genes related to aluminum toxicity stress at germination stage via RNA-seq and QTL mapping in Brassica napus L. [J]. Acta Agronomica Sinica, 2021, 47(12): 2407-2422.
[13] LI Peng, LIU Che, SONG Hao, YAO Pan-Pan, SU Pei-Lin, WEI Yao-Wei, YANG Yong-Xia, LI Qing-Chang. Identification and analysis of non-specific lipid transfer protein family in tobacco [J]. Acta Agronomica Sinica, 2021, 47(11): 2184-2198.
[14] HUANG Su-Hua, LIN Xi-Yue, LEI Zheng-Ping, DING Zai-Song, ZHAO Ming. Physiological characters of carbon, nitrogen, and hormones in ratooning rice cultivars with strong regeneration ability [J]. Acta Agronomica Sinica, 2021, 47(11): 2278-2289.
[15] GUO Qing-Qing, ZHOU Rong, CHEN Xue, CHEN Lei, LI Jia-Na, WANG Rui. Location and InDel markers for candidate interval of the orange petal gene in Brassica napus L. by next generation sequencing [J]. Acta Agronomica Sinica, 2021, 47(11): 2163-2172.
Full text



No Suggested Reading articles found!