Abstract：Hypericum perforatum is an important perennial medicinal plant in the form of herb in China. APETALA2 (AP2) is a special transcription factor family in plants, which plays a key role in plant growth and development. H. perforatum, as a sequenced species in Malpighiales, its AP2 gene family has not been analyzed. In order to reveal the characteristics and functions of HpAP2 gene family, a total of 21 AP2 transcription factors were identified using bioinformatics methods based on the transcriptome sequencing data in different tissues of H. perforatum, and their physicochemical properties, gene structure, conserved motifs, phylogenetic relationship, cis-acting elements and expression pattern were analyzed. In addition, the expression of selected 5 genes in H. perforatum under the treatment of 3 kinds of hormones were detected by qRT-PCR. Results showed that physicochemical properties of HpAP2 family were different, but they all belonged to hydrophilic protein. In the secondary structure of AP2 protein, the proportion of random coil and α-helix was large, while that of extended strand and β-turn was small. The HpAP2 family was subdivided into the eu-AINTEGUMENTA (euANT), euAP2, and basalANT groups according to the phylogenetic relationship. The same subfamily members shared similar gene structure and conserved motifs. Cis-acting elements in the promoter region of HpAP2 genes included 4 categories responding to transcription, development, abiotic or biological stress and hormone. Transcriptome data showed that most AP2 family genes (57.14%) were expressed in 4 tissues of root, stem, leaf and flower, but there were significant differences in expression levels. qRT-PCR analyses demonstrated that the 5 HpAP2 genes were differentially expressed under the treatment of 3 kinds of hormones. Among them, the gene expression of HpAP2-5, HpAP2-11, and HpAP2-18 were significantly upregulated when treated by abscisic acid (ABA), salicylic acid (SA), and gibberellin (GA), respectively, suggesting that these genes might play roles in the response to stress-related phytohormones. This study laid a foundation for further study on the function of HpAP2 genes and genetic improvement of H. perforatum. This study provides reference for further exploring the function of HpAP2 gene family and analyzing its regulatory mechanism in stress responses.
[1] 付春, 唐易, 江纳, 等. 2020. 原始小球藻 AP2 转录因子的生物信息学分析[J]. 广西植物, 40(12): 1800-1815. (Fu C, Tang Y, Jiang N, et al. 2020. Bioinformatics analysis of AP2 transcription factor in Auxenochlorella protothecoides[J]. Guangxi Plants, 40(12): 1800-1815. ) [2] 郭海滨, 郝立冬, 魏雅冬. 2021. 全基因组鉴定和表达模式分析玉米 AP2 转录因子[J]. 分子植物育种, 19(05): 1402-1408. (Guo H B, Hao L D, Wei Y D. 2021. Genome-wide identification and expression profile analysis of maize AP2 transcription factors[J]. Molecular Plant Breeding, 19(05): 1402-1408. ) [3] 黄霞, 柯笑楠, 胡海超, 等. 2021. 全基因组鉴定和分析水稻AP2 基因家族[J]. 分子植物育种, 19(11): 3489-3498. (Huang X, Ke X N, Hu H C, et al. 2021. Genome-wide identification and characterization of the AP2 gene fami-ly in Oryza staiva[J]. Molecular Plant Breeding, 19(11): 3489-3498. [4] 欧阳辉, 谢丽艳, 黄小方, 等. 2010. 贯叶连翘的化学成分与药理研究进展及前景展望[J]. 江西中医药, 41(7): 78-80. (Ou Y H, Xie L Y, Huang X F, et al. 2010. Research progress and prospect of chemical constituents and phar-macology of Hypericum perforatum L[J]. Jiangxi Journal of Traditional Chinese Medicine, 41(7): 78-80. ) [5] 乔永刚, 陈亮, 崔芬芬, 等. 2019. 基于转录组金银花 AP2 基因家族的生物信息学及表达分析[J]. 核农学报, 33(09): 1698-1706. (Qiao Y G, Chen L, Cui F F, et al. 2019. Bioinformatics and expression analysis of AP2 gene family based on transcriptome of Lonicera japonica Thunb[J]. Journal of Nuclear Agricultural Sciences, 33(9): 1698-1706. ) [6] 孙滨, 占小登, 曹立勇, 等. 2017. 水稻 AP2/ERF 转录因子的研究进展[J]. 农业生物技术学报, 25(11): 1860-1869. (Sun B, Zhan X D, Cao L Y, et al. 2017. Research prog-ress of AP2/ERF transcription factor in rice (Oryza sati-va)[J]. Journal of Agricultural Biotechnology, 25(11): 1860-1869. ) [7] 王莉莉. 2016. 甘蓝型油菜 AP2 亚家族转录因子及植物中 WRI1 的基因组学分析[D]. 硕士学位论文, 西北农林科技大学, 导师: 张猛, pp. 10-25. (Wang L L. 2016. Ge-nome-wide investigation of AP2 subfamily in Brassica napus and Wri1 in plants[D]. Thesis for M. S., Northwest Agriculture and Forestry University, Supervisor: Zhang M, pp. 10-25. ) [8] Chinnusamy V, Zhu J H, Zhu J K. 2007. Cold stress regula-tion of gene expression in plants[J]. Trends in Plant Sci-ence, 12(10): 444-451. [9] Gil-Humanes J, Pistón F, Martín A, et al. 2009. Comparative genomic analysis and expression of the APETALA2-like genes from barley, wheat, and barley-wheat amphi-ploids[J]. BMC Plant Biology, 9(1): 66-79. [10] Guo J K, Wu J, Ji Q, et al. 2008. Genome-wide analysis of heat shock transcription factor families in rice and Arabi-dopsis[J]. Journal of Genetics and Genomics, 35(2): 105-118. [11] Hou W, Shakya P, Franklin G. 2016. A perspective on Hyperi-cum perforatum genetic transformation[J]. Frontiers in Plant Science, 7: 879-891. [12] Hu B, Jin J, Guo A Y, et al. 2015. GSDS 2. 0: An upgraded gene feature visualization server[J]. Bioinformatics, 31(8): 1296-1297. [13] Kong X P, Tian H Y, Yu Q Q, et al. 2018. PHB3 maintains root stem cell niche identity through ROS-responsive AP2/ERF transcription factors in Arabidopsis[J]. Cell Re-ports, 22(5): 1350-1363. [14] Krizek B A. 2011. Aintegumenta and aintegumenta-like 6 reg-ulate auxin-mediated flower development in Arabidopsis [J]. BMC Research Notes, 4(1): 176-184. [15] Krizek B A. 2015. AINTEGUMENTA-LIKE genes have part-ly overlapping functions with AINTEGUMENTA but make distinct contributions to Arabidopsis thaliana flow-er development[J]. Journal of Experimental Botany, 66(15): 4537-4549. [16] Kunst L, Klenz J E, Martinez-Zapater J, et al. 1989. AP2 gene determines the identity of perianth organs in flowers of Arabidopsis thaliana[J]. Plant Cell, 1(12): 1196-1208. [17] Lata C, Mishra A K, Muthamilarasan M, et al. 2014. Genome-wide investigation and expression profiling of AP2/ERF transcription factor superfamily in foxtail millet (Setaria italica L. )[J]. PLOS ONE, 9(11): 113092-113106. [18] Lecharny A, Boudet N, Gy I, et al. 2003. Introns in, introns out in plant gene families: A genomic approach of the dynamics of gene structure[J]. Journal of Structural and Functional Genomics, 3(1-4): 111-116. [19] Lescot M, Déhais P, Thijs G, et al. 2002. PlantCARE, a data-base of plant cis-acting regulatory elements and a portal to tools for in silico analysis of promoter sequences[J]. Nucleic Acids Research, 30(1): 325-327. [20] Li M Y, Wang F, Jiang Q, et al. 2013. Genome-wide analysis of the distribution of AP2/ERF transcription factors re-veals duplication and elucidates their potential function in Chinese cabbage (Brassica rapa ssp. pekinensis)[J]. Plant Molecular Biology Reporter, 31(4): 1002-1011. Licausi F, Ohme-Takagi M, Perata P. 2013. APETALA2/ethyl-ene responsive factor (AP2/ERF) transcription factors: Mediators of stress responses and developmental pro-grams[J]. New Phytologist, 199(3): 639-649. [21] Liu L, White M J, MacRae T H. 1999. Transcription factors and their genes in higher plants functional domains, evo-lution and regulation[J]. FEBS Journal, 262(2): 247-257. [22] Lynch M. 2002. Genomics. Gene duplication and evolution[J]. Science, 297(5583): 945-947. [23] Meng L S, Wang Z B, Yao S Q, et al. 2015. The ARF2-ANT-COR15A gene cascade regulates ABA-signaling-mediat-ed resistance of large seeds to drought in Arabidopsis[J]. Journal of Cell Science, 128(21): 3922-3932. [24] Nole-Wilson S, Tranby T L, Krizek B A. 2005. AINTEGU-MENTA-like (AIL) genes are expressed in young tis-sues and may specify meristematic or division-compe-tent states[J]. Plant Molecular Biology, 57(5): 613-628. [25] Riaz M W, Lu J, Shah L, et al. 2021. Expansion and molecu-lar characterization of AP2/ERF gene family in wheat (Triticum aestivum L. )[J]. Frontiers in Genetics, 12: 632155. [26] Rogozin I B, Sverdlov A V, Babenko V N, et al. 2005. Analy-sis of evolution of exon-intron structure of eukaryotic genes[J]. Briefings in Bioinformatics, 6(2): 118-134. [27] Sakuma Y, Liu Q, Dubouzet J G, et al. 2002. DNA-binding specificity of the ERF/AP2 domain of Arabidopsis DREBs, transcription factors involved in dehydration-and cold-inducible gene expression[J]. Biochemical and Biophysical Research Communications, 290(3): 998-1009. [28] Schmittgen T D, Livak K J. 2008. Analyzing real-time PCR data by the comparative CT method[J]. Nature Proto-cols, 3(6): 1101-1108. [29] Shannon S, Meekswagner D R. 1993. Genetic interactions that regulate inflorescence development in Arabidopsis [J]. Plant Cell, 5(6): 639-655. [30] Sirvent T M, Krasnoff S B, Gibson D M. 2003. Induction of hypericins and hyperforins in Hypericum perforatum in response to damage by herbivores[J]. Journal of Chemi-cal Ecology, 29(12): 2667-2681. [31] Toshitsugu N, Kaoru S, Tatsuhito F, et al. 2006. Genome-wide analysis of the ERF gene family in Arabidopsis and rice[J]. Plant Physiology, 140(2): 411-432. [32] Wang P, Cheng T, Lu M, et al. 2016. Expansion and function-al divergence of AP2 group genes in spermatophytes de-termined by molecular evolution and Arabidopsis mutant analysis[J]. Frontiers in Plant Science, 7: 1383. [33] Wang T Y, Ping X K, Cao Y R, et al. 2019. Genomewide ex-ploration and characterization of miR172/eu-AP2 genes in Brassica napus L. for likely role in flower organ devel-opment[J]. BMC Plant Biolgy, 19: 336. [34] Weber W, Stoep A V, Rachelle L, et al. 2008. Hypericum perfo-ratum (St John's Wort) for attention-deficit/hyperactivity disorder in children and adolescents: A randomized con-trolled trial[J]. Journal of the American Medical Associ-ation, 299(22): 2633-2641. [35] Wessler S R. 2005. Homing into the origin of the AP2 DNA binding domain[J]. Trends in Plant Science, 10(2): 54-56. [36] Wolfe K, Ó'hUigín C. 2016. Significance of positive selection and gene duplication in adaptive evolution: In memory of Austin L. Hughes[J]. Immunogenetics, 68(10): 749-753. [37] Yang Z, Chen J, Li C, et al. 2021. Identification and analysis of the AP2 subfamily transcription factors in the pecan (Carya illinoinensis)[J]. International Journal of Molecu-lar Sciences, 22(24): 13568. [38] Zahra K, Tourandokht B, Roghani M. 2016. Hypericum perfo-ratum hydroalcoholic extract mitigates motor dysfunc-tion and is neuroprotective in intrastriatal 6-hydroxydo-pamine rat model of Parkinson's disease[J]. Cellular and Molecular Neurobiology, 36(4): 521-530. [39] Zhang G Y, Chen M, Chen X P, et al. 2008. Phylogeny, gene structures, and expression patterns of the ERF gene fam-ily in soybean (Glycine max L. )[J]. Journal of Experi-mental Botany, 59(15): 4095-4107. [40] Zhang Q, Zhou W, Li B, et al. 2021. Genome-wide analysis and the expression pattern of the ERF gene family in Hy-pericum perforatum[J]. Plants, 10(1): 133. [41] Zhao Y, Ma R Y, Xu D L, et al. 2019. Genome-wide identifica-tion and analysis of the AP2 transcription factor gene family in wheat (Triticum aestivum L. )[J]. Frontiers in Plant Science, 10: 1286. [42] Zhou L, Yu X D, Wang D H, et al. 2021. Genome-wide identi-fication, classification and expression profile analysis of the HSF gene family in Hypericum perforatum[J]. PeerJ, 9: e11345. [43] Zhou W, Wang Y, Li B, et al. 2021. Whole genome sequence data of Hypericum perforatum and functional character-ization of melatonin biosynthesis by N-acetylserotonin O-methyltransferase[J]. Journal of Pineal Research, 70: e12709. [44] Zhuang J, Peng R H, Cheng Z M, et al. 2009. Genome-wide analysis of the putative AP2/ERF family genes in Vitis vinifera[J]. Scientia Horticulturae, 123(1): 73-81.
