Cloning and Expression Analysis of ARP Gene in Capsicum annuum L.var.fasciculatum

doi:10.7668/hbnxb.20194037

Abstract

Abstract:

In order to explore the response of ARP to low temperature stress in ornamental pepper,and provide a theoretical basis for subsequent research on gene function in ornamental pepper under low temperature stress.This study cloned homologous ARP gene from ornamental pepper using RT-PCR technology,named CfARP,and analyzed their expression in different tissues of ornamental peppers and under low temperature treatment;Using bioinformatics analysis to study gene protein coding,physicochemical properties,and genetic relationships,and using qRT-PCR technology to detect the expression level of CfARP in different tissues of ornamental pepper and under low temperature treatment.The results showed that the CDS sequence of the CfARP gene was 342 bp,with 100% homology with Zunla 1.It could encode 113 amino acids and contained a conserved Auxin-repressed domain;protein physicochemical analysis revealed that the molecular weight of CfARP protein was 12.156 ku,its isoelectric point was 10.22,and the average coefficient of hydrophilicity was -0.913.It was preliminarily predicted that CfARP was a hydrophilic protein;compared with other species' ARP amino acid sequences,it was found that CfARP was highly conserved in Solanaceae plants.The Real-time Fluorescence Quantitative PCR results showed that the expression level of CfARP gene was the highest in the stems of ornamental pepper,followed by roots,and relatively less in leaves and flowers;the expression level of CfARP gene continuously increased with the increase of low-temperature treatment duration.It was preliminarily speculated that CfARP had a certain role in the response of ornamental pepper to low temperature stress.

Key words: Capsicum annuum L.var.fasciculatum, CfARP, Gene cloning, Bioinformatics analysis, qRT-PCR, Cold stress

HUANG Miao, LUO Youyou, NI Rui, ZHAO Jin. Cloning and Expression Analysis of ARP Gene in Capsicum annuum L.var.fasciculatum[J]. Acta Agriculturae Boreali-Sinica, 2023, 38(5): 69-76. doi: 10.7668/hbnxb.20194037.

/ / Recommend / Download Citations

URL: http://www.hbnxb.net/EN/10.7668/hbnxb.20194037

http://www.hbnxb.net/EN/Y2023/V38/I5/69

Figures/Tables 10

Fig.1 Sequences alignment of CaARP,SlARP and StARP Primer sequences are marked in red.

Fig.2 Agarose gel electrophoresis of the PCR products of CfARP M.DNA Marker;1,2.CfARP。

Fig.3 Gene and amino acid sequence of CfARP

Fig.4 Protein tertiary structure of CfARP

Fig.5 Sequences alignment of ARP

Fig.6 Multiple alignment results of the amino acid sequence of ARP

Fig.7 Phylogenetic tree of ARP

Fig.8 The prediction of CfARP conserved domain

Fig.9 Expression analysis of CfARP in different organs Different lowercase letters represent significant differences in relative expression at the 0.05 level.The same as Fig.10.

Fig.10 Expression analysis of CfARP at different time under low temperature treatment

References 35

[1]	Nascimento M F, do Rêgo E R, do Nascimento N F F, da Silva Leite P S, Finger F L, Bruckner C H, do Rêgo M M. Heritability of morpho-agronomic traits in ornamental pepper[J]. Crop Breeding and Applied Biotechnology, 2019, 19(3):253-261.doi:10.1590/1984-70332019v19n3a36.
[2]	Zhang Z Z, Lan M F, Han X Y, Wu J H, Wang-Pruski G. Response of ornamental pepper to high-temperature stress and role of exogenous salicylic acid in mitigating high temperature[J]. Journal of Plant Growth Regulation, 2020, 39(1):133-146.doi:10.1007/s00344-019-09969-y.
[3]	Guo Y M, Bai J J, Duan X D, Wang J. Accumulation characteristics of carotenoids and adaptive fruit color variation in ornamental pepper[J]. Scientia Horticulturae, 2021, 275:109699.doi:10.1016/j.scienta.2020.109699. URL
[4]	Baruah P M, Krishnatreya D B, Bordoloi K S, Gill S S, Agarwala N. Genome wide identification and characterization of abiotic stress responsive lncRNAs in Capsicum annuum[J]. Plant Physiology and Biochemistry, 2021, 162:221-236.doi:10.1016/j.plaphy.2021.02.031. pmid: 33706183
[5]	Zhang R X, Zhu W C, Cheng G X, Yu Y N, Li Q H, Haq S U, Said F, Gong Z H. A novel gene,CaATHB-12,negatively regulates fruit carotenoid content under cold stress in Capsicum annuum[J]. Food & Nutrition Research, 2020, 64:3729.doi:10.29219/fnr.v64.3729.
[6]	裴红霞, 高晶霞, 颜秀娟, 冯海萍, 赵云霞, 桑婷, 王学梅. 开花期低温胁迫对辣椒生长和生理特性的影响[J]. 安徽农业科学, 2020, 48(17):59-62,127.doi:10.3969/j.issn.0517-6611.2020.17.018.
	Pei H X, Gao J X, Yan X J, Feng H P, Zhao Y X, Sang T, Wang X M. Effects of cold stress on growth and physiological characteristics of pepper at flowering stage[J]. Journal of Anhui Agricultural Sciences, 2020, 48(17):59-62,127.
[7]	龙佳丽, 邹奕, 邳植, 吴则东. 转录组测序揭示激素介导的信号通路参与甜菜低温应答[J]. 中国农学通报, 2021, 37(31):5-14.doi:10.15889/j.issn.1002-1302.2020.17.010.
	Long J L, Zou Y, Pi Z, Wu Z D. Transcriptome analysis reveals hormone-mediated signaling pathways involved in low temperature response in sugar beet[J]. Chinese Agricultural Science Bulletin, 2021, 37(31):5-14.
[8]	Aslam M, Fakher B, Ashraf M A, Cheng Y, Wang B R, Qin Y. Plant low-temperature stress:Signaling and response[J]. Agronomy, 2022, 12(3):702.doi:10.3390/agronomy12030702. URL
[9]	呼芳娣, 刘丽君, 马骊, 牛早霞, 孙柏林, 徐佳, 杨雯昕, 刘博, 蒲媛媛, 武军艳, 方彦, 李学才, 沈金雄, 孙万仓. 低温胁迫下甘蓝型冬油菜BnYUCCA8基因差异表达及内源生长素含量变化[J]. 干旱地区农业研究, 2022, 40(3):1-10.doi:10.7606/j.issn.1000-7601.2022.03.01.
	Hu F D, Liu L J, Ma L, Niu Z X, Sun B L, Xu J, Yang W X, Liu B, Pu Y Y, Wu J Y, Fang Y, Li X C, Shen J X, Sun W C. Differential expression of BnYUCCA8 gene and changes of endogenous auxin content in Brassica napus L.under low temperature stress[J]. Agricultural Research in the Arid Areas, 2022, 40(3):1-10.
[10]	李琦瑶, 王树声, 刘光亮, 周培禄, 郑璇, 曾文龙, 陈志厚, 陈爱国. 低温胁迫及恢复生长后烟苗叶形指数及生长素的响应[J]. 江苏农业科学, 2019, 47(3):60-65.doi:10.15889/j.issn.1002-1302.2019.03.014.
	Li Q Y, Wang S S, Liu G L, Zhou P L, Zheng X, Zeng W L, Chen Z H, Chen A G. Response of leaf shape index and auxin of tobacco seedlings after low temperature stress and recovery of growth[J]. Jiangsu Agricultural Sciences, 2019, 47(3):60-65.
[11]	陈静, 江玲, 胡晓辉, 翟虎渠, 万建民. 花生生长素抑制蛋白基因AhARP的表达分析[J]. 核农学报, 2016, 30(4):638-645.doi:10.11869/j.issn.100-8551.2016.04.0638.
	Chen J, Jiang L, Hu X H, Zhai H Q, Wan J M. Expression analysis of auxin-repressed protein gene AhARP in peanut[J]. Journal of Nuclear Agricultural Sciences, 2016, 30(4):638-645.
[12]	王玲. 水稻突变体库创制及OsARP基因功能的初步研究[D]. 天津: 天津农学院, 2020.doi:10.11869/j.issn.100-8551.2016.04.0638.
	Wang L. Preliminary study on the creation of rice mutant library and the function of OsARP gene[D]. Tianjin: Tianjin Agricultural University, 2020.
[13]	牛歆雨, 德庆措姆, 朗杰, 梁东. 葡萄ARP/DRM基因家族克隆及其在休眠解除过程中的表达[J]. 分子植物育种, 2018, 16(18):5897-5903.doi:10.13271/j.mpb.016.005897.
	Niu X Y, De Q C M, Lang J, Liang D. Cloning of ARP/DRM gene family from grape and its expression during the process of dormancy-release[J]. Molecular Plant Breeding, 2018, 16(18):5897-5903.
[14]	Wu L M, Yu M, Holowachuk J, Sharpe A, Lydiate D, Hegedus D, Gruber M. Evaluation of a Brassica napus auxin-repressed gene induced by flea beetle damage and Sclerotinia sclerotiorum infection[J]. American Journal of Plant Sciences, 2017, 8(8):1921-1952.doi:10.4236/ajps.2017.88130.
[15]	杨艳. 低温及生长素处理下牡丹花芽生长素抑制蛋白基因的表达差异分析[D]. 青岛: 青岛农业大学, 2010.doi:10.7666/d.Y2332491.
	Yang Y. Differential expression analysis of auxin inhibitory protein gene in peony flower bud under low temperature and auxin treatment[D]. Qingdao: Qingdao Agricultural University, 2010.
[16]	Park S, Han K H. An auxin-repressed gene(RpARP)from black locust(Robinia pseudoacacia)is posttranscriptionally regulated and negatively associated with shoot elongation[J]. Tree Physiology, 2003, 23(12):815-823.doi:10.1093/treephys/23.12.815. URL
[17]	崔维芳. 生长素对苹果糖代谢的影响及苹果MdARP功能的初步研究[D]. 杨凌: 西北农林科技大学, 2019.
	Cui W F. Effect of auxin on sugar metabolism of apple and preliminary study on the function of apple MdARP[D]. Yangling: Northwest A&F University, 2019.
[18]	Īlter Ī B, Öner E T, Çiftçi Y Ö. Determination of the potential of fructants in plant cryopreservation applications[J]. Cryobiology, 2021, 103:170.doi:10.1016/j.cryobiol.2021.11.050.
[19]	于树涛, 吴金桐, 孙泓希, 任亮, 于洪波, 尤淑丽, 孙全喜, 王传堂. 花生生长素抑制蛋白AhARP1的克隆与表达分析[J]. 分子植物育种, 2016, 14(1):26-30.doi:10.13271/j.mpb.014.000026.
	Yu S T, Wu J T, Sun H X, Ren L, Yu H B, You S L, Sun Q X, Wang C T. Cloning and expression analysis of auxin-repressed protein gene AhARP1 in peanuts(Arachis hypogaea L.)[J]. Molecular Plant Breeding, 2016, 14(1):26-30.
[20]	Hwang E W, Kim K A, Park S C, Jeong M J, Byun M O, Kwon H B. Expression profiles of hot pepper(Capsicum annuum)genes under cold stress conditions[J]. Journal of Biosciences, 2005, 30(5):657-667.doi:10.1007/BF02703566. URL
[21]	Felsenstein J. Confidence limits on phylogenies:An approach using the bootstrap[J]. Evolution, 1985, 39(4):783-791.doi:10.1111/j.1558-5646.1985.tb00420.x.
[22]	Saitou N, Nei M. The neighbor-joining method:A new method for reconstructing phylogenetic trees[J]. Molecular Biology and Evolution, 1987, 4(4):406-425.doi:10.1093/oxfordjournals.molbev.a040454. pmid: 3447015
[23]	Tamura K, Nei M, Kumar S. Prospects for inferring very large phylogenies by using the neighbor-joining method[J]. Proceedings of the National Academy of Sciences of the United States of America, 2004, 101(30):11030-11035.doi:10.1073/pnas.0404206101. pmid: 15258291
[24]	Kumar S, Stecher G, Tamura K. MEGA7:Molecular evolutionary genetics analysis version 7.0 for bigger datasets[J]. Molecular Biology and Evolution, 2016, 33(7):1870-1874.doi:10.1093/molbev/msw054.
[25]	Calderón Villalobos L I A, Lee S, De Oliveira C, Ivetac A, Brandt W, Armitage L, Sheard L B, Tan X, Parry G, Mao H B, Zheng N, Napier R, Kepinski S, Estelle M. A combinatorial TIR1/AFB-Aux/IAA co-receptor system for differential sensing of auxin[J]. Nature Chemical Biology, 2012, 8(5):477-485.doi:10.1038/nchembio.926. pmid: 22466420
[26]	Liscum E, Reed J W. Genetics of Aux/IAA and ARF action in plant growth and development[J]. Plant Molecular Biology, 2002, 49(3/4):387-400.doi:10.1023/A:1015255030047. URL
[27]	Tiwari S B, Hagen G, Guilfoyle T. The roles of auxin response factor domains in auxin-responsive transcription[J]. The Plant Cell, 2003, 15(2):533-543.doi:10.1105/tpc.008417. URL
[28]	de Souza G B, de Oliveira Mendes T A, Fontes P P, de Almeida Barros V, Gonçalves A B, de Freitas Ferreira T, Dal-Bianco Lamas Costa M, Alves M S, Fietto L G. Genome-wide identification and expression analysis of dormancy-associated gene 1/auxin repressed protein(DRM1/ARP)gene family in Glycine max[J]. Progress in Biophysics and Molecular Biology, 2019, 146:134-141.doi:10.1016/j.pbiomolbio.2019.03.006. URL
[29]	Zhao Y Y, Li C, Ge J, Xu M Y, Zhu Q, Wu T Q, Guo A, Xie J Y, Dong H S. Recessive mutation identifies auxin-repressed protein ARP1,which regulates growth and disease resistance in tobacco[J]. Molecular Plant-Microbe Interactions, 2014, 27(7):638-654.doi:10.1094/mpmi-08-13-0250-r. URL
[30]	文友义. OsARP1生长素抑制蛋白基因对水稻种子萌发和幼苗生长的影响[D]. 南昌: 南昌大学, 2019.doi:10.27232/d.cnki.gnchu.2019.001849.
	Wen Y Y. Effects of OsARP1 auxin inhibitory protein gene on rice seed germination and seedling growth[D]. Nanchang: Nanchang University, 2019.
[31]	Lee J, Han C T, Hur Y. Molecular characterization of the Brassica rapa auxin-repressed,superfamily genes,BrARP1 and BrDRM1[J]. Molecular Biology Reports, 2013, 40(1):197-209.doi:10.1007/s11033-012-2050-9. URL
[32]	Steiner C, Bauer J, Amrhein N, Bucher M. Two novel genes are differentially expressed during early germination of the male gametophyte of Nicotiana tabacum[J]. Biochimica et Biophysica Acta (BBA)-Gene Structure and Expression, 2003, 1625(2):123-133.doi:10.1016/s0167-4781(02)00598-5.
[33]	王新超, 马春雷, 杨亚军, 姚明哲, 金基强. 茶树生长素抑制蛋白基因CsARP1的克隆与表达分析[J]. 核农学报, 2011, 25(5):910-915,921.doi:10.3969/j.issn.1000-369X.2006.01.002.
	Wang X C, Ma C L, Yang Y J, Yao M Z, Jin J Q. Cloning and expression analysis of auxin-repressed protein gene Csarp1 in tea plant(Camellia sinensis)[J]. Acta Agriculturae Nucleatae Sinica, 2011, 25(5):910-915,921.
[34]	刁桂萍, 柳燕, 宋佳, 林佳忱, 刘志华. 二色补血草LbARP基因的克隆及其表达分析[J]. 安徽农业科学, 2014, 42(32):11250-11252.doi:10.3969/j.issn.0517-6611.2014.32.008.
	Diao G P, Liu Y, Song J, Lin J C, Liu Z H. Cloning and expression of an auxin-repressed protein gene LbARP from limonium bicolor[J]. Journal of Anhui Agricultural Sciences, 2014, 42(32):11250-11252.
[35]	Song J, Niu Z B, Li Q Q, Bao Y G, Ma X, Wang H G, Kong L R, Feng D S. Isolation and identification of differentially expressed genes from wheat in response to Blumeria graminis f.sp.tritici (Bgt)[J]. Plant Molecular Biology Reporter, 2015, 33(5):1371-1380.doi:10.1007/s11105-014-0838-6. URL

Please choose a citation manager

Content to export