香石竹<em>DcHsfA4</em>基因克隆及表达特性分析

doi:10.3969/j.issn.1674-7968.2019.10.005

摘要
图/表
参考文献
相关文章 (15)

全文: PDF (7454 KB) HTML (1 KB)
输出: BibTeX | EndNote (RIS)

摘要热激转录因子(heat shock transcription factors, Hsfs)是响应逆境信号的重要元件。以香石竹(Dianthus caryophyllus)品种'Fancy'为材料,克隆得到热激转录因子基因DcHsfA4的完整编码序列(GenBank No. MN096327),序列全长1 173 bp,编码390个氨基酸。生物信息学分析结果显示,DcHsfA4蛋白分子式为C₁₉₆₃H₃₀₅₁N₅₇₇O₆₂₅S₈,分子量为44.99 kD,理论等电点为5.48,脂肪系数71.95,不稳定系数49.72,预测为不稳定蛋白。疏水性/亲水性预测表明DcHsfA4蛋白为亲水蛋白。蛋白跨膜性分析显示DcHsfA4属于非跨膜蛋白。二级结构预测显示,DcHsfA4氨基酸组成包括α-螺旋(42.56%)、延伸链(6.67%)、β-折叠(3.08%)和无规则卷曲(47.69%),属于不规则结构。氨基酸序列比对显示,DcHsfA4含有高度保守的DNA结合域,此外,还包含疏水7肽重复区,核定位信号序列和C末端激活域。系统进化分析表明,DcHsfA4与拟南芥(Arabidopsis thaliana) AtHsfA4同源性最高。利用qRT-PCR技术分析了DcHsfA4在常温或不同非生物胁迫处理下的表达特性,结果显示,在42 ℃胁迫、ABA或甘露醇分别处理后DcHsfA4的表达水平显著提高(P<0.05),4 ℃胁迫下,不同时间点DcHsfA4的表达模式不同,干旱处理24 h或NaCl处理12 h诱导了DcHsfA4的转录本上调表达。该研究结果为进一步探究香石竹热激转录因子的特性及响应逆境的生物学功能提供了依据。

	服务

	把本文推荐给朋友
	加入我的书架
	加入引用管理器
	E-mail Alert
	RSS
	作者相关文章
	万雪丽
	冯依
	刘庆华
	王奎玲

关键词 ：香石竹, 香石竹热激转录因子A4 (DcHsfA4), qRT-PCR, 非生物胁迫

Abstract：Heat shock transcription factors (Hsfs) are important components in response to stress signals. Carnation (Dianthus caryophyllus) variety 'Fancy' was used as experimental material to clone the complete coding sequence of DcHsfA4 (GenBank No. MN096327). The full length of the sequence was 1 173 bp and encoded 390 amino acid. Bioinformatics analysis showed that the molecular formula of DcHsfA4 protein was C₁₉₆₃H₃₀₅₁N₅₇₇O₆₂₅S₈, the molecular weight was 44.99 kD, the theoretical isoelectric point was 5.48, the fat coefficient was 71.95, and the instability coefficient was 49.72, which was predicted to be unstable protein. Hydrophobic/hydrophilic prediction indicated that the DcHsfA4 protein was a hydrophilic protein. Protein transmembrane analysis revealed that DcHsfA4 was a non-transmembrane protein. The secondary structure prediction showed that the amino acid composition of DcHsfA4 included α-helix (42.56%), extended chain (6.67%), β-sheet (3.08%), and irregular curl (47.69%), which belonged to an irregular structure. Amino acid sequence alignment revealed that DcHsfA4 contained a highly conserved DNA binding domain, in addition, two hydrophobic hepated repeat, a nuclear localization signal sequence and a C-terminal activation domain. Phylogenetic analysis indicated that DcHsfA4 had the highest homology with Arabidopsis thaliana AtHsfA4. The expression characteristics of DcHsfA4 under different abiotic stress treatments were analyzed by qRT-PCR. The results showed that the 42 ℃ stress, ABA or mannitol treatment significantly increased the expression level of DcHsfA4. Moreover, the expression pattern of DcHsfA4 was different at 4 ℃ stress. The transcripts of DcHsfA4 were up-regulated by drought treatment for 24 h and NaCl treatment for 12 h. The results provide a basis for further exploration of the characteristics of carnation heat shock transcription factors and the biological functions in response to stress.

Key words： Carnation Carnation heat shock transcription factor A4 (DcHsfA4) qRT-PCR Abiotic stress

收稿日期: 2019-04-04

ZTFLH:	S682.1+9
	Q785

基金资助:国家自然科学基金(No. 31700623)、山东省高等学校科技计划项目(No. J17KA144)和青岛农业大学高层次人才科研基金(No. 663/1116016)

通讯作者: wkl6310@163.com

引用本文:

万雪丽, 冯依, 刘庆华, 王奎玲. 香石竹DcHsfA4基因克隆及表达特性分析[J]. 农业生物技术学报, 2019, 27(10): 1751-1760.
WAN Xue-Li, FENG Yi, LIU Qing-Hua, WANG Kui-Ling. Cloning and Expressional Characteristics Analysis of DcHsfA4 in Dianthus caryophyllus. 农业生物技术学报, 2019, 27(10): 1751-1760.

链接本文:

http://journal05.magtech.org.cn/Jwk_ny/CN/10.3969/j.issn.1674-7968.2019.10.005 或 http://journal05.magtech.org.cn/Jwk_ny/CN/Y2019/V27/I10/1751

1 Baniwal S K, Bharti K, Chan K Y, et al.2004. Heat stress response in plants: A complex game with chaperones and more than twenty heat stress transcription factors[J]. Journal of Biosciences, 29(4): 471-487.
2 Baniwal S K, Chan K Y, Scharf K D, et al.2006. Role of heat stress transcription factor HsfA5 as specific repressor of HsfA4[J]. Journal of Biological Chemistry, 282(6): 3605-3613.
3 Bechtold U, Albihlal W S, Lawson T, et al.2013. Arabidopsis heat shock transcription factorA1b overexpression enhances water productivity, resistance to drought, and infection[J]. Journal of Experimental Botany, 64(11): 3467-3481.
4 Boxriker M, Boehm R, Krezdorn N, et al.2017. Comparative transcriptome analysis of vase life and carnation type in Dianthus caryophyllus L.[J]. Scientia Horticulturae, 217: 61-72.
5 Chen S S, Jiang J, Han X J, et al.2018. Identification, expression analysis of the Hsf Family, and characterization of class A4 in Sedum alfredii Hance under cadmium stress[J]. International Journal of Molecular Sciences, 19(4): 1216.
6 Czarnecka-Verner E, Pan S, Salem T, et al.2004. Plant class B HSFs inhibit transcription and exhibit affinity for TFIIB and TBP[J]. Plant Molecular Biology, 56(1): 57-75.
7 Fu Z D, Wang H N, Liu J X, et al.2011. Molecular cloning and characterization of carnation EBF1 gene during flower senescence and upon ethylene exposure and sugar[J]. Agricultural Sciences in China, 10(12): 1872-1880.
8 Huang Y, Li M Y, Wang F, et al.2015. Heat shock factors in carrot: Genome-wide identification, classification, and expression profiles response to abiotic stress[J]. Molecular Biology Reports, 42(5): 893-905.
9 In B C, Binder B M, Falbel T G, et al.2013. Analysis of gene expression during the transition to climacteric phase in carnation flowers (Dianthus caryophyllus L.)[J]. Journal of Experimental Botany, 64(16): 4923-4937.
10 Iordachescu M, Verlinden S.2005. Transcriptional regulation of three EIN3-like genes of carnation (Dianthus caryophyllus L. cv. Improved White Sim) during flower development and upon wounding, pollination, and ethylene exposure[J]. Journal of Experimental Botany, 56(418): 2011-2018.
11 Jung H S, Crisp P A, Estavillo G M, et al.2013. Subset of heat-shock transcription factors required for the early response of Arabidopsis to excess light[J]. Proceedings of the National Academy of Sciences of the USA, 110(35): 14474-14479.
12 Kong W L, Bendahmane M, Fu X P.2018. Genome-wide identiﬁcation and characterization of aquaporins and their role in the flower opening processes in carnation (Dianthus caryophyllus)[J]. Molecules, 23: 189.
13 Kotak S, Port M, Ganguli A, et al.2004. Characterization of C-terminal domains of Arabidopsis heat stress transcription factors (Hsfs) and identification of a new signature combination of plant class A Hsfs with AHA and NES motifs essential for activator function and intracellular localization[J]. Plant Journal, 39: 98-112.
14 Li F, Zhang H, Zhao H, et al.2017. Chrysanthemum CmHSFA4 gene positively regulates salt stress tolerance in transgenic chrysanthemum[J]. Plant Biotechnology Journal, 16(7).
15 Larkindale J, Vierling E.2008. Core genome responses involved in acclimation to high temperature[J]. Plant Physiology, 146(2): 748-761.
16 Leonard R T, Alexander A M, Nell T A.2011. Postharvest performance of selected Colombian cut flowers after three transport systems to the United States[J]. Horttechnology, 21: 435-442.
17 Lim T.2014. Edible Medicinal and Non-Medicinal Plants[M]. New York: Springer Science+Business Media Dordrecht, pp. 684-693.
18 Lin Y X, Jiang H Y, Chu Z X, et al.2011. Genome-wide identification, classification and analysis of heat shock transcription factor family in maize[J]. BMC Genomics,12(1): 76.
19 Liu A L, Zou J, Zhang X W, et al.2010. Expression profiles of class a rice heat shock transcription factor genes under abiotic stresses[J]. Journal of Plant Biology, 53(2): 142-149.
20 Miller G, Mittler R.2006. Could heat shock transcription factors function as hydrogen peroxide sensors in plants?[J] Annals of Botany, 98(2): 279-288.
21 Mittal D, Chakrabarti S, Sarkar A, et al.2009. Heat shock factor gene family in rice: Genomic organization and transcript expression profiling in response to high temperature, low temperature and oxidative stresses[J]. Plant Physiology and Biochemistry, 47(9): 785-795.
22 Nishizawa-Yokoi A, Nosaka R, Hayashi H, et al.2011. HsfA1d and HsfA1e involved in the transcriptional regulation of HsfA2 function as key regulators for the Hsf signaling network in response to environmental stress[J]. Plant & Cell Physiology, 52(5): 933-945.
23 Nover L, Bharti K, Döring P, et al.2001. Arabidopsis and the heat stress transcription factor world: How many heat stress transcription factors do we need?[J] Cell Stress & Chaperones, 6(3): 177-189.
24 Ogawa D, Yamaguchi K, Nishiuchi T.2007. High level overexpression of the Arabidopsis HsfA2 gene confers not only increased thermotolerance but also salt/osmotic stress tolerance and enhanced callus growth[J]. Journal of Experimental Botany, 58(12): 3373-3383.
25 Ohama N, Kusakabe K, Mizoi J, et al.2016. The transcriptional cascade in the heat stress response of Arabidopsis is strictly regulated at the level of transcription factor expression[J]. Plant Cell, 28(1): 181-201.
26 Perez-Salamo I, Papdi C, Rigo G, et al.2014. The heat shock factor A4A confers salt tolerance and is regulated by oxidative stress and the mitogen-activated protein kinases MPK3 and MPK6[J]. Plant Physiology, 165(1): 319-334.
27 Satoh S, Shibuya K, Waki K, et al.2005. Mechanism of senescence in carnation flowers[J]. Acta Horticulturae, 669: 191-198.
28 Scharf K D, Berberich T, Ebersberger I, et al.2012. The plant heat stress transcription factor (Hsf) family: Structure, function and evolution[J]. Biochimica Et Biophysica Acta, 1819(2): 104-119.
29 Scharf K D, Heider H, Hohfeld I, et al.1998. The tomato Hsf system: HsfA2 needs interaction with HsfA1 for efficient nuclear import and may be localized in cytoplasmic heat stress granules[J]. Molecular and Cellular Biology, 18(4): 2240-2251.
30 Schramm F, Ganguli A, Kiehlmann E, et al.2006. The heat stress transcription factor HsfA2 serves as a regulatory amplifier of a subset of genes in the heat stress response in Arabidopsis[J]. Plant Molecular Biology, 60(5): 759-772.
31 Tanase K, Otsu S, Satoh S, et al.2015. Expression levels of ethylene biosynthetic genes and senescence-related genes in carnation (Dianthus caryophyllus L.) with ultra-long-life flowers[J]. Scientia Horticulturae, 183: 31-38.
32 Tang R, Zhu W, Song X,et al.2016. Genome-wide identification and function analyses of heat shock transcription factors in potato[J]. Frontiers in Plant Science, 7: 490.
33 Verlinden S, Garcia J J V.2004. Sucrose loading decreases ethylene responsiveness in carnation (Dianthus caryophyllus cv. White Sim) petals[J]. Postharvest Biology and Technology, 31(3): 305-312.
34 Wan X L, Zhou Q, Wang Y Y, et al.2015. Identification of heat-responsive genes in carnation (Dianthus caryophyllus L.) by RNA-seq[J]. Frontiers in Plant Science, 6: 519.
35 Wang X, Huang W, Liu J, et al.2017. Molecular regulation and physiological functions of a novel FaHsfA2c cloned from tall fescue conferring plant tolerance to heat stress[J]. Plant Biotechnology Journal, 15(2): 237-248.
36 Xiang J, Ran J, Zou J, et al.2013. Heat shock factor OsHsfB2b negatively regulates drought and salt tolerance in rice[J]. Plant Cell Reports, 32(11): 1795-1806.
37 Yagi M, Kosugi S, Hirakawa H, et al.2014. Sequence analysis of the genome of carnation (Dianthus caryophyllus L.)[J]. DNA Research, 21(3): 231-241.
38 Yokotani N, Ichikawa T, Kondou Y, et al.2008. Expression of rice heat stress transcription factor OsHsfA2e enhances tolerance to environmental stresses in transgenic Arabidopsis[J]. Planta, 227(5): 957-967.
39 Yoshida T, Ohama N, Nakajima J, et al.2011. Arabidopsis HsfA1 transcription factors function as the main positive regulators in heat shock-responsive gene expression[J]. Molecular Genetics and Genomics, 286(5-6): 321-332.
40 Zhang S, Xu Z S, Li P, et al.2013. Overexpression of TaHSF3 in transgenic Arabidopsis enhances tolerance to extreme temperatures[J]. Plant Molecular Biology Reporter, 31(3): 688-697.
41 Zhang X, Wang Q, Yang S, et al.2018. Identification and characterization of the MADS-Box genes and their contribution to flower organ in carnation (Dianthus caryophyllus L.)[J]. Genes, 9(4): 193.