香石竹热激转录因子基因DcHsfB1的克隆及其对不同非生物胁迫的表达响应
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摘要
热激转录因子(heat shock transcription factors, Hsfs)在植物遭受非生物胁迫时调控热激蛋白(heat shock protein, Hsp)和其他热激诱导的转录本的表达,参与响应热胁迫进程。本文以香石竹‘想象’作为实验材料,对基因DcHsfB1开放阅读框进行克隆。氨基酸序列的组成成分及理化性质分析结果表明, DcHsfB1含有一个804 bp的开放阅读框,编码267个氨基酸,分子量为30.78 kDa,理论等电点为5.84。DcHsfB1蛋白的二级及三级结构预测表明,α-螺旋占36.36%,延伸链占13.82%,β-折叠链占4.0%,无规则卷曲占45.82%,无规则卷曲构成了蛋白质的主要骨架。系统进化树分析结果显示DcHsfB1与AtHsfB1同源性最高。实时定量PCR分析DcHsfB1在各种非生物胁迫下的相对表达量。结果表明,在42°C胁迫2 h、ABA处理3 h和干旱胁迫3 h的Dc Hsf B1相对表达量显著上调;在甘露醇、NaCl和4°C胁迫下DcHsfB1的相对表达量也有不同程度的上升。
Heat shock transcription factors(Hsfs) regulate the expression of heat shock proteins(Hsps) and other heat shock-induced transcripts when plants are subjected to abiotic stress, and participate in the response to heat stress. In this paper, carnation ‘Fancy' was used as experimental material to clone the open reading frame of DcHsfB1. The composition and physicochemical properties analysis of the amino acid sequence showed that DcHsfB1 contained a 804 bp open reading frame encoding 267 amino acids with a molecular weight of 30.78 kDa and a theoretical isoelectric point of 5.84. The secondary structure and tertiary structure prediction of the protein showed that, α-helix accounted for 36.36%, extended chain accounted for 13.82%, β-folded chain accounted for 4.0%, and irregular curl accounted for 45.82%. Irregular curl constituted the main skeleton of the protein. Phylogenetic tree analysis showed that DcHsfB1 had the highest homology with AtHsfB1. The relative expressions of DcHsfB1 under various abiotic stresses were analyzed by quantitative real-time PCR. The results showed that the relative expression of the gene were significantly up-regulated under 42°C for 2 h, ABA treatment for 3 h and drought stress for 3 h. The relative expression of DcHsfB1 under mannitol and NaCl and 4°C also increased in different degrees.
Heat shock transcription factors(Hsfs) regulate the expression of heat shock proteins(Hsps) and other heat shock-induced transcripts when plants are subjected to abiotic stress, and participate in the response to heat stress. In this paper, carnation ‘Fancy' was used as experimental material to clone the open reading frame of DcHsfB1. The composition and physicochemical properties analysis of the amino acid sequence showed that DcHsfB1 contained a 804 bp open reading frame encoding 267 amino acids with a molecular weight of 30.78 kDa and a theoretical isoelectric point of 5.84. The secondary structure and tertiary structure prediction of the protein showed that, α-helix accounted for 36.36%, extended chain accounted for 13.82%, β-folded chain accounted for 4.0%, and irregular curl accounted for 45.82%. Irregular curl constituted the main skeleton of the protein. Phylogenetic tree analysis showed that DcHsfB1 had the highest homology with AtHsfB1. The relative expressions of DcHsfB1 under various abiotic stresses were analyzed by quantitative real-time PCR. The results showed that the relative expression of the gene were significantly up-regulated under 42°C for 2 h, ABA treatment for 3 h and drought stress for 3 h. The relative expression of DcHsfB1 under mannitol and NaCl and 4°C also increased in different degrees.
引文
Baniwal SK,Bhartii K,Chan KY,et al(2004).Heat stress response in plants:a complex game with chaperones and more than twenty heat stress transcription factors.J Biosci,29(4):471-487
Boxriker M,Boehm R,Krezdorn N,et al(2017).Comparative transcriptome analysis of vase life and carnation type in Dianthus caryophyllus L.Sci Hortic,217:61-72
Boxriker M,M?hring J,Piepho HP(2018).Genetic and phenotypic correlation for breeding relevant traits in Dianthus caryophyllus L.Postharvest Biol Tec,43:129-136
Chung E,Kim KM,Lee JH(2013).Genome-wide analysis and molecularcharacterization of heat shock transcription factor family in Glycine max.J Genet Genomics,40(3):127-135
Czarnecka-Verner E,Yuan CX,Scharf KD,et al(2000).Plants contain a novel multi-member class of heat shock factors without transcriptional activator potential.Plant Mol Biol,43:459-471
Hu XJ,Chen DD,Mclntyre L,et al(2017).Heat shock factor C2a serves as a proactive mechanism for heat protection in developing grains in wheat via an ABA-mediated regulatory pathway.Plant Cell Environ,41(1):79-98
Ikeda M,Mitsuda N,Ohme-Takagi M(2011).Arabidopsis HsfB1 and HsfB2b act as repressors of the expression of heat-inducible Hsfs but positively regulate the acquired thermotolerance.Plant Physiol,157(3):1243-1254
Kong WL,Hu R,Bao MZ,et al(2017).Cloning and expression analysis of the aquaporin genes in carnation.Acta Hortic Sin,44(3):515-527(in Chinese with English abstract)[,,(2017).abstract)[孔维龙,胡瑞,包满珠等(2017).香石竹水孔蛋白基因的克隆及表达分析.园艺学报,44(3):515-527]
Kong WL,Wang C,Dan NZ,et al(2018).Agrobacterium-mediated genetic transformation systeem of carnation.JChina Agric Univ,23(4):34-44(in Chinese with English abstract)[孔维龙,王翠,但乃震等(2018).根癌农杆菌介导的香石竹遗传转化体系的建立.中国农业大学学报,23(4):34-44]
Kumar M,Buschb W,Birkec H,et al(2009).Heat shock factors HsfB1 and HsfB2b are involved in the regulation of Pdf1.2 expression and pathogen resistance in Arabidopsis.Mol Plant,2(1):152-165
Lin KF,Tsai MY,Lu CA,et al(2018).The roles of Arabidopsis HSFA2,HSFA4a,and HSFA7a in the heat shock response and cytosolic protein response.Bot Stud,59(1):1-9
Liu GT,Chai FM,Wang Y,et al(2018).Genome-wide identification and classification of HSF family in grape,and their transcriptional analysis under heat acclimation and heat stress.Hotric Plant J,4(4):133-143
Nover L,Bharti K,Mishra SK,et al(2001).Arabidopsis and the heat stress transcription factor world:how many heat stress transcription factors do we need?Cell Stress Chaperon,6(3):177-189
Scharf KD,Berberich T,Ebersberger I,et al(2012).The plant heat stress transcription factor(Hsf)family:structure,function and evolution.Biochim Biophys Acta,1819(2):104-119
Wu Z,Liang JH,Wang CP,et al(2018).Overexpression of lily HsfA3s in Arabidopsis confers increased thermotolerance and salt sensitivity via alterations in proline catabolism.JExp Bot,69(8):2005-2021
Xie J,Liang XJ,Weng LF,et al(2018).Estimation on multicolor dye formulae and compound preservatives for carnation cut flowers.Hunan Agric Sci,(10):92-94(in Chinese with English abstract)[谢婕,梁雄基,翁柳芳等(2018).香石竹切花多彩染色及复合保鲜剂研究.湖南农业科学,(10):92-94]
Xin HB,Zhang H,Zhong XH,et al(2017).Over-expression of LlHsfA2b,a lily heat shock transcription factor lacking trans-activation activity in yeast,can enhance tolerance to heat and oxidative stress in transgenic Arabidopsis seedlings.Plant Cell Tiss Org Cult,130:617-629
Zhang XN,Wang QJ,Yang SZ,et al(2018).Identification and characterization of the mads-box genes and their contribution to flower organ in carnation(Dianthus caryophyllus L.).Genes,9(4):193
Zhu XJ,Thalor SK,Takahashi Y,et al(2012).An inhibitory effect of the sequence-conserv ed upstream open-reading frame on the translation of themain open-reading frame of HsfB1 transcripts in Arabidopsis.Plant Cell Environ,35:2014-2030
Boxriker M,Boehm R,Krezdorn N,et al(2017).Comparative transcriptome analysis of vase life and carnation type in Dianthus caryophyllus L.Sci Hortic,217:61-72
Boxriker M,M?hring J,Piepho HP(2018).Genetic and phenotypic correlation for breeding relevant traits in Dianthus caryophyllus L.Postharvest Biol Tec,43:129-136
Chung E,Kim KM,Lee JH(2013).Genome-wide analysis and molecularcharacterization of heat shock transcription factor family in Glycine max.J Genet Genomics,40(3):127-135
Czarnecka-Verner E,Yuan CX,Scharf KD,et al(2000).Plants contain a novel multi-member class of heat shock factors without transcriptional activator potential.Plant Mol Biol,43:459-471
Hu XJ,Chen DD,Mclntyre L,et al(2017).Heat shock factor C2a serves as a proactive mechanism for heat protection in developing grains in wheat via an ABA-mediated regulatory pathway.Plant Cell Environ,41(1):79-98
Ikeda M,Mitsuda N,Ohme-Takagi M(2011).Arabidopsis HsfB1 and HsfB2b act as repressors of the expression of heat-inducible Hsfs but positively regulate the acquired thermotolerance.Plant Physiol,157(3):1243-1254
Kong WL,Hu R,Bao MZ,et al(2017).Cloning and expression analysis of the aquaporin genes in carnation.Acta Hortic Sin,44(3):515-527(in Chinese with English abstract)[,,(2017).abstract)[孔维龙,胡瑞,包满珠等(2017).香石竹水孔蛋白基因的克隆及表达分析.园艺学报,44(3):515-527]
Kong WL,Wang C,Dan NZ,et al(2018).Agrobacterium-mediated genetic transformation systeem of carnation.JChina Agric Univ,23(4):34-44(in Chinese with English abstract)[孔维龙,王翠,但乃震等(2018).根癌农杆菌介导的香石竹遗传转化体系的建立.中国农业大学学报,23(4):34-44]
Kumar M,Buschb W,Birkec H,et al(2009).Heat shock factors HsfB1 and HsfB2b are involved in the regulation of Pdf1.2 expression and pathogen resistance in Arabidopsis.Mol Plant,2(1):152-165
Lin KF,Tsai MY,Lu CA,et al(2018).The roles of Arabidopsis HSFA2,HSFA4a,and HSFA7a in the heat shock response and cytosolic protein response.Bot Stud,59(1):1-9
Liu GT,Chai FM,Wang Y,et al(2018).Genome-wide identification and classification of HSF family in grape,and their transcriptional analysis under heat acclimation and heat stress.Hotric Plant J,4(4):133-143
Nover L,Bharti K,Mishra SK,et al(2001).Arabidopsis and the heat stress transcription factor world:how many heat stress transcription factors do we need?Cell Stress Chaperon,6(3):177-189
Scharf KD,Berberich T,Ebersberger I,et al(2012).The plant heat stress transcription factor(Hsf)family:structure,function and evolution.Biochim Biophys Acta,1819(2):104-119
Wu Z,Liang JH,Wang CP,et al(2018).Overexpression of lily HsfA3s in Arabidopsis confers increased thermotolerance and salt sensitivity via alterations in proline catabolism.JExp Bot,69(8):2005-2021
Xie J,Liang XJ,Weng LF,et al(2018).Estimation on multicolor dye formulae and compound preservatives for carnation cut flowers.Hunan Agric Sci,(10):92-94(in Chinese with English abstract)[谢婕,梁雄基,翁柳芳等(2018).香石竹切花多彩染色及复合保鲜剂研究.湖南农业科学,(10):92-94]
Xin HB,Zhang H,Zhong XH,et al(2017).Over-expression of LlHsfA2b,a lily heat shock transcription factor lacking trans-activation activity in yeast,can enhance tolerance to heat and oxidative stress in transgenic Arabidopsis seedlings.Plant Cell Tiss Org Cult,130:617-629
Zhang XN,Wang QJ,Yang SZ,et al(2018).Identification and characterization of the mads-box genes and their contribution to flower organ in carnation(Dianthus caryophyllus L.).Genes,9(4):193
Zhu XJ,Thalor SK,Takahashi Y,et al(2012).An inhibitory effect of the sequence-conserv ed upstream open-reading frame on the translation of themain open-reading frame of HsfB1 transcripts in Arabidopsis.Plant Cell Environ,35:2014-2030