中国南瓜CmHSP70基因的克隆及表达分析
|
摘要
为了探究中国南瓜(Cucurbita moschata)热激蛋白70基因的功能,该研究采用转录组测序和RT-PCR方法,从中国南瓜中分离获得3个HSP70基因的开放阅读框(ORF)全长,分别命名为CmHSP70-1、CmHSP70-2和CmHSP70-3,三者的序列长度分别为1 998、1 941和2 118 bp,编码666、647和706个氨基酸。蛋白序列分析显示,3个CmHSP70蛋白均属于亲水性蛋白,都含有典型的NBD和SBD保守结构域;CmHSP70-1蛋白含有信号肽和跨膜结构,亚细胞主要定位于内质网;CmHSP70-2蛋白和CmHSP70-3蛋白不含信号肽和跨膜结构,亚细胞主要定位于细胞质和叶绿体。同源比对和进化分析发现,3个CmHSP70蛋白与苦瓜、甜瓜和黄瓜的HSP70蛋白的相似性最高且遗传距离最近。qRT-PCR分析显示,42℃处理能够诱导3个CmHSP70基因在根、茎、嫩叶和成熟叶中表达,并且表达量存在明显的组织特异性,成熟叶中基因的表达量最高;高温条件下,成熟叶中3个CmHSP70基因均能够在短时间内(处理0～2 h)响应热胁迫而大量表达,尤其是CmHSP70-2基因,推测该基因可能在中国南瓜热胁迫过程发挥重要的调节作用。该研究结果为深入了解HSP70基因功能以及阐明中国南瓜的耐热机制提供理论依据。
In order to investigate the function of heat shock protein gene in Cucurbita moschata, we isolated the open reading frames(ORFs) of three HSP70 genes from C. moschata using transcriptome sequencing and RT-PCR methods and named CmHSP70-1, CmHSP70-2 and CmHSP70-3. Their sequence length were 1 998, 1 941 and 2 118 bp, encoding 666, 647 and 706 amino acids, respectively. Protein sequence analysis showed that all three CmHSP70 were hydrophilic proteins with typical conserved domains of NBD and SBD. CmHSP70-1 contained signal peptide and transmembrane structure, and it was mainly located in endoplasmic reticulum. CmHSP70-2 and CmHSP70-3 did not contain signal peptide and transmembrane structure, and they were mainly located in cytoplasm and chloroplast, respectively. Homologous comparison and evolutionary analysis revealed that three CmHSP70 had the highest consistency with the HSP70 of Momordica charantia, Cucumis melo and Cucumis sativus, and the genetic distance was the closest. The qRT-PCR analysis showed that three CmHSP70 could be induced in roots, stems, immature leaves and mature leaves by 42 ℃, and the expression level had obvious tissue specificity, with the highest in mature leaves. Under high temperature conditions, three CmHSP70 in mature leaves could be expressed in response to heat stress in a short time(0-2 h), especially CmHSP70-2. It is speculated that CmHSP70-2 may play an important regulatory role in the process of heat stress in C. moschata. The results provided a theoretical basis for further understanding the function of HSP70 and elucidating the thermotolerance mechanism of C. moschata.
In order to investigate the function of heat shock protein gene in Cucurbita moschata, we isolated the open reading frames(ORFs) of three HSP70 genes from C. moschata using transcriptome sequencing and RT-PCR methods and named CmHSP70-1, CmHSP70-2 and CmHSP70-3. Their sequence length were 1 998, 1 941 and 2 118 bp, encoding 666, 647 and 706 amino acids, respectively. Protein sequence analysis showed that all three CmHSP70 were hydrophilic proteins with typical conserved domains of NBD and SBD. CmHSP70-1 contained signal peptide and transmembrane structure, and it was mainly located in endoplasmic reticulum. CmHSP70-2 and CmHSP70-3 did not contain signal peptide and transmembrane structure, and they were mainly located in cytoplasm and chloroplast, respectively. Homologous comparison and evolutionary analysis revealed that three CmHSP70 had the highest consistency with the HSP70 of Momordica charantia, Cucumis melo and Cucumis sativus, and the genetic distance was the closest. The qRT-PCR analysis showed that three CmHSP70 could be induced in roots, stems, immature leaves and mature leaves by 42 ℃, and the expression level had obvious tissue specificity, with the highest in mature leaves. Under high temperature conditions, three CmHSP70 in mature leaves could be expressed in response to heat stress in a short time(0-2 h), especially CmHSP70-2. It is speculated that CmHSP70-2 may play an important regulatory role in the process of heat stress in C. moschata. The results provided a theoretical basis for further understanding the function of HSP70 and elucidating the thermotolerance mechanism of C. moschata.
引文
[1] RITOSSA F.A new puffing pattern induced by temperature shock and DNP in drosophila[J].Experientia,1962,18(12):571-573.
[2] 王明强,张道远.植物热激蛋白70基因家族及其生物学功能研究进展[J].基因组学与应用生物学,2015,34(2):421-428.WANG M Q,ZHANG D Y.Research advance of heat shock protein 70 gene family and its biological functions in plant[J].Genomics and Applied Biology,2015,34(2):421-428.
[3] QI Y C,WANG H J,ZOU Y,et al.Over-expression of mitochondrial heat shock protein 70 suppresses programmed cell death in rice[J].FEBS Letters,2011,585(1):231-239.
[4] 李雅博,李婷,韩莹琰,等.叶用莴苣热激蛋白基因LsHsp70-2711的克隆及高温胁迫下的功能分析[J].中国农业科学,2017,50(8):1 486-1 494.LI Y B,LI T,HAN Y Y,et al.Cloning and function analysis of heat-shock-protein LsHsp70-2711 gene under high temperature stress in leaf lettuce (Lactuca sativa L.)[J].Scientia Agricultura Sinica,2017,50(8):1 486-1 494.
[5] SONG A P,ZHU X R CHEN F D,et al.A chrysanthemum heat shock protein confers tolerance to abiotic stress[J].International Journal of Molecular Sciences,2014,15(3):5 063-5 078.
[6] 胡秀丽,李艳辉,杨海荣,等.HSP70可提高干旱高温复合胁迫诱导的玉米叶片抗氧化防护能力[J].作物学报,2010,36(4):636-644.HU X L,LI Y H,YANG H R,et al.Heat shock protein 70 may improve the ability of antioxidant defense induced by the combination of drought and heat in maize leaves[J].Acta Agronomica Sinica,2010,36(4):636-644.
[7] CHEN X,WANG Y,LI J,et al.Mitochondrial proteome during salt stress-induced programmed cell death in rice[J].Plant Physiology and Biochemistry:PPB,2009,47(5):407-415.
[8] ANARAKI Z E,TAFRESHI S A H,SHARIATI M.Transient silencing of heat shock proteins showed remarkable roles for HSP70 during adaptation to stress in plants[J].Environmental and Experimental Botany,2018,155:142-157.
[9] KANZAKI H,SAITOH H,ITO A,et al.Cytosolic HSP90 and HSP70 are essential components of INF1-mediated hypersensitive response and non-host resistance to Pseudomonas cichorii in Nicotiana benthamiana[J].Molecular Plant Pathology,2003,4(5):383-391.
[10] JUNGKUNZ I,LINK K,VOGEL F,et al.AtHsp70-15-deficient Arabidopsis plants are characterized by reduced growth,a constitutive cytosolic protein response and enhanced resistance to TuMV[J].The Plant Journal:for Cell and Molecular Biology,2011,66(6):983-995.
[11] 陈旭,石垒,朱璐,等.植物HSP70蛋白家族分子进化特征及其表达模式分析[J].基因组学与应用生物学,2017,36(10):4 282-4 294.CHEN X,SHI L,ZHU L,et al.Molecular evolution characteristics and expression pattern analysis of the heat shock protein 70(HSP70) gene superfamily in plant[J].Genomics and Applied Biology,2017,36(10):4 282-4 294.
[12] 郭丽红,杨晓虹,刘开庆,等.拟南芥热激蛋白HSP70基因片段克隆实验体系的建立[J].昆明学院学报,2009,31(6):46-48.GUO L H,YANG X H,LIU K Q,et al.Establishment on cloning experimental system of heat shock protein HSP70 in Arabidopsis thaliana[J].Journal of Kunming University,2009,31(6):46-48.
[13] 李慧聪,郭秀林,王冬梅,等.玉米热激蛋白70基因对温度胁迫的响应[J].河北农业大学学报,2010,33(6):12-15,25.LI H C,GUO X L,WANG D M,et al.Responses of HSP70 gene expression to temperature stresses in maize (Zea mays L.)[J].Journal of Agricultural University of Hebei,2010,33(6):12-15,25.
[14] 安艳秋,蔺瑞明,冯晶,等.小麦热激蛋白基因TaHSP70克隆及其在植物防卫和抗逆反应中的表达分析[J].分子植物育种,2011,9(4):402-409.AN Y Q,LIN R M,FENG J,et al.Molecular cloning of wheat heat shock protein gene TaHSP70 and expression analysis in plant defense and stress responses[J].Molecular Plant Breeding,2011,9(4):402-409.
[15] 王涛,唐颂豪,何梅,等.热激蛋白与植物耐热性关系的研究进展[J].西北林学院学报,2014,29(6):72-79.WANG T,TANG S H,HE M,et al.Advances in relationship between heat shock proteins and thermo-tolerance of plants[J].Journal of Northwest Forestry University,2014,29(6):72-79.
[16] 王荣青,万红建,李志邈,等.番茄Hsp70基因鉴定及系统发育关系分析[J].核农学报,2014,28(3):378-385.WANG R Q,WAN H J,LI Z M,et al.Identification and phylogenetic relationships of hsp70 genes in tomato[J].Journal of Nuclear Agricultural Sciences,2014,28(3):378-385.
[17] 宋晋辉,马海莲,瓮巧云,等.玉米HSP70基因家族的全基因组鉴定与分析[J].核农学报,2017,31(7):1 245-1 254.SONG J H,MA H L,WENG Q Y,et al.Genome-wide identification and analysis of HSP70 gene family in maize[J].Journal of Nuclear Agricultural Sciences,2017,31(7):1 245-1 254.
[18] 徐剑文,赵君,刘剑光,等.芝麻全基因组Hsp70基因的鉴定与生物信息学分析[J].江苏农业学报,2017,33(3):493-502.XU J W,ZHAO J,LIU J G,et al.Genome-wide identification and bioinformatics analysis of Hsp70 gene family in Sesamum indicum[J].Jiangsu Journal of Agricultural Sciences,2017,33(3):493-502.
[19] GUY C L,LI Q B.The organization and evolution of the spinach stress 70 molecular chaperone gene family[J].The Plant Cell,1998,10(4):539-556.
[20] 张侠,尹海波,熊冬金,等.植物热激蛋白70[J].植物生理学通讯,2004,40(4):500-504.ZHANG X,YIN H B,XIONG D J,et al.The progress in heat shock protein 70[J].Plant Physiology Communications,2004,40(4):500-504.
[21] 陈兰英,朱泮民,李冰冰,等.HSP70的结构、特性和功能研究进展[J].平顶山工学院学报,2004,13(2):41-43.CHEN L Y,ZHU P M,LI B B,et al.Research progress of structure,features and function of HSP70[J].Journal of Pingdingshan Institute of Technology,2004,13(2):41-43.
[22] 阮文进,门维婷,马婧,等.蜡梅热激蛋白基因CpHSP70-1的克隆、亚细胞定位与表达分析[J].西南大学学报(自然科学版),2016,38(1):43-52.RUAN W J,MEN W T,MA J,et al.Cloning,subcellular localization and expression analysis of heat shock protein gene CpHSP70-1 from Chimonanthus praecox (L.)[J].Journal of Southwest University (Natural Science Edition),2016,38(1):43-52.
[23] KIMPEL J A,NAGAO R T,GOEKJIAN V,et al.Regulation of the heat shock response in soybean seedlings[J].Plant Physiology,1990,94(3):988-995.
[24] SUNG D Y,VIERLING E,GUY C L.Comprehensive expression profile analysis of the Arabidopsis Hsp70 gene family[J].Plant Physiology,2001,126(2):789-800.
[25] 李翠,侯蕾,任丽,等.花生热激蛋白 AhHSP70与热激因子 AhHSF 基因的克隆及表达分析[J].山东农业科学,2015,47(4):1-7.LI C,HOU L,REN L,et al.Cloning and expression analysis of AhHSP70 and AhHSF genes in Arachis hypogaea L.[J].Shandong Agricultural Sciences,2015,47(4):1-7.
[26] 陈二龙,范志勇,王松峰,等.烟草Hsp70基因家族的鉴定及NtHsp70Chl基因的表达分析[J].中国烟草科学,2018,39(2):8-16.CHEN E L,FAN Z Y,WANG S F,et al.Bioinformatics of tobacco(Nicotiana tabacum)hsp70 gene family and expression analysis of NtHsp70Chl in midrib[J].Chinese Tobacco Science,2018,39(2):8-15,16.
[27] 罗昌,陈东亮,程曦,等.甘菊ClHSP70与ClHSP90基因的克隆及表达分析[J].西北植物学报,2016,36(7):1 321-1 330.LUO C,CHEN D L,CHENG X,et al.Cloning and expression of ClHSP70 and ClHSP90 gene from Chrysanthemum lavandulifolium [J].Acta Botanica Boreali-Occidentalia Sinica,2016,36(7):1 321-1 330.
[28] KREBS R A,HOLBROOK S H.Reduced enzyme activity following Hsp70 overexpression in Drosophila melanogaster[J].Biochemical Genetics,2001,39(1-2):73-82.
[29] SUNG D Y,GUY C.Physiological and molecular assessment of altered expression of Hsc70-1 in Arabidopsis [J].Plant Physiology,2003,132(2):979-987.
[2] 王明强,张道远.植物热激蛋白70基因家族及其生物学功能研究进展[J].基因组学与应用生物学,2015,34(2):421-428.WANG M Q,ZHANG D Y.Research advance of heat shock protein 70 gene family and its biological functions in plant[J].Genomics and Applied Biology,2015,34(2):421-428.
[3] QI Y C,WANG H J,ZOU Y,et al.Over-expression of mitochondrial heat shock protein 70 suppresses programmed cell death in rice[J].FEBS Letters,2011,585(1):231-239.
[4] 李雅博,李婷,韩莹琰,等.叶用莴苣热激蛋白基因LsHsp70-2711的克隆及高温胁迫下的功能分析[J].中国农业科学,2017,50(8):1 486-1 494.LI Y B,LI T,HAN Y Y,et al.Cloning and function analysis of heat-shock-protein LsHsp70-2711 gene under high temperature stress in leaf lettuce (Lactuca sativa L.)[J].Scientia Agricultura Sinica,2017,50(8):1 486-1 494.
[5] SONG A P,ZHU X R CHEN F D,et al.A chrysanthemum heat shock protein confers tolerance to abiotic stress[J].International Journal of Molecular Sciences,2014,15(3):5 063-5 078.
[6] 胡秀丽,李艳辉,杨海荣,等.HSP70可提高干旱高温复合胁迫诱导的玉米叶片抗氧化防护能力[J].作物学报,2010,36(4):636-644.HU X L,LI Y H,YANG H R,et al.Heat shock protein 70 may improve the ability of antioxidant defense induced by the combination of drought and heat in maize leaves[J].Acta Agronomica Sinica,2010,36(4):636-644.
[7] CHEN X,WANG Y,LI J,et al.Mitochondrial proteome during salt stress-induced programmed cell death in rice[J].Plant Physiology and Biochemistry:PPB,2009,47(5):407-415.
[8] ANARAKI Z E,TAFRESHI S A H,SHARIATI M.Transient silencing of heat shock proteins showed remarkable roles for HSP70 during adaptation to stress in plants[J].Environmental and Experimental Botany,2018,155:142-157.
[9] KANZAKI H,SAITOH H,ITO A,et al.Cytosolic HSP90 and HSP70 are essential components of INF1-mediated hypersensitive response and non-host resistance to Pseudomonas cichorii in Nicotiana benthamiana[J].Molecular Plant Pathology,2003,4(5):383-391.
[10] JUNGKUNZ I,LINK K,VOGEL F,et al.AtHsp70-15-deficient Arabidopsis plants are characterized by reduced growth,a constitutive cytosolic protein response and enhanced resistance to TuMV[J].The Plant Journal:for Cell and Molecular Biology,2011,66(6):983-995.
[11] 陈旭,石垒,朱璐,等.植物HSP70蛋白家族分子进化特征及其表达模式分析[J].基因组学与应用生物学,2017,36(10):4 282-4 294.CHEN X,SHI L,ZHU L,et al.Molecular evolution characteristics and expression pattern analysis of the heat shock protein 70(HSP70) gene superfamily in plant[J].Genomics and Applied Biology,2017,36(10):4 282-4 294.
[12] 郭丽红,杨晓虹,刘开庆,等.拟南芥热激蛋白HSP70基因片段克隆实验体系的建立[J].昆明学院学报,2009,31(6):46-48.GUO L H,YANG X H,LIU K Q,et al.Establishment on cloning experimental system of heat shock protein HSP70 in Arabidopsis thaliana[J].Journal of Kunming University,2009,31(6):46-48.
[13] 李慧聪,郭秀林,王冬梅,等.玉米热激蛋白70基因对温度胁迫的响应[J].河北农业大学学报,2010,33(6):12-15,25.LI H C,GUO X L,WANG D M,et al.Responses of HSP70 gene expression to temperature stresses in maize (Zea mays L.)[J].Journal of Agricultural University of Hebei,2010,33(6):12-15,25.
[14] 安艳秋,蔺瑞明,冯晶,等.小麦热激蛋白基因TaHSP70克隆及其在植物防卫和抗逆反应中的表达分析[J].分子植物育种,2011,9(4):402-409.AN Y Q,LIN R M,FENG J,et al.Molecular cloning of wheat heat shock protein gene TaHSP70 and expression analysis in plant defense and stress responses[J].Molecular Plant Breeding,2011,9(4):402-409.
[15] 王涛,唐颂豪,何梅,等.热激蛋白与植物耐热性关系的研究进展[J].西北林学院学报,2014,29(6):72-79.WANG T,TANG S H,HE M,et al.Advances in relationship between heat shock proteins and thermo-tolerance of plants[J].Journal of Northwest Forestry University,2014,29(6):72-79.
[16] 王荣青,万红建,李志邈,等.番茄Hsp70基因鉴定及系统发育关系分析[J].核农学报,2014,28(3):378-385.WANG R Q,WAN H J,LI Z M,et al.Identification and phylogenetic relationships of hsp70 genes in tomato[J].Journal of Nuclear Agricultural Sciences,2014,28(3):378-385.
[17] 宋晋辉,马海莲,瓮巧云,等.玉米HSP70基因家族的全基因组鉴定与分析[J].核农学报,2017,31(7):1 245-1 254.SONG J H,MA H L,WENG Q Y,et al.Genome-wide identification and analysis of HSP70 gene family in maize[J].Journal of Nuclear Agricultural Sciences,2017,31(7):1 245-1 254.
[18] 徐剑文,赵君,刘剑光,等.芝麻全基因组Hsp70基因的鉴定与生物信息学分析[J].江苏农业学报,2017,33(3):493-502.XU J W,ZHAO J,LIU J G,et al.Genome-wide identification and bioinformatics analysis of Hsp70 gene family in Sesamum indicum[J].Jiangsu Journal of Agricultural Sciences,2017,33(3):493-502.
[19] GUY C L,LI Q B.The organization and evolution of the spinach stress 70 molecular chaperone gene family[J].The Plant Cell,1998,10(4):539-556.
[20] 张侠,尹海波,熊冬金,等.植物热激蛋白70[J].植物生理学通讯,2004,40(4):500-504.ZHANG X,YIN H B,XIONG D J,et al.The progress in heat shock protein 70[J].Plant Physiology Communications,2004,40(4):500-504.
[21] 陈兰英,朱泮民,李冰冰,等.HSP70的结构、特性和功能研究进展[J].平顶山工学院学报,2004,13(2):41-43.CHEN L Y,ZHU P M,LI B B,et al.Research progress of structure,features and function of HSP70[J].Journal of Pingdingshan Institute of Technology,2004,13(2):41-43.
[22] 阮文进,门维婷,马婧,等.蜡梅热激蛋白基因CpHSP70-1的克隆、亚细胞定位与表达分析[J].西南大学学报(自然科学版),2016,38(1):43-52.RUAN W J,MEN W T,MA J,et al.Cloning,subcellular localization and expression analysis of heat shock protein gene CpHSP70-1 from Chimonanthus praecox (L.)[J].Journal of Southwest University (Natural Science Edition),2016,38(1):43-52.
[23] KIMPEL J A,NAGAO R T,GOEKJIAN V,et al.Regulation of the heat shock response in soybean seedlings[J].Plant Physiology,1990,94(3):988-995.
[24] SUNG D Y,VIERLING E,GUY C L.Comprehensive expression profile analysis of the Arabidopsis Hsp70 gene family[J].Plant Physiology,2001,126(2):789-800.
[25] 李翠,侯蕾,任丽,等.花生热激蛋白 AhHSP70与热激因子 AhHSF 基因的克隆及表达分析[J].山东农业科学,2015,47(4):1-7.LI C,HOU L,REN L,et al.Cloning and expression analysis of AhHSP70 and AhHSF genes in Arachis hypogaea L.[J].Shandong Agricultural Sciences,2015,47(4):1-7.
[26] 陈二龙,范志勇,王松峰,等.烟草Hsp70基因家族的鉴定及NtHsp70Chl基因的表达分析[J].中国烟草科学,2018,39(2):8-16.CHEN E L,FAN Z Y,WANG S F,et al.Bioinformatics of tobacco(Nicotiana tabacum)hsp70 gene family and expression analysis of NtHsp70Chl in midrib[J].Chinese Tobacco Science,2018,39(2):8-15,16.
[27] 罗昌,陈东亮,程曦,等.甘菊ClHSP70与ClHSP90基因的克隆及表达分析[J].西北植物学报,2016,36(7):1 321-1 330.LUO C,CHEN D L,CHENG X,et al.Cloning and expression of ClHSP70 and ClHSP90 gene from Chrysanthemum lavandulifolium [J].Acta Botanica Boreali-Occidentalia Sinica,2016,36(7):1 321-1 330.
[28] KREBS R A,HOLBROOK S H.Reduced enzyme activity following Hsp70 overexpression in Drosophila melanogaster[J].Biochemical Genetics,2001,39(1-2):73-82.
[29] SUNG D Y,GUY C.Physiological and molecular assessment of altered expression of Hsc70-1 in Arabidopsis [J].Plant Physiology,2003,132(2):979-987.
© 2004-2018 中国地质图书馆版权所有 京ICP备05064691号 京公网安备11010802017129号 地址：北京市海淀区学院路29号 邮编：100083 电话：办公室：(+86 10)66554848；文献借阅、咨询服务、科技查新：66554700 |