向日葵E3泛素连接酶基因的分析定位和表达鉴定
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摘要
该研究利用前期获得的向日葵耐盐相关基因E3泛素连接酶基因序列(HERC2),构建瞬时表达载体Cam-35S-HERC2-GFP,采用基因枪法转化洋葱表皮细胞进行亚细胞定位;采用RT-PCR技术,分析盐胁迫下HERC2在耐盐品种P50和盐敏感品种P29根、下胚轴和叶中的表达差异;构建HERC2植物表达载体pPZP221-HERC2,采用农杆菌介导法将HERC2导入烟草,进行耐盐功能验证。结果表明:(1)HERC2蛋白定位在细胞膜、细胞质和细胞核中。(2)受到NaCl胁迫后,HERC2基因在耐盐品种P50和盐敏感品种P29中均上调表达,但耐盐品种中的表达量较高。(3)HERC2基因的表达,能够提高转基因烟草的耐盐性。该研究结果为进一步解析向日葵对盐胁迫的响应机制,以及耐盐新品种的选育奠定了基础。
In this study,utilizing the salt tolerant gene E3 ubiquitin ligase HERC2 sequence in sunflower which acquired previously,we constructed the transient expression vector Cam-35 S-HERC2-GFP.Subcellular localization was carried out by transforming onion epidermal cells with particle bombardment.The expression analysis,that of root,hypocotyl and leaf between the salt-tolerant hybrid P50 and the salt-sensitive hybrid P29 under 120 mmol·L-1 NaCl stress,was done by RT-qPCR.Plant expression vector pPZP221-HERC2 was constructed.HERC2 was transferred into tobacco to verify salt-tolerant functionality by Agrobacterium-mediated.The results showed that:(1)subcellular localization showed that HERC2 protein expressed in cell membrane,cytoplasm and nucleus;(2)the expression of HERC2 gene was upregulated between the salt-tolerant hybrid P50 and salt-sensitive hybrid P29 under NaCl stress,but the expression quantity of the former is higher than that of the latter;(3)the salt tolerance of transgenic HERC2 tobacco was stronger than that of the wild type tobacco.The study laid a foundation for analyzing of salt tolerance mechanism in sunflower and breeding salt-tolerant cultivar.
In this study,utilizing the salt tolerant gene E3 ubiquitin ligase HERC2 sequence in sunflower which acquired previously,we constructed the transient expression vector Cam-35 S-HERC2-GFP.Subcellular localization was carried out by transforming onion epidermal cells with particle bombardment.The expression analysis,that of root,hypocotyl and leaf between the salt-tolerant hybrid P50 and the salt-sensitive hybrid P29 under 120 mmol·L-1 NaCl stress,was done by RT-qPCR.Plant expression vector pPZP221-HERC2 was constructed.HERC2 was transferred into tobacco to verify salt-tolerant functionality by Agrobacterium-mediated.The results showed that:(1)subcellular localization showed that HERC2 protein expressed in cell membrane,cytoplasm and nucleus;(2)the expression of HERC2 gene was upregulated between the salt-tolerant hybrid P50 and salt-sensitive hybrid P29 under NaCl stress,but the expression quantity of the former is higher than that of the latter;(3)the salt tolerance of transgenic HERC2 tobacco was stronger than that of the wild type tobacco.The study laid a foundation for analyzing of salt tolerance mechanism in sunflower and breeding salt-tolerant cultivar.
引文
[1]张新民,陈鹏,叶盛,等.蛋白酶体抑制剂减少病毒性心肌炎小鼠炎症因子IL-6和TNF-α的表达[J].中国临床药理学与治疗学,2017,22(4):406-411.ZHANG X M,CHEN P,YE S,et al.Proteasome inhibitors decrease IL-6and TNF-αlevels in mouse acute viral myocarditis[J].Chinese Journal of Clinical Pharmacology and Therapeutics,2017,22(4):406-411.
[2]倪晓光,赵平.泛素-蛋白酶体途径的组成和功能[J].生理科学进展,2006,37(3):255-258.NI X G,ZHAO P.The composition and function of ubiquitin proteasome pathway[J].Progress in Physiological Sciences,2006,37(3):255-258.
[3]李龙.拟南芥E3泛素连接酶PUB30与PUB31耐盐功能分析及互作的14-3-3蛋白的筛选[D].沈阳:沈阳农业大学,2016.
[4]方慧敏.两个E3泛素连接酶基因在水稻耐非生物逆境胁迫中的作用研究[D].南京:南京农业大学,2014.
[5]冷欢.黑果枸杞泛素E3连接酶LrMCIP8-like基因克隆及功能研究[D].兰州:兰州大学,2014.
[6]张艳芳,孙瑞芬,郭树春,等.向日葵E3泛素连接酶基因克隆及表达分析[J].西北植物学报,2016,36(10):1 933-1 940.ZHANG Y F,SUN R F,GUO S C,et al.Cloning and expression analysis of E3ubiquitin ligase gene HERC2of sunflower(Helianthus annuus L.)[J].Acta Botanica BorealiOccidentalia Sinica,2016,36(10):1 933-1 940.
[7]张艳芳,孙瑞芬,郭树春,等.油葵杂交种耐盐性鉴定及幼苗对盐胁迫的生理响应[J].科技导报,2016,34(7):94-102.ZHANG Y F,SUN R F,GUO S C,et al.Identification and screening of salinity oil sunflower hybrids and effects of salt stress on the physiological characteristics at the seedling stage[J].Science&Technology Review,2016,34(7):94-102.
[8]THOMAS DUTRA.Green fluorescent protein[J].Progress in Biochemistry&Biophysics,2010,62(4):651-656.
[9]邢浩然,刘丽娟,刘国振.植物蛋白质的亚细胞定位研究进展[J].华北农学报,2006,21(b11):1-6.XING H R,LIU L J,LIU G Z.Advancement of protein subcellular localization in plants[J].Acta Agriculturae BorealiSinica,2006,21(b11):1-6.
[10]T LOURENCO,H SAPETA,D D FIGUEIREDO,et al.Isolation and characterization of rice(Oryza sativa L.)E3-ubiquitin ligase OsHOS1gene in the modulation of cold stress response[J].Plant Molecular Biology,2013,83(4-5):351-363.
[11]王赛寒.番茄E3泛素连接酶SLBAH1基因的克隆及其功能研究[D].山东泰安:山东农业大学,2014.
[12]李晓辉.黄瓜泛素E3连接酶基因的克隆及表达分析[D].郑州:河南农业大学,2014.
[13]王慰,刘园,曹爱忠,等.簇毛麦中一个具E3连接酶活性和U-box/ARM结构域的Hv-CMPG基因的克隆及其功能分析[C].南京:江苏省遗传学会会员代表大会暨学术研讨会,2010.
[14]钱泽勇,张会龙,荆晓姝,等.胡杨Ring finger E3连接酶PeRH2提高烟草耐旱机制研究[J].基因组学与应用生物学,2015,34(3):454-463.QIAN Z Y,ZHANG H L,JING X S,et al.Overexpression of Ring finger E3-ligase gene PeRH2fromPopulus euphraticain tobacco enhances drought tolerance in transgenic plants[J].Genomics and Applied Biology,2015,34(3):454-463.
[15]D MUKHOPADHYAY,H RIEZMAN.Proteasome-independent functions of ubiquitin in endocytosis and signaling[J].Science,2007,315(5 809):201-205.
[16]刘杰,张美丽,张义,等.人工模拟盐、碱环境对向日葵种子萌发及幼苗生长的影响[J].作物学报,2008,34(10):1 818-1 825.LIU J,ZHANG M L,ZHANG Y.Effects of simulated salt and alkali conditions on seed germination and seedling growth of sunflower(Helianthus annuus L.)[J].Acta Agronomica Sinica,2008,34(10):1 818-1 825.
[17]聂惠,于海峰,刘浩明.向日葵对盐胁迫的反应及其抗盐机理的研究进展[J].内蒙古农业科技,2008,(6):17-19.NIE H,YU H F,LIU H M.Response of sunflower to salt stress and its salt resistance mechanism[J].Inner Mongolia Agricultural Science and Technology,2008,(6):23-25.
[18]胡春霞,王秀芹.外源水杨酸对镉胁迫下油菜渗透调节物质和保护酶活性的影响[J].鞍山师范学院学报,2010,12(2):40-42.HU C X,WANG X Q.Effects of exogenous Salicylic Acid on osmotic and protective enzyme activity of Brassica campestris L.seedlings in Cd+2 stress[J].Journal of Anshan Normal University,2010,12(2):40-42.
[19]田晓艳,刘延吉,郭迎春.盐胁迫对NHC牧草Na+、K+、Pro、可溶性糖及可溶性蛋白的影响[J].草业科学,2008,25(10):34-38.TIAN X Y,LIU Y J,GUO Y C.Effect of salt on Na+,K+,proline,soluble sugar and protein of NHC[J].Pratacultural Science,2008,25(10):34-38.
[20]王波,宋凤斌.燕麦对盐碱胁迫的反应和适应性[J].生态环境学报,2006,15(3):625-629.WANG B,SONG F B.Physiological responses and adaptive capacity of oats to saline-alkali stress[J].Ecology and Environment,2006,15(3):625-629.
[21]杜秀敏,殷文璇,赵彦修,等.植物中活性氧的产生及清除机制[J].生物工程学报,2001,17(2):121-125.DU X M,YIN W X,ZHAO Y X,et al.The production and scavenging of reactive oxygen species in plants[J].Chinese Journal of Biotechnology,2001,17(2):121-125.
[22]张孝廉.大豆E3泛素连接酶基因GmARI的克隆与功能验证[D].南京:南京农业大学,2012.
[23]方坤海.稻瘟病菌若干假定泛素E3连接酶基因的功能研究[D].福州:福建农林大学,2010.
[2]倪晓光,赵平.泛素-蛋白酶体途径的组成和功能[J].生理科学进展,2006,37(3):255-258.NI X G,ZHAO P.The composition and function of ubiquitin proteasome pathway[J].Progress in Physiological Sciences,2006,37(3):255-258.
[3]李龙.拟南芥E3泛素连接酶PUB30与PUB31耐盐功能分析及互作的14-3-3蛋白的筛选[D].沈阳:沈阳农业大学,2016.
[4]方慧敏.两个E3泛素连接酶基因在水稻耐非生物逆境胁迫中的作用研究[D].南京:南京农业大学,2014.
[5]冷欢.黑果枸杞泛素E3连接酶LrMCIP8-like基因克隆及功能研究[D].兰州:兰州大学,2014.
[6]张艳芳,孙瑞芬,郭树春,等.向日葵E3泛素连接酶基因克隆及表达分析[J].西北植物学报,2016,36(10):1 933-1 940.ZHANG Y F,SUN R F,GUO S C,et al.Cloning and expression analysis of E3ubiquitin ligase gene HERC2of sunflower(Helianthus annuus L.)[J].Acta Botanica BorealiOccidentalia Sinica,2016,36(10):1 933-1 940.
[7]张艳芳,孙瑞芬,郭树春,等.油葵杂交种耐盐性鉴定及幼苗对盐胁迫的生理响应[J].科技导报,2016,34(7):94-102.ZHANG Y F,SUN R F,GUO S C,et al.Identification and screening of salinity oil sunflower hybrids and effects of salt stress on the physiological characteristics at the seedling stage[J].Science&Technology Review,2016,34(7):94-102.
[8]THOMAS DUTRA.Green fluorescent protein[J].Progress in Biochemistry&Biophysics,2010,62(4):651-656.
[9]邢浩然,刘丽娟,刘国振.植物蛋白质的亚细胞定位研究进展[J].华北农学报,2006,21(b11):1-6.XING H R,LIU L J,LIU G Z.Advancement of protein subcellular localization in plants[J].Acta Agriculturae BorealiSinica,2006,21(b11):1-6.
[10]T LOURENCO,H SAPETA,D D FIGUEIREDO,et al.Isolation and characterization of rice(Oryza sativa L.)E3-ubiquitin ligase OsHOS1gene in the modulation of cold stress response[J].Plant Molecular Biology,2013,83(4-5):351-363.
[11]王赛寒.番茄E3泛素连接酶SLBAH1基因的克隆及其功能研究[D].山东泰安:山东农业大学,2014.
[12]李晓辉.黄瓜泛素E3连接酶基因的克隆及表达分析[D].郑州:河南农业大学,2014.
[13]王慰,刘园,曹爱忠,等.簇毛麦中一个具E3连接酶活性和U-box/ARM结构域的Hv-CMPG基因的克隆及其功能分析[C].南京:江苏省遗传学会会员代表大会暨学术研讨会,2010.
[14]钱泽勇,张会龙,荆晓姝,等.胡杨Ring finger E3连接酶PeRH2提高烟草耐旱机制研究[J].基因组学与应用生物学,2015,34(3):454-463.QIAN Z Y,ZHANG H L,JING X S,et al.Overexpression of Ring finger E3-ligase gene PeRH2fromPopulus euphraticain tobacco enhances drought tolerance in transgenic plants[J].Genomics and Applied Biology,2015,34(3):454-463.
[15]D MUKHOPADHYAY,H RIEZMAN.Proteasome-independent functions of ubiquitin in endocytosis and signaling[J].Science,2007,315(5 809):201-205.
[16]刘杰,张美丽,张义,等.人工模拟盐、碱环境对向日葵种子萌发及幼苗生长的影响[J].作物学报,2008,34(10):1 818-1 825.LIU J,ZHANG M L,ZHANG Y.Effects of simulated salt and alkali conditions on seed germination and seedling growth of sunflower(Helianthus annuus L.)[J].Acta Agronomica Sinica,2008,34(10):1 818-1 825.
[17]聂惠,于海峰,刘浩明.向日葵对盐胁迫的反应及其抗盐机理的研究进展[J].内蒙古农业科技,2008,(6):17-19.NIE H,YU H F,LIU H M.Response of sunflower to salt stress and its salt resistance mechanism[J].Inner Mongolia Agricultural Science and Technology,2008,(6):23-25.
[18]胡春霞,王秀芹.外源水杨酸对镉胁迫下油菜渗透调节物质和保护酶活性的影响[J].鞍山师范学院学报,2010,12(2):40-42.HU C X,WANG X Q.Effects of exogenous Salicylic Acid on osmotic and protective enzyme activity of Brassica campestris L.seedlings in Cd+2 stress[J].Journal of Anshan Normal University,2010,12(2):40-42.
[19]田晓艳,刘延吉,郭迎春.盐胁迫对NHC牧草Na+、K+、Pro、可溶性糖及可溶性蛋白的影响[J].草业科学,2008,25(10):34-38.TIAN X Y,LIU Y J,GUO Y C.Effect of salt on Na+,K+,proline,soluble sugar and protein of NHC[J].Pratacultural Science,2008,25(10):34-38.
[20]王波,宋凤斌.燕麦对盐碱胁迫的反应和适应性[J].生态环境学报,2006,15(3):625-629.WANG B,SONG F B.Physiological responses and adaptive capacity of oats to saline-alkali stress[J].Ecology and Environment,2006,15(3):625-629.
[21]杜秀敏,殷文璇,赵彦修,等.植物中活性氧的产生及清除机制[J].生物工程学报,2001,17(2):121-125.DU X M,YIN W X,ZHAO Y X,et al.The production and scavenging of reactive oxygen species in plants[J].Chinese Journal of Biotechnology,2001,17(2):121-125.
[22]张孝廉.大豆E3泛素连接酶基因GmARI的克隆与功能验证[D].南京:南京农业大学,2012.
[23]方坤海.稻瘟病菌若干假定泛素E3连接酶基因的功能研究[D].福州:福建农林大学,2010.