甜高粱角蛋白基因的分离及功能分析
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摘要
本研究从甜高粱耐盐自交系M-81E中分离出角蛋白HTV(hornerin transcript variant)基因,我们构建了pROKII-GFP-SbHTV融合表达载体对SbHTV进行亚细胞定位分析,表明该基因编码的蛋白定位于细胞核.将该基因构建过表达载体,转入到野生型拟南芥中获得纯合过表达株系,并筛选出了拟南芥HTV的T-DNA插入纯合突变体.通过测定和分析盐处理下拟南芥野生型、过表达和突变体株系在萌发期、苗期的各项生理指标,我们发现当用100mM NaCl处理时,拟南芥各个株系的萌发率和根长都出现了显著差异,突变体的根长要显著长于野生型,过表达株系的根长要显著小于野生型.并且用100mM NaCl处理后,突变体株系生物量积累的抑制程度明显低于野生型,而过表达株系受抑制的程度要明显高于野生型.这表明SbHTV的过量表达降低植物的抗盐能力,而敲除该基因有利于提高植物抗盐性,初步阐明该基因在植物抗盐方面的作用.
In this study,hornerin transcript variant(HTV)gene was isolated from sweet sorghum of salt-tolerant inbred line M-81 E.We constructed a pROKII-SbHTV-GFP vector for subcellular localization analysis of SbHTV,which indicated that the gene was located in the cell nucleus.Homozygous overexpression lines were obtained,and T-DNA insertion homozygous mutants of Arabidopsis thaliana HTV were screened.The physiological indices of wild type,overexpression and mutant lines at germination and seedling stages under salt stress were determined and analyzed to elucidate the role of the gene under salt stress.We found that when treated with 100 mM NaCl,the germination rate and root length of each Arabidopsis strain were significantly different.The root length of the mutant was significantly longer than that of the wild type,and that of the overexpressed strain was significantly smaller than that of the wild type.After 100 mM NaCl treatment,the inhibition degree of biomass accumulation of mutant lines was significantly lower than that of wild type,while that of overexpressed lines was significantly higher than that of wild type.This indicates that the overexpression of SbHTV reduces the salt tolerance of plants,and the knockout of SbHTV gene is beneficial to improve the salt tolerance of plants.The role of SbHTV gene in salt tolerance of plants is elucidated preliminarily.
In this study,hornerin transcript variant(HTV)gene was isolated from sweet sorghum of salt-tolerant inbred line M-81 E.We constructed a pROKII-SbHTV-GFP vector for subcellular localization analysis of SbHTV,which indicated that the gene was located in the cell nucleus.Homozygous overexpression lines were obtained,and T-DNA insertion homozygous mutants of Arabidopsis thaliana HTV were screened.The physiological indices of wild type,overexpression and mutant lines at germination and seedling stages under salt stress were determined and analyzed to elucidate the role of the gene under salt stress.We found that when treated with 100 mM NaCl,the germination rate and root length of each Arabidopsis strain were significantly different.The root length of the mutant was significantly longer than that of the wild type,and that of the overexpressed strain was significantly smaller than that of the wild type.After 100 mM NaCl treatment,the inhibition degree of biomass accumulation of mutant lines was significantly lower than that of wild type,while that of overexpressed lines was significantly higher than that of wild type.This indicates that the overexpression of SbHTV reduces the salt tolerance of plants,and the knockout of SbHTV gene is beneficial to improve the salt tolerance of plants.The role of SbHTV gene in salt tolerance of plants is elucidated preliminarily.
引文
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[14]姜志磊,刘艳芝,韦正乙,等.玉米耐盐基因ZmHKT1;5在烟草中的功能验证[J].玉米科学,2017(5):32-39.
[15]姚新转,刘洋,赵德刚.高粱Na+转运蛋白基因SbSKC1的克隆及其在烟草中的抗盐功能鉴定[J].作物学报,2017,43(2):190-200.
[16]Rahman S,Matsumuro T,Miyake H,et al.Salinity-induced ultrastructural alterations in leaf cells of rice(Oryza sativa L.)[J].Plant Production Science,2000,3(4):422-429.
[17]Misra N,Dwivedi UN.Genotypic difference in salinity tolerance of green gram cultivars[J].Plant Science,2004,166(5):1135-1142.
[18]Almansouri M,Kinet J M,Lutts S.Effect of salt and osmotic stresses on germination in durum wheat(Triticum durum Desf.)[J].Plant&Soil,2001,231(2):243-254.
[2]苏菲,顾伟,翟中和.植物叶肉细胞的类角蛋白中间纤维[J].中国科学,1990(3):267-270.
[3]赵大中,陈丹英,陈敏,等.拟南芥细胞中存在中间纤维的研究[J].植物学报(英文版),1999,41(8):795-799.
[4]赵大中,陈丹英,杨澄,等.植物细胞类角蛋白的纯化、序列分析与类中间纤维cDNA的克隆[J].中国科学:生命科学,2000,30(3):316-321.
[5]柯丹霞,彭昆鹏,夏远君,等.盐胁迫应答基因GmWRKY6的克隆及转基因百脉根的抗盐分析[J].草业学报,2018,27,157(08):98-109.
[6]金太成,孟大伟,王悦,等.利用AH1基因的遗传转化改良紫花苜蓿耐盐性[J].分子植物育种官方网站,2016,14(12):1724-1729.
[7]管清杰,罗秋香,夏德习,等.水稻OsAPX1基因在烟草中的表达及其抗盐性研究[J].分子植物育种,2007(1):1-7.
[8]Steduto P,Katerji N,Puertos-Molina H,et al.Water-use efficiency of sweet sorghum under water stress conditions Gas-exchange investigations at leaf and canopy scales[J].Field Crops Research,1997,54(2-3):221-234.
[9]Almodares A,Hadi M R,Kholdebarin B,et al.The response of sweet sorghum cultivars to salt stress and accumulation of Na+,Cl-and K+ions in relation to salinity[J].Journal of Environmental Biology,2014,35(4):733-739.
[10]Xie T,Su P.Canopy and leaf photosynthetic characteristics and water use efficiency of sweet sorghum under drought stress[J].Russian Journal of Plant Physiology,2012,59(2):224-234.
[11]Han G,Wang M,Yuan F,et al.The CCCH zinc finger protein gene AtZFP1,improves salt resistance in Arabidopsis thaliana[J].Plant Molecular Biology,2014,86(3):237-253.
[12]黎茵.农杆菌注射渗透法转化烟草实验研究[J].实验技术与管理,2010,27(11):50-52.
[13]王为,潘宗瑾,潘群斌.作物耐盐性状研究进展[J].江西农业学报,2009,21(2):30-33.
[14]姜志磊,刘艳芝,韦正乙,等.玉米耐盐基因ZmHKT1;5在烟草中的功能验证[J].玉米科学,2017(5):32-39.
[15]姚新转,刘洋,赵德刚.高粱Na+转运蛋白基因SbSKC1的克隆及其在烟草中的抗盐功能鉴定[J].作物学报,2017,43(2):190-200.
[16]Rahman S,Matsumuro T,Miyake H,et al.Salinity-induced ultrastructural alterations in leaf cells of rice(Oryza sativa L.)[J].Plant Production Science,2000,3(4):422-429.
[17]Misra N,Dwivedi UN.Genotypic difference in salinity tolerance of green gram cultivars[J].Plant Science,2004,166(5):1135-1142.
[18]Almansouri M,Kinet J M,Lutts S.Effect of salt and osmotic stresses on germination in durum wheat(Triticum durum Desf.)[J].Plant&Soil,2001,231(2):243-254.