异源表达伴矿景天SpHMA2基因提高拟南芥对重金属镉的耐性及地上部分积累

Ectopic expression of SpHMA2 of Sedum plumbizincicola enhanced cadmium tolerance and shoot accumulation in Arabidopsis thaliana

植物修复技术(phytoremediation)是指使用超富集植物去除环境污染物的技术,但是野生超富集植物往往具有生物量低、生长缓慢等缺点,因此限制了该技术的广泛应用.从野生重金属耐性植物伴矿景天(Sedum plumbizincicola)中克隆到一个重金属响应基因,分析序列和系统进化关系,并探讨其在大肠杆菌和模式植物拟南芥中的表达能否提高重金属镉(Cd)耐性及积累量.该基因表达的蛋白是一个典型的重金属转运ATP酶(heavy metal ATPase,HMA),将该基因命名为SpHMA2.SpHMA2基因在伴矿景天根、茎、叶中均表达并受Cd处理的诱导,Cd处理后各组织中表达量提高了0.50-1.45倍.大肠杆菌Cd耐性及积累试验显示,转SpHMA2基因的大肠杆菌在0.8 mmol/L Cd胁迫处理时细胞的生长明显好于对照菌株,且吸附量提升了2.54倍.进一步采用基因工程的手段将SpHMA2转化拟南芥发现,转基因植株在75、150μmol/L Cd胁迫处理下地上部分生物量、根长、发芽率及吸附量普遍高于野生型(wild type,WT).在75μmol/L Cd处理时,转SpHMA2拟南芥株系M3、M4和M5的根长是WT的1.34-1.63倍,发芽率是WT的1.16-1.23倍,M3、M4吸附量较WT分别提升了1.93、0.34倍;150μmol/L Cd处理时,转基因株系根长是WT的1.89-9.43倍,发芽率是WT的1.73-2.02倍,吸附量较WT提升了0.18-1.47倍.50μmol/L Cd处理24 h,转基因植株叶片超氧化物歧化酶(SOD)、过氧化物酶(POD)和过氧化氢酶(CAT)酶活性及H_(2)O_(2)、O_(2)^(-)含量整体低于WT.上述研究结果表明,SpHMA2在拟南芥中一定水平的高表达量能赋予植株更高的Cd耐性和地上部分积累,是Cd地下向地上部转运的重要基因,可作为重金属污染植物修复的候选基因.(图7表1参41)

Phytoremediation refers to the technology that uses hyperaccumulators to remove environmental pollutants;however,hyperaccumulators have many shortcomings,such as low biomass and slow growth,which limit the wide application of this technology.In this study,we cloned a Cd-responsive gene,SpHMA2,in Sedum plumbizincicola and analyzed its encoding protein sequence and phylogenetic relationships in plants.SpHMA2 is a typical heavy-metal ATPase(HMA).SpHMA2 is expressed in the roots,stems,and leaves of S.plumbizincicola,and its expression in each tissue can be increased 0.50-1.45 times by Cd treatment as compared to the control.When SpHMA2 was transformed into Escherichia coli cells,the growth of the transgenic E.coli in the medium was found to be better than that of the control cells under 0.8 mmol/L Cd treatment,and Cd accumulation was increased by 2.54 times.When SpHMA2 was transformed into Arabidopsis thaliana,the transgenic plants exhibited markedly higher biomass,root length,and seed germination rate than the wild-type(WT)plants when grown with 75 and 150μmol/L Cd.Transgenic Arabidopsis overexpressing SpHMA2 plants also showed higher tolerance to and shoot accumulation of Cd than did WT plants.Under 75μmol/L Cd treatment,compared with the WT,in the transgenic Arabidopsis lines,the root length was increased 1.34-1.63 times,the seed germination rate was increased 1.16-1.23 times,and the shoot accumulation was increased by 1.93 and 0.34 times in the transgenic plant lines M3 and M4,respectively.Under 150μmol/L Cd treatment,the root length of the three transgenic lines was 1.89-9.43 times that of the WT,the seed germination rate in the three transgenic lines was 1.73-2.02 times that of the WT,and the shoot accumulation was increased by 0.18-1.47 times in the transgenic plant lines.Under 50μmol/L Cd treatment,SOD,POD,and CAT activities,and H_(2)O_(2) and O_(2)^(-) levels of leaves of the transgenic lines were lower than those of the WT.The results of these studies indicate that SpHMA2 can be used to improve Arabidopsis thaliana tolerance to and accumulation of Cd,and therefore,it can be applied for phytoremediation of heavy metal pollution.