摘要
通过构建融合番茄RuBP羧化酶小亚基转运肽基因(rbcS-3)和CAT基因编码阅读框(ORF)的双元表达载体,采用农杆菌介导的叶圆盘转化法将融合基因转入烟草,使其能够定向导入叶绿体中发挥作用。在含有50mg/L潮霉素的培养基上筛选获得转CAT烟草30多个株系,并对其进行了分子生物学的验证和生理指标的检测。对获得的抗性植株用PCR、RT-PCR、植株总蛋白Western blot和叶绿体蛋白Western blot分析表明,目的基因已经整合到烟草基因组中,并能正常表达,且在叶绿体rbcS-3转运肽的作用下能定向进入叶绿体中。对转基因植株生理指标的检测发现,在20%PEG6000模拟干旱条件下,野生型烟草的相对电导率提高幅度为43.4%,而转CAT植株的相对电导率仅提高8.8%,表明在干旱胁迫下转CAT烟草的质膜透性小于野生型烟草;经20%PEG6000处理后,野生型和转CAT基因烟草的叶绿素含量都下降,下降幅度分别为68.0%和20.4%;另外,经20%PEG6000处理的野生型烟草叶片的Fv/Fm下降幅度为5.3%,而转CAT基因烟草叶片的Fv/Fm下降幅度0.9%,这些结果表明,在叶绿体中过量表达CAT对干旱胁迫下的细胞质膜、叶绿素和PSⅡ具有一定的保护作用。此外,经150μmol/L百草枯处理后发现,处理3h后,野生型烟草和转CAT烟草的相对电导率分别比对照提高67.9%和13.5%,而野生型和转CAT烟草的Fv/Fm都下降,降幅分别为23.7%和3.9%,这表明在百草枯氧化胁迫下转CAT烟草的质膜和PSⅡ的损伤程度都小于野生型烟草。总之,豌豆CAT基因在烟草叶绿体中过量表达,提高了转基因烟草的抗旱性和抗氧化性。
In order to study the effects of overexpressing pea catalase（CAT） in tobacco chloroplast on drought and photooxidation tolerance,a binary vector harboring the gene fusion of tomato rbcS-3 and pea CAT was constructed,and the transgenic tobacco plants with high expression levels of rbcS-3 and CAT in chloroplast were generated by Agrobacterium tumerficians-mediated transformation.Transformants were selected on the medium containing hygromycin（50 mg/L）,and over 30 transgenic lines were obtained and used for further molecular and physiological determination.Analysis of the transgenic plants was performed by using PCR,RT-PCR and Western blotting approaches.The result indicated that the genes of rbcS-3 and CAT were successfully integrated into tobacco genome,and the fusion protein was overexpressed in tobacco chloroplast by the help of rbcS-3 targeting.The permeability of plasma membrane,Fv/Fm and the content of chlorophyll in tobacco leaves treated with 20% PEG6000 and 150μmol/L paraquat were measured.Compared to 43.4% increase of the electrical conductivity in wild type plants under the drought stress,the electrical conductivity was only increased 8.8% in the transgenic plants,indicating that the permeability of plasma membrane in transgenic plants was dramatically reduced.Although the chlorophyll levels in tobacco leaves were all decreased when treated with 20% PEG6000,but they were decreased by 68.0% and 20.4% in the wild type and transgenic plants respectively.In addition,under the drought stress,the Fv/Fm decreased 5.3% in wild type plants,but the decrease of Fv/Fm in transgenic plants was only 0.9%.These results indicated that overexpression of the pea CAT in chloroplast could protect plasma membrane,chlorophyll and PSⅡ from injury under drought stress.Moreover,after 3h treatment with paraquat,the relative electrical conductivity in wild type plants was increased by 67.9%,but it was only increased by 13.5% in the transgenic plants.Whereas the Fv/Fm all decreased in wild type and transgenic plants after 3h treatment with paraquat,but the decrease was 23.7% in the wild type and 3.9% in the transgenic plants respectively.These data suggest that the extent of injury in transgenic plants was largely reduced under the paraquat stress.Overall,our results in the present study demonstrate that the transgenic tobacco plants overexpressing pea CAT in chloroplast enhance the drought and photooxidation tolerance.
出处
《生态学报》
CAS
CSCD
北大核心
2011年第4期1058-1063,共6页
Acta Ecologica Sinica
基金
国家自然科学基金项目(30170556
30970240)
山东省自然科学基金项目(2009ZRB019E7)
植物生理生化国家重点实验室开放课题(PPB08005)
青岛农业大学高层次人才基金(630743)
