摘要
松叶猪毛菜(Salsola laricifolia)是典型的荒漠C3-C4中间型植物,隶属于藜科(Chenopodiaceae)猪毛菜属,是研究C4途径进化以及光呼吸减少机制的最佳物种。以松叶猪毛菜无菌苗为试验材料,通过农杆菌介导的瞬时转化方法,分别对质粒载体、苗龄、农杆菌侵染浓度、侵染时间和乙酰丁香酮浓度进行研究,探究各因素对松叶猪毛菜植株瞬时遗传转化效率的影响,以确立最佳的瞬时转化体系;并在上述体系的基础上,过表达松叶猪毛菜叶绿体型NADP-苹果酸酶基因(SaNADP-ME1),通过GUS组织化学染色和荧光定量PCR验证瞬时转化效果。结果表明:(1)采用pCAMBIA1301构建表达载体,以30 d苗龄植株作为转化受体,在侵染液中添加200μmol·L^-1乙酰丁香酮,农杆菌菌液OD600为0.8,侵染6 h条件下,瞬时转化效率最佳(92%);(2)构建过表达载体并瞬时转化松叶猪毛菜植株,通过染色检测到GUS活性,并且荧光定量PCR检测到SaNADP-ME1的表达量相比对照组显著增高,该基因成功瞬时过表达转化至植株中。综上所述,本试验在松叶猪毛菜植株中建立了一种快速基因功能验证方法,为以后研究松叶猪毛菜功能基因奠定了基础。
Salsola laricifolia is an intermediate type of C3-C4 of the genus Chenopodiaceae,which is an ideal material for studying the evolution of the C4 pathway and the mechanism of photorespiration reduction.In this study,sterile seedlings of S.laricifolia were used as experimental materials to investigate the transient expression mediated by Agrobacterium.Effects of the plasmid vector,seedling age,Agrobacterium(containing pCAMBIA1301 plasmid)concentration,infection time,and acetosyringone(AS)concentration were analyzed to determine the optimal transformation condition.Under these optimal conditions,the chloroplast-type NADP-malic enzyme gene(SaNADP-ME1)was over expressed in the S.laricifolia,as detected by the GUS histochemical staining and real-time fluorescent quantitative polymerase chain reaction(PCR).(1)pCAMBIA1301-gus was used to construct the expression vector,and the 30-day-old seedling was used as the transformation receptor.By adding 200μmol·L^-1 of AS to the immersion solution,the OD600 of Agrobacterium was 0.8,and by transforming for 6 h,the transient transformation efficiency was the best(92%).(2)The overexpression vector was constructed and transiently transformed into the S.laricifolia plant.GUS activity was detected by staining,and the quantitative expression of SaNADP-ME1 detected by real-time PCR was significantly higher than that of the control group,indicating that the gene was successfully transiently overexpressed into the plant.In summary,this experiment established a rapid gene function verification method in S.laricifolia,which laid a foundation for studying the gene function of S.laricifolia.
作者
夏春兰
李亚楠
闻志彬
XIA Chun-lan;LI Ya-nan;WEN Zhi-bin(Key Laboratory of Biogeography and Bioresource in Arid Land of Chinese Academy of Sciences,Xinjiang Institute of Ecology and Geography,Chinese Academy of Sciences,Urumqi 830011,Xinjiang,China;University of Chinese Academy of Sciences,Beijing 100049,China;College of Life Sciences,Shihezi University,Shihezi 832003,Xinjiang,China)
出处
《干旱区研究》
CSCD
北大核心
2020年第5期1317-1326,共10页
Arid Zone Research
基金
国家自然科学基金(31670339,31970354)
中国科学院战略生物资源服务网络计划生物标本馆经典分类学青年人才项目(ZSBR-007)资助
