摘要
喜树是中国传统的珍贵药用植物,因其含有抗癌、抗病毒药物喜树碱而被广泛关注。然而喜树碱在喜树中的低积累量限制了其在市场上的应用,因此喜树碱生物合成途径关键酶基因功能的鉴定及其代谢途径的解析变得尤为重要。稳定遗传转化因其周期长、效率低而无法快速鉴定相关基因,瞬时转化则具有高效、方便、易操作等特点。利用瞬时转化可望更快速地解析未知代谢途径,进而调控喜树碱的合成、提高喜树碱的含量。目前喜树中瞬时转化研究较少,并且在前期实验中发现,选取同时期不同个体的喜树叶片进行瞬时转化,会因生理生化差异导致实验数据误差较大,进而增加实验结果分析的难度。所以,建立一种降低个体差异的喜树叶片瞬时转化方法具有重要的理论价值和现实意义。本研究以转化p BI121GD(GUS DELETED)和p BI121(35S::GUS)的根瘤农杆菌GV3101分别侵染喜树同一叶片左右部分,在侵染后不同时期进行GUS染色以检测同一叶片的左右部分GUS蛋白的表达差异,并利用半定量RT-PCR来检测GUS基因的转录水平。结果表明:与对照组比较,在侵染后的第7天实验组中的GUS基因和GUS蛋白均有较高的表达。上述结果表明,成功的在喜树中建立了一种降低个体差异的喜树叶片瞬时转化方法。
Camptotheca acuminata is a traditional Chinese medicinal plant,which attracts many concerns for its producing Camptothecin（CPT）,an effective anti-cancer and anti-viral drug.However,the low accumulation of CPT in C.acuminata plant limits CPT application in the market.Therefore,elucidate of CPT biosynthetic pathway should be very important for possible promoting CPT production by pathway regulation.Although stable genetic transformation is considered to be the best approach for functional identification of candidate pathway genes,low transformation efficiency and hard regeneration process extremely limit its further application.Alternatively,transient transformation is thought to be good approach to identify functions of unknown genes according to its easy and quick manipulation.In this work,we found that C.acuminata leaves collected from different plants with same developmental age showing huge physiological and biochemical differences,which leads to difficult analysis for evaluating further experimental data.Therefore,establishment of transient transformation of C.acuminata leaves with reduced variation of each plant should be very useful for CPT biosynthesis pathway elucidation.In thisexperiment,Agrobacterium tumefaciens GV3101 harboring pBI121 GD（GUS DELETED） and p BI121（35 S：：GUS）vectors were used to infect the left and right parts of same C.acuminata leaf.GUS staining was performed to detect the production of GUS protein,while semi-quantitative RT-PCR was used to examine GUS transcripts levels.Results showed that the highest GUS expression at both translation and transcription levels was observed at the 7 th day after Agrobacterium treatment.These results showed that a new instantaneous transformation method was successfully established to reduce the individual differences in C.acuminata.
作者
王碧莹
谷金芸
霍春红
常纯皓
于放
Wang Biying;Gu Jinyun;Huo Chunhong;Chang Chunhao;Yu Fang(School of Biological Engineering,Dalian Polytechnic University,Dalian,116034)
出处
《分子植物育种》
CAS
CSCD
北大核心
2018年第17期5624-5630,共7页
Molecular Plant Breeding
基金
国家自然科学基金项目(31570303)资助
关键词
喜树
农杆菌介导
瞬时转化
GUS染色
个体差异
Camptotheca acuminata
Agrobacterium tumefaciens mediated
Transient transformation
GUS staining
Individual differences
