摘要
喜树是一种具有药用价值的木本植物,其主要次级代谢产物喜树碱具有良好的抗癌功效。本研究主要通过病毒诱导的基因沉默(virus induced gene silencing,VIGS)技术对喜树碱生物合成途径中的关键酶基因CaCYC1环烯醚萜合酶(Camptotheca acuminata iridoid synthase,CaCYC1)进行瞬时沉默,进而研究该基因对喜树碱生物合成的影响。以喜树叶片总RNA为模板,通过RT-PCR技术克隆CaCYC1基因片段并构建TRV-VIGS重组质粒,转化农杆菌GV3101后侵染喜树叶片,培养20 d后利用qRT-PCR与HPLC技术分析沉默对CaCYC1基因转录水平及喜树碱合成的影响。结果表明:与对照组相比,实验组中CaCYC1基因的相对表达量显著下降49%,叶片中喜树碱的积累量显著下降35%。上述结果表明,本研究所构建的由TRV介导的VIGS体系可有效沉默内源基因的表达,CaCYC1基因可正向调控喜树叶片中喜树碱的生物合成。
Camptotheca acuminata Decne is a kind of woody plant with medicinal value.Its main secondary metabolite camptothecin has good anticancer effect.The virus-induced gene silencing(VIGS)technique was used to transiently silence the key enzyme gene CaCYC1 in the camptothecin biosynthesis pathway to study the effect of this gene on camptothecin biosynthesis.The total RNA was used as a template,and the CaCYC1 gene fragment was cloned by RT-PCR.The recombinant plasmid of TRV-VIGS was constructed and transiently transformed to C.acuminata leaves mediated by Agrobacterium.After 20 days of culture,the silencing of CaCYC1 was utilized to analyze the level of CaCYC1 gene transcription and the effect on camptothecin synthesis by qRT-PCR and HPLC techniques.The results showed that compared with the control group,the relative expression of CaCYC1 gene in the experimental group decreased significantly by 49%,and the accumulation of camptothecin in the leaves decreased significantly by 35%.The above results show that the TRV-mediated VIGS system constructed in the paper can effectively silence the expression of endogenous genes,CaCYC1 gene can positively regulate the biosynthesis of camptothecin in leaves of Camptotheca acuminata.
作者
万如意
丛云寒
姜珊珊
金朝霞
Wan Ruyi;Cong Yunhan;Jiang Shanshan;Jin Zhaoxia(School of Biological Engineering,Dalian Polytechnic University,Dalian,116034)
出处
《分子植物育种》
CAS
CSCD
北大核心
2020年第1期159-167,共9页
Molecular Plant Breeding
基金
国家自然科学基金项目(31670604
31570303)
辽宁省自然科学基金(20180550237)
国家级大学生创新训练项目(201810152002)共同资助
