蝴蝶兰花瓣瞬时转化体系建立

摘要

蝴蝶兰花器官中基因功能的研究受遗传转化效率低和遗传转化周期长的制约,而花瓣瞬时表达体系是一种快速分析基因功能的有效手段.该研究以蝴蝶兰'大辣椒'花瓣和萼片为实验材料,通过农杆菌介导的瞬时转化方法,分析了侵染的菌液浓度、侵染时间、乙酰丁香酮浓度和共培养时间等4个因素对β-葡糖醛酸酶(GUS)报告基因表达效率的影响,以探寻其瞬时表达的最佳条件;并将查尔酮合成酶( chalcone synthase ,CHS )基因RNAi干扰载体瞬时转化蝴蝶兰花瓣,共培养3 d后观察转化材料中花色表型以及色素的变化,并利用半定量 RT-PCR来检测C H S基因转录水平的表达.结果表明:(1 )农杆菌菌液 OD600为0 .6 、侵染时间60 s ,在重悬液中添加150 μmol/L乙酰丁香酮,共培养3 d ,GUS瞬时表达率最高(85 .01%) . (2)转基因蝴蝶兰花瓣颜色明显变淡,色素含量降低. (3)半定量PCR检测表明,C H S基因的转录活性相比于对照组显著降低.该实验成功的在蝴蝶兰花器官中建立了一种快速基因功能验证方法,为后期蝴蝶兰基因功能研究和育种工作提供技术支持.%The study of gene function in Phalaenopsis aphrodite is hampered by the low efficiency of trans-formation systems and the long time span needed for the generation of transgenic plants.Therefore ,the es-tablishment of transient expression system of Phalaenopsis aphrodite petals will be an effective method for gene function analysis.An experiment was conducted to investigate the transient expression mediated by Agrobacterium tumefaciens using the sepals and petals of Phalaenopsis aphrodite (P.'Big chili'). Effects of A.tume faciens concentration ,infection time ,acetosyringone concentration ,and co-culture length on the transient expression of β-glucuronidase(GUS)gene were analyzed to determine the optimal transfor-mation condition.The chalcone synthetase gene RNAi interference vector was transiently transformed into the petals of Phalaenopsis aphrodite.After 3 days of co-cultivation ,changes in the phenotype and pig-mentation of the flower color were observed ,and RT-PCR was used to detect the expression level of CHS gene transcription.(1)A higher transient expression level of GUS gene could be obtained as OD600 value of A.tume faciens 0.6 ,infiltrating for 60 seconds ,adding 150 μmol/L acetosyringone to co-culture medium , and co-culture for 3 days in darkness.It reaches 85.01%.(2)The color of the petals of transgenic Phalae-nopsis aphrodite was significantly lighter and pigment content decreased.(3 ) Semi-quantitative PCR showed that the transcriptional activity of CHS gene was significantly lower than that of the control group.The experiment successfully established a rapid gene function verification method ,providing techni-cal support for the study of gene function and breeding work of the Phalaenopsis aphrodite.