杜氏盐藻番茄红素ε-环化酶基因DsLYCE表达载体构建及其在烟草中瞬时表达分析

Expression vector construction of Dunaliella Salina Lycopeneε-cyclase gene(DsLCYE)and its transient expression in tobacco leaves

[目的]ε-环化酶(lycopeneε-cyclase,LYCE)可将番茄红素环化,并与番茄红素β-环化酶共同作用合成α-胡萝卜素,参与植物体内类胡萝卜素的合成途径。富含β-胡萝卜素的杜氏盐藻(Dunaliella Salina)LYCE功能还未解析。[方法]本文利用RT-PCR分离杜氏盐藻DsLYCE基因ORF,并克隆到植物表达载体pCAMBIA1303、构建DsLYCE组成型表达载体pCAMBIA1303-DsLYCE。通过农杆菌介导渗入法在烟草叶组织中瞬时表达DsLYCE,鉴定DsLYCE功能。[结果]烟叶生理生化分析显示,与野生型和空载体对照相比,DsLYCE瞬时表达的烟叶组织中类胡萝卜素积累显著提高(P<0.05),含量增加了24%。相似地,转DsLYCE基因的烟草叶片中叶绿素a和叶绿素b含量也明显高于野生型空载对照烟草。这表明杜氏盐藻DsLYCE基因异源表达不仅能促进宿主细胞类胡萝卜素的合成与积累,而且还可以改善宿主细胞的光合作用。[结论]本研究为深入解析杜氏盐藻DsLYCE参与类胡萝卜素生物合成的分子机制以及应用于其他植物的色素代谢遗传改良提供科学参考。

[Objective]Lycopeneε-cyclase(LYCE),an important player in carotenoid biosynthesis pathway in plants,can catalyze lycopene by cyclization,and co-function with lycopeneβ-cyclase in synthesis ofα-carotene.However,the function of LYCE of Dunaliella Salina rich inβ-carotene remains to be addressed.[Methods]The ORF of DsLYCE in D.Salina was isolated by RT-PCR,and inserted into the vector pCAMBIA1303 for the construction of the constitutive expression vector of DsLYCE(pCAMBIA1303-DsLYCE)in plants.Agobacterium-mediated infiltration method was employed to ensure DsLYCE gene transiently expressed in tobacco(Nicotiana benthamiana)leaves and to conduct a functional analysis of this gene.[Results]Physiological and biochemical analysis on tobacco leaves indicated that the amount of accumulated carotenoids in DsLYCE-transgenic leaves was significantly increased(P<0.05)by 24%compared to that in the wild-type and empty-vector controls.Similarly,the contents of chlorophyll a and chlorophyll b in the DsLYCE-expressed tobacco leaves were also significantly higher than those in the control groups,which indicated that the heterologous expression of DsLYCE could not only boost the biosynthesis and accumulation of carotenoids but also improve the photosynthesis of the host tissues.[Conclusion]The present data provided the scientific references for in-depth elucidation the molecular mechanism of DsLYCE-involved carotenoid biosynthesis and the application of DsLYCE in genetic improvement of carotenoid-related pigment metabolism of high plants.