烟草ζ-胡萝卜素脱氢酶基因的克隆和功能分析

Cloning and Functional Analysis of ζ-Carotene Desaturase Gene in Nicotiana tabacum

为揭示烟草ζ-胡萝卜素脱氢酶(ZDS)基因的功能,采用同源克隆的方法从普通烟草(Nicotiana tabacum)中克隆了Nt ZDS基因,并进行了遗传进化分析。同时通过荧光定量PCR技术分析Nt ZDS的时空表达模式、利用烟草脆裂病毒诱导的基因沉默(VIGS)体系抑制本氏烟草中ZDS基因的表达,在该基因沉默后再通过定量PCR技术分析Nt ZDS上下游基因的表达量变化,并检测了烟株色素含量的变化。结果显示:Nt ZDS基因在普通烟草中至少存在两个同源拷贝,其编码序列与番茄、马铃薯的ZDS基因相似度高于90%。Nt ZDS在烟草盛花期的叶和花中表达量最高。与对照比较,该基因沉默后,烟株新生叶片出现严重白化现象,能够为VIGS体系提供一个良好的候选报告基因;同时类胡萝卜素合成通路其他基因的表达量和烟株类胡萝卜素、叶绿素含量都显著降低,表明ZDS基因在烟草生长发育及类胡萝卜素合成过程中发挥着重要作用。

To reveal the function ofζ-carotene desaturase（ZDS） in Nicotiana tabacum（N.tabacum）,Nt ZDS gene was homologously cloned from N.tabacum and its phylogenetic analysis was conducted.Fluorescence quantitative PCR technology was used to analyze the temporal and spatial expression patterns of Nt ZDS and tobacco rattle virus induced gene silencing（VIGS） system was employed to inhibit the expression of ZDS in Nicotiana benthamiana.The expression levels of upstream and downstream genes were further analyzed by fluorescence quantitative PCR,and the changes of pigment contents in tobaccoplants were determined.The results showed that there existed at least two homologous copies of Nt ZDS gene in N.tabacum with the similarity of more than 90% in coding sequence to ZDS genes in tomato and potato.The expression level of Nt ZDS was the highest in leaves and flowers at flowering stage.Comparing with the control,the leaves of tobacco plants presented severe bleaching phenotype after Nt ZDS silencing,it provided a good candidate for reporter gene for VIGS system; meanwhile the expression levels of upstream and downstream genes and pigment contents（carotenoid and chlorophyll） significantly decreased,it implied that ZDS gene played an important role in the course of growth,development and carotenoid formation of N.tabacum.