‘西伯利亚’百合LiCMK基因克隆及功能分析

Cloning and Functional Analysis of LiCMK Gene in Lilium 'Siberia'

4-二磷酸胞苷-2-C-甲基赤藓糖激酶(CMK)是萜类花香合成甲基赤藓糖醇磷酸(MEP)途径的关键酶,为揭示其对百合花香的调控作用,从‘西伯利亚’百合(Lilium‘Siberia’)中克隆LiCMK基因,进行生物信息学分析,利用亚细胞定位确定蛋白位置,采用荧光定量PCR技术检测基因时空表达模式,并运用病毒诱导基因沉默(VIGS)方法瞬时沉默LiCMK,验证其功能。结果显示,LiCMK全长1200 bp,编码399个氨基酸,属于IspE家族,与油棕(Elaeis guineensis)中的CMK相似度最高。LiCMK的表达随花期呈现先上升后降低的规律,在盛花期的表达量达到峰值,在花器官中的表达量明显高于茎、叶中的表达量。LiCMK蛋白定位于叶绿体中。LiCMK在百合中瞬时沉默后,LiCMK表达水平下降了约84%,主要的单萜合成基因表达量明显下降,月桂烯合酶基因(MYS)、罗勒烯合酶基因(OCS)和芳樟醇合酶基因(LIS)分别降低了57%、59%和63%,主要的单萜类化合物月桂烯、罗勒烯和芳樟醇释放量显著下降。结果表明,LiCMK基因对‘西伯利亚’百合单萜化合物的合成与花香释放有重要作用,为后续深入研究百合单萜合成及其花香释放的调控机制奠定基础。

4-diphosphocytidyl-2-C-methyl-D-erythritol kinase(CMK)is a crucial enzyme for terpenoid synthesis in the methylerythritol phosphate(MEP)pathway.To uncover its regulating effect on the aroma of the lilies,gene LiCMK was cloned from Lilium 'Siberia' and analyzed by bioinformatics.Specifically,the protein was located by subcellular localization and spatial-temporal expression patterns of LiCMK gene were explored by means of fluorescence quantitative PCR.And the validation was performed by instantaneously silenced LiCMK through viral induced gene silencing(VIGS).As a result,LiCMK was 1 200 bp in length and encoded 399 amino acids,belonging to IspE family,which was relatively similar to CMK in Elaeis guineensis.The expression of LiCMK rose and fell corresponding to the flowering and reached the peak when it came to full flowering stage.Moreover,the expression in flower was significantly higher than that in stems and leaves.In addition,LiCMK protein was located in chloroplast.LiCMK expression dropped by about 84% after LiCMK was silenced instantaneously in Lilium 'Siberia',leading to the expressions of major monoterpene synthesis genes decreased,i.e.,myrcene synthase gene(MYS),ocimene synthase gene(OCS)and linalool synthase gene(LIS)plunged by 57%,59% and 63%,respectively,and the release of major monoterpenes,myrcene,ocimene and linalool significantly decreased.To conclude,LiCMK gene is indispensable to the synthesis of monoterpenoid compounds and floral release in Lilium 'Siberia',which lays the foundation for future researches about the synthesis and regulatory mechanism of monoterpenoid compounds and the floral scent release.