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百合萜烯合成相关基因LiGGPPS大小亚基基因的克隆、表达及功能分析
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引用本文:刘旭平,王浩楠,张 茜,冷平生,胡增辉.百合萜烯合成相关基因LiGGPPS大小亚基基因的克隆、表达及功能分析[J].西北农业学报,2024,(3):503~510
DOI:10.7606/j.issn.1004-1389.2024.03.012
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作者单位
刘旭平,王浩楠,张 茜,冷平生,胡增辉 (1.北京林木分子设计育种高精尖创新中心北京农学院北京 1022062.北京市乡村景观规划设计工程技术研究中心/园林学院北京农学院北京 102206) 
基金项目:国家重点研发计划(2018YFD1000400);国家自然科学基金(31201645);北京市自然科学基金(6172006)。
中文摘要:香叶基香叶基焦磷酸合酶(GGPPS)是调节萜类化合物生物合成的关键酶,为了研究GGPPS基因在百合萜类物质合成中的作用,本试验以‘西伯利亚’百合(Lilium ‘Siberia’)为试材,克隆了GGPPS大亚基(LiGGPPS.LSU)和小亚基(LiGGPPS.SSU)基因,并分析其表达和功能。通过生物信息学分析、相对表达量分析、荧光定量PCR、亚细胞定位、基因沉默(virus induced gene silencing,VIGS)预测并验证其特征及功能,揭示其表达模式。结果表明,LiGGPPS.LSULiGGPPS.SSU分别为1 100 bp和1 040 bp,LiGGPPS.LSU和LiGGPPS.SSU的氨基酸序列与其他物种的GGPPS.LSU和GGPPS.SSU的氨基酸序列具有较高的一致性,系统进化树显示它们分别与薯蓣(Dioscorea cayenensis subsp. rotundata)和深圳拟兰(Apostasia shenzhenica)亲缘关系最近。LiGGPPS.LSULiGGPPS.SSU基因均定位在叶绿体中,表达量随百合的生长发育呈现先上升后下降的趋势。LiGGPPS.LSU在花被片中高量表达,LiGGPPS.SSU在花药中显著高量表达。VIGS试验显示芳樟醇、罗勒烯、月桂烯释放量下降。LiGGPPS.LSULiGGPPS.SSU基因对百合花香单萜类物质合成具有重要作用,本试验为进一步研究百合GGPPS以及相关代谢途径提供理论基础。
中文关键词:‘西伯利亚’百合  GGPPS  萜烯化合物  基因克隆  表达分析
 
Cloning, Expression and Functional Analysis of LiGGPPS.LSU and LiGGPPS.SSU Involved in Flower Fragrance Synthesis in Lilium
Abstract:Geranylgeranyl pyrophosphate synthase(GGPPS) is a crutial enzyme that regulats terpenoid biosynthesis in plants. To investigate the role of GGPPS genes in lily terpenoid synthesis, we cloned LiGGPPS. LSU and LiGGPPS.SSU genes and analyzed their expression and function using ‘Siberian’ lily as test material. Bioinformatics analysis, relative expression analysis, fluorescence quantitative PCR, subcellular localization, VIGS(virus induced gene silencing) and yeast two-hybrid were used to predict and verify their characteristics and functions, and to reveal their expression patterns. Results showed that LiGGPPS.LSU and LiGGPPS.SSU were 1 100 bp and 1 040 bp,respectively, with the amino acid sequences that had high similarity with those of other species.Phylogenetic analysis showed that LiGGPPS.LSU and LiGGPPS.SSU had the closest relationship with DcGGPPS.LSU and AsGGPPS.SSU in Dioscorea cayenensis subsp.rotundata and Apostasia shenzhenica. Both LiGGPPS.LSU and LiGGPPS.SSU were localized in the chloroplasts, and their expression showed an rising trend, followed by declining trend as the flower grew and developed. LiGGPPS.LSU was highly expressed in the tepals,while LiGGPPS.SSU was highly expressed in the anthers. The VIGS assay showed a decrease in the release of linalool, basilene and lauricene. The findings suggest that LiGGPPS.LSU and LiGGPPS.SSU genes play an important role in the synthesis of monoterpenes in lily flowers,providing a theoretical basis for further research on LiGGPPS and related metabolic pathways.
keywords:Lilium ‘Siberia’  GGPPS  Terpene compounds  Gene cloning  Expression analysis
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