烟草牻牛儿基牻牛儿基焦磷酸合成酶类似基因的克隆及其功能研究

利用同源克隆法从普通烟草(Nicotiana tabacum)红花大金元的cDNA 中克隆到一个新基因NtGGPPSL(Geranylgeranylpyrophosphate synthase-like),其编码区全长为813 bp。Blast 结果显示该基因与林烟草(Nicotiana sylvestris)、绒毛状烟草(Nicotiana tomentosiformis)和番茄(Solanum lycopersicum)的GGPPS 小亚基基因的相似度分别达到了99%、91% 和82%,但其编码的蛋白质序列缺失了GGPPS小亚基的关键功能域“DDXXXXD”,这表明NtGGPPSL 基因的功能可能与GGPPS 小亚基基因不同。为了深入研究NtGGPPSL 基因的功能,通过实时荧光定量PCR(Real-time PCR)分析NtGGPPSL 基因在普通烟草不同时期和器官中的表达差异。利用TRV 病毒诱导的基因沉默(VIGS)体系抑制本氏烟草(Nicotiana benthamiana)中NtGGPPSL基因的表达,在成功沉默该基因之后,检测烟草中质体色素(新黄质、紫黄质、叶黄素、叶绿素a/b、β-胡萝卜素)含量的变化。结果显示,NtGGPPSL 基因在茎和根中的表达量明显高于其他器官。与对照组相比,在NtGGPPSL 基因沉默后,烟草叶片发生明显的褪绿现象,质体色素含量显著降低,表明该基因参与了叶绿素的合成,这为VIGS 体系提供了一个可参考的指示基因,也为烟草的遗传改良和基因功能研究提供了理论依据。 

Cloning and functional analysis of GGPPSL in Nicotiana tabacum

Coding DNA sequence of one new gene NtGGPPSL (813 bp) was successfully obtained from cDNA of Nicotiana tabacum with the method of homology cloning. Blast results showed that the gene was 99%, 91% and 82% homologous to Nicotiana sylvestris, Nicotiana tomentosiformis and Solanum lycopersicum GGPPS small subunit gene, respectively. But the protein sequence encoded by NtGGPPSL did not have the key motif (DDXXXXD) of GGPPS small subunit, which suggested that the function of NtGGPPSL might be different. In order to explore the function of NtGGPPSL, expression level of NtGGPPSL in different stages and organs was studied by real-time PCR. NtGGPPSL was depressed through virus induced gene silencing strategy in Nicotiana benthamiana and content of plastid pigments (neoxanthin, violaxanthin, lutein, chlorophyll a/b and β-carotene) was detected. Results showed that expression level of NtGGPPSL in stems and roots was significantly higher than other organs such as leaf. After NtGGPPSL was suppressed, Nicotiana benthamiana leaves became albescent obviously and the content of plastid pigments was significantly decreased compared with controls, suggesting that NtGGPPSL was involved in the synthesis of chlorophyll. This research not only provided a reference reporter gene, it also lay foundation for genetic improvement and gene function research.