油茶种子法尼基焦磷酸酶基因的克隆与表达模式分析  

Cloning and expression pattern analysis of CoFPPS gene in Camellia oleifera seed

作　　者：代忠迪 李思颖 丁健 DAI Zhongdi;LI Siying;DING Jian(Tongren Forestry Bureau,Tongren 554300,Guizhou,China;Tongren Academy of Forestry,Tongren 554300,Guizhou,China;Dalian Minzu University,Dalian 116600,Liaoning,China)

机构地区：[1]铜仁市林业局,贵州铜仁554300 [2]铜仁市林业科学院,贵州铜仁554300 [3]大连民族大学,辽宁大连116600

出　　处：《经济林研究》2024年第4期34-40,共7页Non-wood Forest Research

基　　金：铜仁市科技计划项目(铜市科研〔2020〕53号);贵州省科技计划项目(黔科合支撑〔2022〕重点017号)。

摘　　要：【目的】解析油茶CoFPPS基因的生物学特征和功能,及其在油茶种子角鲨烯合成过程中的作用机制。【方法】以高角鲨烯油茶品系‘WBL’为材料,利用转录组测序、qRT-PCR和气相色谱串联质谱等分析方法,挖掘油茶CoFPPS基因,通过基因克隆、生物信息学分析、基因表达量分析和角鲨烯含量测定,解析CoFPPS基因的理化性质及其表达对种子角鲨烯含量的影响。【结果】油茶CoFPPS基因与NCBI数据库中茶FPPS1基因(XM_028249399.1)同源相似性达99.13%,CoFPPS开放阅读框为1029 bp,编码342个氨基酸,其蛋白相对分子质量为39332.14 Da,为酸性稳定蛋白质,肽链中无明显跨膜区;定位于细胞质,无信号肽;蛋白质高级结构以α-螺旋为主(222个,占64.91%),其三级结构的序列相似值为82.94%,QMEANDisCo Global值为0.81±0.05。油茶CoFPPS蛋白与中华猕猴桃、喜马拉雅凤仙花、香叶天竺葵、胡桃、花生和大豆的同源蛋白具有较高的相似性,且均属于Isoprenoid-Biosyn-C1超家族;油茶CoFPPS蛋白与狭叶油茶、朱红大杜鹃、羊踯躅、杜鹃、越橘、三七和竹节参的FPPS蛋白处于同一分支。qRT-PCR分析结果表明,高角鲨烯油茶品系发育期间种仁CoFPPS基因表达量呈现上升趋势,且与角鲨烯含量变化规律相似。【结论】油茶CoFPPS为类异戊二烯生物合成酶,与其他物种的FPPS蛋白可能具有相似的生物学功能,而且CoFPPS基因表达与油茶种子角鲨烯合成密切相关。【Objective】For understanding the biological characteristics and functions of CoFPPS gene in Camellia oleifera,and the regulatory mechanism of CoFPPS gene in C.oleifera seed squalene synthesis pathway.【Method】C.oleifera germplasm‘WBL’with high squalene content were used as experimental materials.Based on the RNA-seq,qRT-PCR and gas chromatography tandem mass spectrometry,FPPS genes were discovered from C.oleifera seed.Meanwhile,gene cloning,bioinformatics,gene expression and squalene content were analyzed,to understand the physicochemical properties of CoFPPS gene,and the relationship of expression pattern of CoFPPS gene and squalene content.【Result】The coefficient of homology similarity between CoFPPS gene and FPPS1 gene(XM_028249399.1,C.sinensis)was 99.13%in NCBI database,the open reading frame of CoFPPS was 1029 bp,encoding 342 amino acids,no signal peptide,and located to the cytoplasm.The relative molecular mass of CoFPPS was 39332.14 Da,which was acidic stable protein with no obvious transmembrane domain in the peptide chain.In addition,the secondary structure was mainly based on alpha helix(222,64.91%),the sequence similarity of protein tertiary structure was 82.94%,the value of QMEANDisCo Global was 0.81±0.05.The amino acid sequence of CoFPPS shared high homology with FPPS from Actinidia chinensis var.chinensis,Impatiens glandulifera,Pelargonium graveolens,Juglans regia L.,Arachis ipaensis and Glycine max,and they belong to the Isoprenoid-Biosyn-C1 superfamily.CoFPPS,ClFPPS,RgFPPS,RmFPPS,RsFPPS,VdFPPS,PnFPPS and PjFPPS were classified into the same branch.qRT-PCR results revealed that CoFPPS gene was significantly up-regulated expression during developing period in seed,and which was consistent with the dynamic change pattern of squalene contents.【Conclusion】CoFPPS is an isoprene synthetase,which may have similar biological functions with FPPS proteins of other species,and CoFPPS gene expression is associated with the biosynthesis of squalene in C.oleifera seeds.

关 键 词：法尼基焦磷酸酶 角鲨烯 油茶 基因克隆 基因表达分析 

分 类 号：S601[农业科学—园艺学]