摘要
目的克隆茅苍术Atractylodes lancea倍半萜类成分生物合成关键酶法呢基焦磷酸合酶(FPPS)基因全长并分析其表达规律。方法以茅苍术总RNA为模板,运用同源克隆法和RACE技术克隆茅苍术FPPS基因(Al FPPS)的c DNA全长,并进行生物信息学分析;用q RT-PCR和GC-MS分析不同生长阶段FPPS在茅苍术根茎中的表达及倍半萜类成分量变化,并分析二者的相关性。结果获得Al FPPS基因全长c DNA为1 320 bp(Gen Bank accession no.KX443242),含一个长1 029 bp的开放阅读框,编码342个氨基酸,包含5个保守功能域,其中2个富含天冬氨酸(DXXDD)。q RT-PCR及GC-MS结果显示不同时期茅苍术Al FPPS基因表达量与倍半萜类成分量呈显著正相关。结论首次克隆获得Al FPPS基因的全长c DNA,初步证明Al FPPS基因是茅苍术倍半萜类成分生物合成途径中的重要调控位点,为茅苍术倍半萜类成分生物合成途径阐明与生物工程应用提供科学依据。
Objective To clone the full-length cDNA encoding farnesyl pyrophosphate synthase （FPPS） from Atractylodes lancea and analyze its expression. Methods The full-length cDNA of FPPS in A. lancea was cloned via homology-based cloning and rapid amplification of cDNA ends approach. Also, the characterization of gene was revealed by bioinformatic analysis. The expression of FPPS was determined by qRT-PCR while the content of sesquiterpenes of rhizome in different growth stages in A. lancea was measured by GC-MS. Meanwhile the correlation between them was analyzed. Results The full-length cDNA （1 320 bp） of FPPS gene was obtained （AlFPPS, GenBank accession number KX443242）, with an open reading frame of 1 029 bp, encoding 342 amino acids. The deduced AlFPPS protein sequence contained five conserved motifs, two of which is full of Asp （DDXXD）. qRT-PCR and GC-MS results showed a significant positive correlation between the content of sesquiterpenes and AlFPPS expression level. Conclusion It can be primarily deduced that AlFPPS gene should be an important control point in the biosynthetic pathway of sesquiterpenes in A. lancea. This work provides scientific basis for clarifying the biosynthetic pathway of sesquiterpenes and application of biological engineering.
作者
蒋玲
谷巍
巢建国
桑晓华
韩赟
刘青芝
席彩彩
JIANG Ling GU Wei CHAO Jian-guo SANG Xiao-hua HAN Yun LIU Qing-zhi XI Cai-cai(School of Pharmacy, Nanjing University of Chinese Medicine, Nanjing 210023, China)
出处
《中草药》
CAS
CSCD
北大核心
2017年第4期760-766,共7页
Chinese Traditional and Herbal Drugs
基金
国家自然科学基金资助项目(81573520)
江苏省中药优势学科II期建设(ysxk-2014)
"六大人才高峰"高层次人才项目(2012-YY-009)
江苏省"333高层次人才培养工程"
江苏省中药资源产业化过程协同创新中心资助项目
关键词
茅苍术
法尼基焦磷酸合酶
基因克隆
基因表达
倍半萜
Atractylodes lancea （Thunb.） DC.
famesyl diphosphate synthase
gene clone
gene expression
sesquiterpene
