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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 of key enzyme pyrophosphorylase synthase (FPPS) gene from the biosynthesis of Atractylodes lancea sesquiterpenoids and to analyze its expression pattern. Methods The total RNA of AFP (AFPPS) of Atractylodes lancea was cloned by using the total RNA of Atractylodes macrocephala as a template. The full length c cDNA of AFP (AFPPS) was cloned by using homologous cloning and RACE techniques and analyzed by bioinformatics. The expression of FPPS in the growth stages of Atractylodes lancea and the changes of sesquiterpenoids in the roots of Atractylodes lancea and the correlation between them were analyzed. Results The full-length c DNA of Al FPPS gene was 1 320 bp (GenBank accession no. KX443242). It contained a 1 029 bp open reading frame encoding 342 amino acids and contained 5 conserved domains, of which 2 were rich in Aspartic acid (DXXDD). q RT-PCR and GC-MS results showed that there was a significant positive correlation between Al FPPS gene expression and sesquiterpenoid content in different stages of Atractylodes. Conclusion The full-length c DNA of Al FPPS gene was cloned for the first time. Al FPPS gene was initially proved to be an important regulatory site in the biosynthesis pathway of sesquiterpene constituents of Atractylodes lancea. Engineering application to provide a scientific basis.