中国现代中药2025，Vol.27Issue(12)：2219-2228,10.DOI:10.13313/j.issn.1673-4890.20250318001
茅苍术3个CYP71家族基因的克隆、亚细胞定位与表达分析
Cloning,Subcellular Localization,and Expression Analysis of Three CYP71 Family Genes from Atractylodes lancea
摘要
Abstract
Objective:To clone cytochrome P450 71(CYP71)genes from Atractylodes lancea and analyze their bioinformatics and expression characteristics.Methods:Primers were designed based on CYP71 genes sequences obtained from A.lancea transcriptome data.The target genes were amplified by PCR,cloned,and sequenced.The bioinformatics analysis of the proteins encoded by the target genes was carried out via a variety of software such as ExPASy,ExPasy-ProtScale,NetPhos 3.1 Server,SOPMA,and SWISS-MODEL.Fusion expression vectors of the target genes and pCN-green fluorescent protein(GFP)were constructed and transfected into tobacco epidermal cells via the Agrobacterium-mediated transformation.The fluorescent signal was observed to determine the subcellular localization of the proteins encoded by the genes.qRT-PCR was utilized to analyze the expression levels of these genes in tender rhizomes,old rhizomes,stems,and leaves.Results:The open reading frames(ORFs)of AlCYP71BZ36v1,AlCYP71BZ36v2,and AlCYP71BZ36v3 were 1 296,1 503,and 1 032 bp in length,respectively,encoding 431,500,and 343 amino acid residues.These genes belonged to the cytochrome P450 superfamily and were members of the CYP71-like family,containing the conserved domains GGxDTS,ExxR,PERF,and FxxGxRxCxG.The proteins encoded by AlCYP71BZ36v1,AlCYP71BZ36v2,and AlCYP71BZ36v3 are hydrophilic.Prediction of transmembrane domains indicated that all three proteins were secretory proteins.AlCYP71BZ36v1 and AlCYP71BZ36v3 lacked transmembrane regions,while AlCYP71BZ36v2 had a transmembrane region located between amino acids 5 and 22.The results of qRT-PCR showed that the expression trends of AlCYP71BZ36v2 and AlCYP71BZ36v3 were relatively consistent,with the highest expression in tender rhizomes,whereas AlCYP71BZ36v1 had the highest expression in stems.Conclusion:AlCYP71BZ36v1,AlCYP71BZ36v2,and AlCYP71BZ36v3 were successfully cloned from A.lancea,laying a foundation for studying the functions of the CYP71 family and elucidating the biosynthesis of sesquiterpenoids in A.lancea.关键词
茅苍术/CYP71家族/基因克隆/亚细胞定位/生物信息学分析Key words
Atractylodes lancea(Thunb.)DC./CYP71 family/gene cloning/subcellular localization/bioinformatics analysis分类
医药卫生引用本文复制引用
GAO Bingyan,CHEN Chang,ZHANG Baiyi,ZHENG Kunpeng,YANG Cheng,YU Kun,GONG Ling..茅苍术3个CYP71家族基因的克隆、亚细胞定位与表达分析[J].中国现代中药,2025,27(12):2219-2228,10.基金项目
湖北省自然科学基金项目(2023AFB972) （2023AFB972）
中国博士后一般面上基金项目(2022M71223) （2022M71223）
国家自然科学基金项目(32000254) （32000254）
