目的 克隆药用真菌猪苓细胞色素P450基因并进行生物信息学分析。方法 利用RT-PCR技术取得基因 cDNA 全长;利用在线工具预测蛋白的理化性质、结构域等分子特征;用MEGA 6. 0分别对氨基酸进行多序列比对和进化关系分析。结果 本实验从中国野生猪苓菌核(Polyporus umbellatus)中克隆得到 11个P450超家族基因,该11个基因 cDNA包含的完整开放阅读框在1 326~1 635 bp之间,编码蛋白含442~545个氨基酸之间,相对分子质量在(48.9~61.5)×103之间,理论等电点在6.3~8.9之间。序列分析发现其具有保守的P450结构域。氨基酸序列多重比对及系统发育树结果显示这11个细胞色素P450基因都隶属于担子菌类。实时荧光定量 RT-PCR 分析表明,这9个基因在不同的菌核部分都有表达,除基因comp18720、comp26906、comp32003及comp33717在被蜜环菌侵染部位的表达量要远远低于未被蜜环侵染的猪苓菌核,其他7个基因都是表达显著上调。结论 药用真菌猪苓11种P450酶基因的分子特征为进一步研究其在猪苓菌核防御蜜环菌侵染的作用奠定理论基础。
Abstract
OBJECTIVE To clone the cytochrome P450 genes from a medicinal fungus Polyporus umbellatus and carry out bioinformatic analysis.METHODS The full length cDNAs of the genes were obtained by RT-PCR.Some bioinformatic tools were used to characterize the physiochemical properties of the 11 deduced protein.The analyses of multiple alignment and phylogenetic trees were performed with MEGA 6.0 software.RESULTS Eleven P450 genes were cloned using RT-PCR method from the Polyporus umbellatus sclerotium. The full open reading frame cDNA sequence of these eleven genes was between 1 326 and 1 635 bp, the putative encoding proteins were between 442 and 545 amino acids, the molecular weight was between 48.9×103 and 61.5×103, and the theoretical pI was between 6.3 and 8.9. Protein sequence analysis found that there was conserved P450 domain in the 11 genes. Phylogenetic tree analysis indicated that all these 11 P450 genes belonged to basidiomycetes. Quantitative real-time PCR showed that these 11 genes were expressed in both the infected partand non-infectedpart. Meanwhile,the expressions of seven genes were significantly up-regulated in the infected part except comp18720, comp26906, comp32003 or comp33717.CONCLUSION Molecular characterization of the 11Cytochrome P450 genes will be useful for further functional determination of the genes involved in the defense progress of Polyporus umbellatus.
关键词
猪苓 /
细胞色素P450 /
基因克隆 /
特异性表达
{{custom_keyword}} /
Key words
Polyporus umbellatus /
cytochrome P450 /
gene clone /
specific expression
{{custom_keyword}} /
中图分类号：
R284
{{custom_clc.code}}
({{custom_clc.text}})
{{custom_sec.title}}
{{custom_sec.title}}
{{custom_sec.content}}
参考文献
[1] NELSON D R, KOYMANS L, KAMATAKI T, et al. P450 superfamily: update on new sequences, gene mapping, accession numbers and nomenclature [J]. Pharmacogenetics Genomics, 1996, 6(1): 1-42.
[2] MEIJER A H, SOUER E, VERPOORTE R, et al. Isolation of cytochrome P-450 cDNA clones from the higher plant Catharanthus roseus by a PCR strategy [J]. Plant Molecul Biol, 1993, 22(2): 379-383.
[3] WERCK-REICHHART D. Cytochromes P450 in phenylpropanoid metabolism [J]. Drug Metab Drug Interact, 1995, 12(3-4): 221-244.
[4] MIZUTANI M, SATO F. Unusual P450 reactions in plant secondary metabolism [J]. Arch Biochem Biophys, 2011, 507(1): 194-203.
[5] MIZUTANI M. Impacts of diversification of cytochrome P450 on plant metabolism [J]. Biol Pharm Bull, 2012, 35(6): 824-832.
[6] PAN Y, MICHAEL T P, HUDSON M E, et al. Cytochrome P450 monooxygenases as reporters for circadian-regulated pathways [J]. Plant Physiol, 2009, 150(2): 858-878.
[7] LI H, PINOT F, SAUVEPLANE V, et al. Cytochrome P450 family member CYP704B2 catalyzes the ω-hydroxylation of fatty acids and is required for anther cutin biosynthesis and pollen exine formation in rice [J]. Plant Cell, 2010, 22(1): 173-190.
[8] NARUSAKA M, SEKI M, UMEZAWA T, et al. Crosstalk in the responses to abiotic and biotic stresses in Arabidopsis: analysis of gene expression in cytochrome P450 gene superfamily by cDNA microarray [J]. Plant Mol Biol, 2004, 55(3): 327-332.
[9] KONG L, ANDERSON J M, OHM H W. Induction of wheat defense and stress-related genes in response to Fusarium graminearum [J]. Genome, 2005, 48(1): 29-40.
[10] LI X, ZHANG J, SONG B, et al. Resistance to Fusarium head blight and seedling blight in wheat is associated with activation of a cytochrome P450 gene [J]. Phytopathology, 2010, 100(2): 183-191.
[11] MORANT M, J RGENSEN K, SCHALLER H, et al. CYP703 is an ancient cytochrome P450 in land plants catalyzing in-chain hydroxylation of lauric acid to provide building blocks for sporopollenin synthesis in pollen [J]. The Plant Cell, 2007, 19(5): 1473-1487.
[12] MACHIDA M, ASAI K, SANO M, et al. Genome sequencing and analysis of Aspergillus oryzae [J]. Nature, 2005, 438(7071): 1157-1161.
[13] KELLY D E, KRAEVEC N, MULLINS J, et al. The CYPome (cytochrome P450 complement) of Aspergillus nidulans [J]. Fungal Genet Biol, 2009, 46(1): S53-S61.
[14] DODDAPANENI H, SUBRAMANIAN V, YADAV J S. Physiological regulation, xenobiotic induction, and heterologous expression of P450 monooxygenase gene pc-3 (CYP63A3), a new member of the CYP63 gene cluster in the white-rot fungus Phanerochaete chrysosporium [J]. Curr Microbiol, 2005, 50(6): 292-298.
[15] DA SILVA M, CERNIGLIA C E, POTHULURI J V, et al. Screening filamentous fungi isolated from estuarine sediments for the ability to oxidize polycyclic aromatic hydrocarbons [J]. World J Microbiol Biotechnol, 2003, 19(4): 399-405.
[16] HUNTER A C, BERGIN-SIMPSON H. Distinct metabolic handling of 3β-hydroxy-17a-oxa-D-homo-5α-androstan-17-one by the filamentous fungus Aspergillus tamarii KITA: evidence in support of steroid/hydroxylase binding hypothesis [J]. Biochim Biophys Acta (BBA)-Molecular and Cell Biology of Lipids, 2007, 1771(9): 1254-1261.
[17] KASAI N, IKUSHIRO S I, HIROSUE S, et al. Enzymatic properties of cytochrome P450 catalyzing 3′-hydroxylation of naringenin from the white-rot fungus Phanerochaete chrysosporium [J]. Biochem Biophys Res Commun, 2009, 387(1): 103-108.
[18] KULLMAN S W, MATSUMURA F. Metabolic pathways utilized by Phanerochaete chrysosporium for degradation of the cyclodiene pesticide endosulfan [J]. Appl Environ Microbiol, 1996, 62(2): 593-600.
[19] LIVAK K J, SCHMITTGEN T D. Analysis of relative gene expression data using real-time quantitative PCR and the 2-ΔΔCTmethod [J]. Methods, 2001, 25(4): 402-408.
[20] WANG Y, LI Q, HE Z. Blast fungus-induction and developmental and tissue-specific expression of a rice P450CYP72A gene cluster [J]. Chin Sci Bull(科学通报), 2004, 49(2): 131-135.
[21] GU Y Z, MORAN S, HOGENESCH J B, et al. Molecular characterization and chromosomal localization of a third alpha-class hypoxia inducible factor subunit, HIF3alpha [J]. Gene Expression, 1997, 7(3): 205-213.
{{custom_fnGroup.title_cn}}
脚注
{{custom_fn.content}}
基金
国家重点基础研究发展计划资助(2014CB13830X);国家自然科学基金资助项目(31201666);山西省煤基重点科技攻关项目资助项目(FT2014-03);河北山区特色中药材种质资源评价与仿野生栽培技术研究与示范资助项目(16232503D)
{{custom_fund}}
PDF(2271 KB)
