云南切梢小蠹超氧化物歧化酶基因的克隆与序列分析  

Molecular cloning and sequence analysis of superoxide dismutase gene from Tomicus yunnanensis

作　　者：赵琰明 朱家颖[1] 泽桑梓 赵宁[1] 杨斌[1] 

机构地区：[1]西南林业大学云南省森林灾害预警与控制重点实验室,昆明650224 [2]云南省林业职业技术学院,昆明650224

出　　处：《植物保护》2015年第5期32-38,共7页Plant Protection

基　　金：国家自然科学基金项目(31260179);国家林业局林业公益性行业科研专项(201004067)

摘　　要：超氧化物歧化酶（superoxide dismutase,SOD）是一种广泛存在于生物体各组织,在机体抗氧化过程中起着重要作用的抗氧化酶。采用 RACE 技术,克隆获得了云南切梢小蠹超氧化物歧化酶基因。该基因 cDNA 序列全长768 bp,开放阅读框为462 bp,编码153个氨基酸。预测蛋白质分子量为15.8 ku,等电点为5.68。同源性比对分析发现,云南切梢小蠹 SOD 与中欧山松大小蠹 SOD 在氨基酸水平上相似性高达93%,与赤拟谷盗、点蜂缘蝽、侵扰锥猎蝽、丽蝇蛹集金小蜂、佛罗里达弓背蚁、柑橘凤蝶、棉铃虫、黑果蝇、家蝇的相似性也都在67%～71%之间。其推导的氨基酸序列中含有2个 Cu/Zn-SOD 的特异序列（GFHIHEFGDNT4252和 GNAGGRLACAVI136147）,Cu 原子分别与 His44、His46、His61和 His118配位,Zn 原子分别与 His61、His69、His78和 Asp81配位,半胱氨酸 Cys55和Cys144基之间形成 Cu/Zn-SOD 链内二硫键。系统进化树分析表明,克隆获得的云南切梢小蠹 SOD 隶属于铜锌SOD 家族（icCu/Zn-SOD）。实时荧光定量 PCR 表明,icCu/Zn-SOD 基因在云南切梢小蠹各发育阶段和不同部位均有表达,但表达量各不相同。Superoxide dismutase （SOD）is a kind of antioxidant enzymes,existing widely in biological groups.It plays a key role in insects’antioxidant protection system.A cDNA encoding Cu/Zn-SOD was first isolated from Tomicus yunnanensis using rapid amplification of cDNA ends （RACE）strategy.This gene was 768 bp in full-length with an open reading frame （ORF）of 41 1 bp,which encoded 1 53 amino acid residues.Its predicted molec-ular weight and isoelectric point were 1 5.8 ku and 5.68,respectively.The deduced amino acid sequence of T .yunnanensis SOD showed the highest similarity （93%）with that of Dendroctonus ponderosae ,and had 67%-71% similarity with other insects,including Tribolium castaneum ,Riptortus pedestris ,Triatoma infestans ,Naso-nia vitripennis ,Camponotus floridanus ,Papilio xuthus ,Helicoverpa armigera ,Drosophila virilis and Musca dom-estica .Two typical structure and functional domains of Cu/Zn-SOD （GFHIHEFGDNT42 5 2 and GNAGGRLA-CAVI1 3 6 1 47 ）were located in the deduced amino acid sequence.Several highly conserved motifs including Cu,Zn binding sites H（44）,H（46）,H（61）,H（1 18）for Cu binding,and H（61）,H（69）,H（78）,D（81）for Zn binding, and Cys55 and Cys144 for the only intrachain disulfide bond were also identified in T .yunnanensis Cu/Zn-SOD. Phylogenetic tree indicated that T .yunnanensis Cu/Zn-SOD belonged to icCu/Zn-SOD family.The real-time quantitative PCR showed that icCu/Zn-SOD gene expressed at different developmental stages and in different tis-sues of T .yunnanensis ,but the expression profiles were different.

关 键 词：云南切梢小蠹 超氧化物歧化酶 基因克隆 序列分析 

分 类 号：S763.3[农业科学—森林保护学]