植物保护
植物保護
식물보호
Plant Protection
2015年
5期
32-38
,共7页
赵琰明%朱家颖%泽桑梓%赵宁%杨斌
趙琰明%硃傢穎%澤桑梓%趙寧%楊斌
조염명%주가영%택상재%조저%양빈
云南切梢小蠹%超氧化物歧化酶%基因克隆%序列分析%实时荧光定量 PCR
雲南切梢小蠹%超氧化物歧化酶%基因剋隆%序列分析%實時熒光定量 PCR
운남절소소두%초양화물기화매%기인극륭%서렬분석%실시형광정량 PCR
Tomicus yunnanensis%superoxide dismutase%gene clone%sequence analysis%real-time quantitative PCR
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。
超氧化物歧化酶(superoxide dismutase,SOD)是一種廣汎存在于生物體各組織,在機體抗氧化過程中起著重要作用的抗氧化酶。採用 RACE 技術,剋隆穫得瞭雲南切梢小蠹超氧化物歧化酶基因。該基因 cDNA 序列全長768 bp,開放閱讀框為462 bp,編碼153箇氨基痠。預測蛋白質分子量為15.8 ku,等電點為5.68。同源性比對分析髮現,雲南切梢小蠹 SOD 與中歐山鬆大小蠹 SOD 在氨基痠水平上相似性高達93%,與赤擬穀盜、點蜂緣蝽、侵擾錐獵蝽、麗蠅蛹集金小蜂、彿囉裏達弓揹蟻、柑橘鳳蝶、棉鈴蟲、黑果蠅、傢蠅的相似性也都在67%~71%之間。其推導的氨基痠序列中含有2箇 Cu/Zn-SOD 的特異序列(GFHIHEFGDNT4252和 GNAGGRLACAVI136147),Cu 原子分彆與 His44、His46、His61和 His118配位,Zn 原子分彆與 His61、His69、His78和 Asp81配位,半胱氨痠 Cys55和Cys144基之間形成 Cu/Zn-SOD 鏈內二硫鍵。繫統進化樹分析錶明,剋隆穫得的雲南切梢小蠹 SOD 隸屬于銅鋅SOD 傢族(icCu/Zn-SOD)。實時熒光定量 PCR 錶明,icCu/Zn-SOD 基因在雲南切梢小蠹各髮育階段和不同部位均有錶達,但錶達量各不相同。
초양화물기화매(superoxide dismutase,SOD)시일충엄범존재우생물체각조직,재궤체항양화과정중기착중요작용적항양화매。채용 RACE 기술,극륭획득료운남절소소두초양화물기화매기인。해기인 cDNA 서렬전장768 bp,개방열독광위462 bp,편마153개안기산。예측단백질분자량위15.8 ku,등전점위5.68。동원성비대분석발현,운남절소소두 SOD 여중구산송대소두 SOD 재안기산수평상상사성고체93%,여적의곡도、점봉연춘、침우추작춘、려승용집금소봉、불라리체궁배의、감귤봉접、면령충、흑과승、가승적상사성야도재67%~71%지간。기추도적안기산서렬중함유2개 Cu/Zn-SOD 적특이서렬(GFHIHEFGDNT4252화 GNAGGRLACAVI136147),Cu 원자분별여 His44、His46、His61화 His118배위,Zn 원자분별여 His61、His69、His78화 Asp81배위,반광안산 Cys55화Cys144기지간형성 Cu/Zn-SOD 련내이류건。계통진화수분석표명,극륭획득적운남절소소두 SOD 대속우동자SOD 가족(icCu/Zn-SOD)。실시형광정량 PCR 표명,icCu/Zn-SOD 기인재운남절소소두각발육계단화불동부위균유표체,단표체량각불상동。
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.
