桑氧化固醇结合相关蛋白的基因克隆及序列与表达分析  

Cloning,Sequencing and Expression Analyses of Mulberry ORP Gene

作　　者：师彗云[1,2] 王恒[3] 赵卫国[3] 胡兴明[1] 尚亚君 刘利[3] 邓文[1] 叶楚华[1] 

机构地区：[1]湖北省农业科学院经济作物研究所,武汉430064 [2]武汉大学生命科学学院,武汉430072 [3]江苏科技大学生物技术学院,江苏镇江212018

出　　处：《湖北农业科学》2014年第24期6030-6037,共8页Hubei Agricultural Sciences

基　　金：国家现代农业产业技术体系建设专项(CARS-22)

摘　　要：氧化固醇结合蛋白家族是一类存在于真核生物中的能结合氧化固醇的保守蛋白,主要作用是参与细胞中的脂类代谢、囊泡运输及信号转导等方面。试验利用RT-PCR和RACE技术从桑树(Morus L.)鲁桑系品种育71-1(M.multicaulis Perr.cv.Yu 71-1)中克隆出一个氧化固醇结合蛋白相关蛋白基因,该c DNA全长1 974 bp,含有一个完整的全长1 371 bp的读码框(ORF),内有220 bp的5’非翻译区(5’UTR)和383 bp的3’非翻译区(3’UTR),3’端以Poly(A)15结束。其开放读码框共编码456个氨基酸,分子量为51.97 k D,等电点为5.143。利用Pfam和PROSITE在线分别进行了蛋白质的结构及功能位点和motif预测,结果显示,该蛋白为氧化固醇结合蛋白相关蛋白家族的成员,具有氧化固醇结合蛋白相关结构域,且含有该家族蛋白特有的极其保守的指纹基序"EQVSHHPP"。多重序列比对结果表明,该蛋白高度保守,在系统进化树中桑树与野草莓(Fragaria vesca subsp.vesca L.)、碧桃(Prunus persica Batsch.var.duplex Rehd.)等的亲缘关系较近。Real-time PCR分析表明,该基因在花、幼果、成熟果的表达量比根、茎和芽中的表达量要高。在高盐胁迫下,桑树Ma-acyin-ORP基因的表达量先升高、后下降,初步推断该基因可能参与某种压力胁迫的生理反应。Oxysterol-binding protein （OSBP） and its homologues constitute a protein family in many eukaryotes, which is highly conservative, with a high-affinity receptor protein for oxysterols （oxygenated cholesterols）, involving in cellular lipid metabolism, vesicle transport and signal transduction. A full-lenth eDNA sequence encoding ORP （Oxysterol binding protein- related proteins） was cloned with RT-PCR and rapid amplification of eDNA ends from the mulberry （Morus L.） variety M. multieaulis Pert. cv. Yu 71-1. The sequence was 1 974 bp and contained a 220 bp 5＂-UTR and a 383 bp 3＂-UTR, had an ORF of 1 371 bp encoding for 456 amino acids with predicted molecular weight of 51.97 kD and theoretical pI of 5.143. On- line forecasting protein structural and functional sites and motif with PROSITE and Pfam showed that the protein was be- longed to ORP family, having only ORD domain （OSBP-related ligand-binding domain） and containing the highly conserved fingerprint motif ＂EQVSHHPP＂. Multiple sequence alignment showed that the protein was highly conserved. Phylogenetic tree analysis showed that the muberry had a closer genetic relationship with Fragaria vesca subsp, vesca L. and Prunus persica Batsch. var. duplex Rehd. Real-time PCR analysis showed that the expression level was higher in flower, young fruit, and ripe fruit than that in roots, stems and buds. Under salt stress, the expression of gene ORP increased first and then began to decline. It is indicated that the gene may be involved in some physiological responses to stress.

关 键 词：桑树(Morus L) 氧化固醇结合蛋白相关蛋白 基因克隆 序列特征 基因表达 

分 类 号：S888.2[农业科学—特种经济动物饲养] S813.3[农业科学—畜牧兽医]