转细胞凋亡抑制基因OpIAP和p35增强小麦对纹枯病的抗性

Expression of Apoptosis Inhibitor Genes OpIAP and p35 Enhances Resistance to Rhizoctonia cerealis in Transgenic Wheat

OpIAP（Orgyiapseudotsugatainhibitorofapoptosisprotein）是黄杉毒蛾核型多角体病毒中编码细胞凋亡抑制蛋白（inhibitorofapoptosisprotein,IAP）的基因,p35基因编码苜蓿斜纹夜蛾核型多角体病毒中具细胞凋亡抑制作用的35kDa蛋白。本研究人工合成了OpIAP和p35基因,构建了同时含有OpIAP和p35表达盒的转双价基因载体pUbi：p35-RSS1P：Myc-OpIAP,两基因分别由玉米泛素基因启动子（Ubiquitin,Ubi）和水稻蔗糖合酶-1启动子（sucrosesynthase-1promoter,RSS1P）驱动。通过基因枪介导法将该载体导入小麦品种扬麦16,获得双价转基因小麦。对T0-T2代植株,利用PCR、RT-PCR、qRT-PCR及Westernblot分析,确认导入的外源OpIAP和p35基因能够在4个转双价基因小麦株系中遗传并表达。用来源不同、致病力不同的禾谷丝核菌强致病株型R0301和WK207对转双价基因小麦的T1、T2代植株分别进行纹枯病抗性鉴定,结果表明,与受体扬麦16相比,双价转基因小麦的T1、T2代植株对纹枯病的抗性明显提高,说明OpIAP和p35基因的表达可以增强转基因小麦对来源不同、致病力不同的禾谷丝核菌的抗性。

OpIAP （Orgyia pseudotsugata inhibitor of apoptosis protein） gene encodes an inhibitor of apoptosis protein （IAP） which comes fromOrgyia pseudotsugatamulticapsid polyhedrosis virus.p35geneisolated from anAutographa californicanu-cleo-polyhedron virus encodes a 35 kDa apoptosis protein inhibitor. The expression of both genes displays enhanced resistance in tobacco, maize and cotton. In this study, the full-length coding sequences ofOpIAPand p35 genes were synthesized, respectively. The transformation vector pUbi：p35-RSS1P：Myc-OpIAP containing two gene expression cassettes was constructed. In the expres-sion vector pUbi：p35-RSS1P：Myc-OpIAP, theOpIAP gene was driven by the rice sucrose synthase-1 promoter and thep35 gene was driven by the maizeubiquitin promoter. Embryo calli of Yangmai 16 were bombarded by the gold particle containing pUbi：p35-RSS1P：Myc-OpIAP vector DNA. Transgenic wheat plants in T0–T2 generations were subjected to PCR, RT-PCR, qRT-PCR, and Western blot analyses. The results indicated that the introducedOpIAPand p35genescould beinherited and ex-pressed in four transgenic wheat lines.Rhizoctonia cerealisisolate R0301 or WK207 was used to inoculate T1 andT2 plants for sharp eyespot severity assessments. The results showed that the transgenic wheat plants expressingOpIAPand p35 displayed sig-nificantly enhanced resistance to sharp eyespotcompared with non-transgenic wheat Yangmai 16. Thus, the introducedOpIAPand p35genes could be used in improving wheat resistance to wheat sharp eyespot caused by differentRhizoctonia cerealisisolates.