抗性稗草1-氨基环丙烷-1-羧酸氧化酶基因的克隆与表达分析  

Identification and Expression Analysis of 1-Aminocyclopropane-1-Carboxylate Oxidase Gene from Quinclorac-Resistant Barnyardgrass(Echinochloa crus-galli)

作　　者：董明超[1,2] 杨霞[2] 张自常[2] 李永丰[2] 管荣展[1] 

机构地区：[1]南京农业大学农学院,南京210095 [2]江苏省农业科学院植物保护研究所,南京210014

出　　处：《中国农业科学》2015年第20期4077-4085,共9页Scientia Agricultura Sinica

基　　金：国家自然科学基金(31371953);国家公益性行业(农业)科研专项(201303031);江苏省农业科技自主创新资金(SCX(13)3063)

摘　　要：【目的】克隆稗草(Echinochloa crus-galli)乙烯生物合成途径关键酶1-氨基环丙烷-1-羧酸氧化酶基因(Ec ACO),对其进行表达分析和酶活性测定,以探究稗草抗二氯喹啉酸的机理。【方法】根据转录组测序所得Ec ACO部分序列设计引物,分别从二氯喹啉酸的抗性和敏感型稗草中克隆Ec ACO的全长序列,用DNAman以及Gene DOC等软件进行序列分析。用q RT-PCR方法分析抗性和敏感性稗草间的Ec ACO表达水平差异。最后分别将抗性和敏感性稗草Ec ACO的开放阅读框(ORF)序列连接至原核表达载体p MAL-c5x中,并转化至大肠杆菌菌株BL21,经终浓度为0.4 mmol·L-1的IPTG于18℃诱导16 h后,检测Ec ACO蛋白的表达情况。采用MBP吸附柱分离纯化Ec ACO蛋白后,通过测定乙烯释放量,测定抗性和敏感稗草Ec ACO蛋白间的活性差异。【结果】克隆得到抗性稗草和敏感稗草Ec ACO,其编码区序列长度为936 bp,预测蛋白含311个氨基酸残基,蛋白分子量大小和理论等电点分别为35 k D和5.4。序列比对表明,抗性稗草Ec ACO氨基酸序列与粟(Setaria italica)、玉米(Zea mays)、高粱(Sorghum bicolor)同源性分别为93%、92%和91%;与敏感性稗草Ec ACO相比,抗性稗草Ec ACO的氨基酸序列存在5个突变位点,其中有3个突变位点位于保守功能域上。q PCR分析显示,Ec ACO在抗性和敏感性稗草中并无明显的表达水平差异。原核蛋白表达和酶活性测定结果表明,敏感型稗草MBP::Ec ACO融合蛋白单位时间内产生的乙烯释放量是抗性稗草MBP::Ec ACO融合蛋白的2.15倍,因而该基因可能解释了稗草的抗药性机理。【结论】从抗二氯喹啉酸的稗草中克隆了Ec ACO,发现了与抗性相关的5个氨基酸突变位点,其中的3个位点突变位于保守结构域,这可能是引起乙烯释放速率降低以及稗草产生二氯喹啉酸抗性的原因。【Objective】The objective of this study is to clone barnyardgrass（Echinochloa crus-galli） 1-aminocyclopropane-1-carboxylate oxidase gene（Ec ACO）, analyze its expression and test its enzyme activity, and to unravel the quinclorac-resistant mechanism of E. crus-galli to quinclorac.【Method】The partial sequence of Ec ACO obtained from E. crus-galli transcriptome pyrosequencing was used to design primers for cloning Ec ACO from quinclorac-resistant and susceptible E. crus-galli. Ec ACO was then cloned and sequenced. The nucleotide and putative amino acid sequence analysis were compared using DNAman and Gen Doc softwares. The transcript levels of Ec ACO between resistant and susceptible biotype E. crus-galli were determined by real-time quantitative PCR（q RT-PCR） with β-actin gene as the reference. Finally, the open reading frame（ORF） sequences of Ec ACO from resistant and susceptible biotypes E. crus-galli were inserted into the expression vector p MAL-c5 x, respectively. After the recombinant plasmids were transformed into Escherichia coli strain BL21, the fusion proteins were expressed by the induction with 0.4 mmol·L^-1 IPTG for 16 h at 18℃. The soluble proteins were purified with MBP column for the measurement of ethylene released from MBP：：Ec ACO fusion protein. 【Result】Ec ACO was isolated from E. crus-galli with quinclorac-resistant and susceptible biotypes of E. crus-galli. The ORF of Ec ACO was 936 bp, encoding 311 amino acids, with p I 5.4 and Mw 35 k D. The deduced amino acid sequences shared high identity with other ACO sequences from Setaria italica（93%）, Zea mays（92%） and Sorghum bicolor（91%）. Compared with Ec ACO from the susceptible biotype, five site mutations of Ec ACO were found in the resistant biotype, of which three site mutations were located in the putative conserved domain. Furthermore, q RT-PCR results showed that there was no significant difference in expression level of Ec ACO between resistant and susceptible biotype. Using the prokaryotic expr

关 键 词：稗草 1-氨基环丙烷-1-羧酸氧化酶 基因克隆 点突变 表达 

分 类 号：S451[农业科学—植物保护] Q943.2[生物学—植物学]