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

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（EcACO），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 EcACO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 EcACO的全长序列，用DNAman以及GeneDOC等软件进行序列分析。用qRT-PCR方法分析抗性和敏感性稗草间的EcACO表达水平差异。最后分别将抗性和敏感性稗草EcACO的开放阅读框（ORF）序列连接至原核表达载体pMAL-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EcACO蛋白的表达情况。采用MBP吸附柱分离纯化EcACO蛋白后，通过测定乙烯释放量，测定抗性和敏感稗草EcACO蛋白间的活性差异。【结果】克隆得到抗性稗草和敏感稗草EcACO，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 kD和5.4。序列比对表明，抗性稗草EcACO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EcACO相比，抗性稗草EcACO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。qPCR分析显示，EcACO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：EcACO融合蛋白单位时间内产生的乙烯释放量是抗性稗草MBP：EcACO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EcACO，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 (EcACO), analyze its expression and test its enzyme activity, and to unravel the quinclorac-resistant mechanism ofE. crus-galli to quinclorac.[Method]The partial sequence ofEcACO obtained fromE. crus-galli transcriptome pyrosequencing was used to design primers for cloning EcACOfrom quinclorac-resistant and susceptibleE. crus-galli. EcACO was then cloned and sequenced. The nucleotide and putative amino acid sequence analysis were compared using DNAman and GenDoc softwares. The transcript levels ofEcACO between resistant and susceptible biotype E. crus-galli were determined by real-time quantitative PCR (qRT-PCR) withβ-actin gene as the reference. Finally, the open reading frame (ORF) sequences ofEcACO from resistant and susceptible biotypes E. crus-galli were inserted into the expression vector pMAL-c5x, respectively. After the recombinant plasmids were transformed intoEscherichia colistrainBL21, 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::EcACO fusion protein.[Result]EcACO was isolated fromE. crus-galli with quinclorac-resistant and susceptible biotypes ofE. crus-galli. The ORF ofEcACO was 936 bp, encoding 311 amino acids, with pI 5.4 and Mw 35 kD. The deduced amino acid sequences shared high identity with other ACO sequences fromSetaria italica (93%), Zea mays(92%) andSorghum bicolor (91%). Compared with EcACO from the susceptible biotype, five site mutations of EcACO were found in the resistant biotype, of which three site mutations were located in the putative conserved domain. Furthermore, qRT-PCR results showed that there was no significant difference in expression level ofEcACObetween resistant and susceptible biotype. Using the prokaryotic expression system and the measurement of MBP::EcACO activity, the released amount of ethylene in the MBP::EcACO from susceptible biotype was 2.15 folds higher than that from resistant biotype.[Conclusion]EcACO was identified from quinclorac-resistant and susceptibleE. crus-galli. Compared with the susceptible biotype, theEcACOfrom the resistant one had five amino acid mutations, of which three site mutations were in the conserved domain. This might probably contribute to the reduction of released amount of ethylene and result in quinclorac resistance ofE. crus-galli.