中国农业科学Issue(20)：4077-4085,9.DOI:10.3864/j.issn.0578-1752.2015.20.009
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)
摘要
Abstract
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.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Key words
Echinochloa crus-galli/1-aminocyclopropane-1-carboxylate oxidase/gene cloning/site mutation/expression引用本文复制引用
董明超,杨霞,张自常,李永丰,管荣展..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[J].中国农业科学,2015,(20):4077-4085,9.基金项目
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 （13）
