Identification and Expression Analysis of 1-Aminocyclopropane-1-Carboxylate Oxidase Gene from Quinclorac-Resistant Barnyardgrass(Echinochloa crus-galli)
DONG Ming-chao;YANG Xia;ZHANG Zi-chang;LI Yong-feng;GUAN Rong-zhan;College of Agriculture, Nanjing Agricultural University;Institute of Plant Protection, Jiangsu Academy of Agricultural Sciences;
【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 expression system and the measurement of MBP::Ec ACO activity, the released amount of ethylene in the MBP::Ec ACO from susceptible biotype was 2.15 folds higher than that from resistant biotype. 【 Conclusion 】 Ec ACO was identified from quinclorac-resistant and susceptible E. crus-galli. Compared with the susceptible biotype, the Ec ACO from 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 of E. crus-galli.
【Fund】： 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)
【CateGory Index】： S451;Q943.2
【CateGory Index】： S451;Q943.2