中國農業科學 ?? 2015, Vol. 48 ?? Issue (20): 4077-4085.doi: 10.3864/j.issn.0578-1752.2015.20.009

? 植物保護 ? 上一篇    下一篇

抗性稗草1-氨基環丙烷-1-羧酸氧化酶基因的克隆與表達分析

 董明超1,2,楊霞2,張自常2,李永豐2,管榮展1
  

  1. 1南京農業大學農學院,南京 210095
    2江蘇省農業科學院植物保護研究所,南京 210014
  • 收稿日期:2015-04-20 出版日期:2015-10-20 發布日期:2015-10-20
  • 通訊作者: 管榮展,E-mail:[email protected]。李永豐,E-mail:[email protected] E-mail:[email protected]; [email protected]
  • 作者簡介:董明超,Tel:15093338059;E-mail:[email protected]
  • 基金資助:
    國家自然科學基金(31371953)、國家公益性行業(農業)科研專項(201303031)、江蘇省農業科技自主創新資金(SCX(13)3063)

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

DONG Ming-chao1,2, YANG Xia2, ZHANG Zi-chang2, LI Yong-feng2, GUAN Rong-zhan1   

  1. 1College of Agriculture, Nanjing Agricultural University, Nanjing 210095
    2Institute of Plant Protection, Jiangsu Academy of Agricultural Sciences, Nanjing 210014
  • Received:2015-04-20 Online:2015-10-20 Published:2015-10-20

摘要: 【目的】克隆稗草(Echinochloa crus-galli)乙烯生物合成途徑關鍵酶1-氨基環丙烷-1-羧酸氧化酶基因(EcACO),對其進行表達分析和酶活性測定,以探究稗草抗二氯喹啉酸的機理。【方法】根據轉錄組測序所得EcACO部分序列設計引物,分別從二氯喹啉酸的抗性和敏感型稗草中克隆EcACO的全長序列,用DNAman以及GeneDOC等軟件進行序列分析。用qRT-PCR方法分析抗性和敏感性稗草間的EcACO表達水平差異。最后分別將抗性和敏感性稗草EcACO的開放閱讀框(ORF)序列連接至原核表達載體pMAL-c5x中,并轉化至大腸桿菌菌株BL21,經終濃度為0.4 mmol·L-1的IPTG于18℃誘導16 h后,檢測EcACO蛋白的表達情況。采用MBP吸附柱分離純化EcACO蛋白后,通過測定乙烯釋放量,測定抗性和敏感稗草EcACO蛋白間的活性差異。【結果】克隆得到抗性稗草和敏感稗草EcACO,其編碼區序列長度為936 bp,預測蛋白含311個氨基酸殘基,蛋白分子量大小和理論等電點分別為35 kD和5.4。序列比對表明,抗性稗草EcACO氨基酸序列與粟(Setaria italica)、玉米(Zea mays)、高粱(Sorghum bicolor)同源性分別為93%、92%和91%;與敏感性稗草EcACO相比,抗性稗草EcACO的氨基酸序列存在5個突變位點,其中有3個突變位點位于保守功能域上。qPCR分析顯示,EcACO在抗性和敏感性稗草中并無明顯的表達水平差異。原核蛋白表達和酶活性測定結果表明,敏感型稗草MBP::EcACO融合蛋白單位時間內產生的乙烯釋放量是抗性稗草MBP::EcACO融合蛋白的2.15倍,因而該基因可能解釋了稗草的抗藥性機理。【結論】從抗二氯喹啉酸的稗草中克隆了EcACO,發現了與抗性相關的5個氨基酸突變位點,其中的3個位點突變位于保守結構域,這可能是引起乙烯釋放速率降低以及稗草產生二氯喹啉酸抗性的原因。

關鍵詞: 稗草, 1-氨基環丙烷-1-羧酸氧化酶, 基因克隆, 點突變, 表達

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 of E. crus-galli to quinclorac.【Method】The partial sequence of EcACO obtained from E. crus-galli transcriptome pyrosequencing was used to design primers for cloning EcACO from quinclorac-resistant and susceptible E. 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 of EcACO between resistant and susceptible biotype E. crus-galli were determined by real-time quantitative PCR (qRT-PCR) with β-actin gene as the reference. Finally, the open reading frame (ORF) sequences of EcACO from resistant and susceptible biotypes E. crus-galli were inserted into the expression vector pMAL-c5x, 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::EcACO fusion protein. 【Result】EcACO was isolated from E. crus-galli with quinclorac-resistant and susceptible biotypes of E. crus-galli. The ORF of EcACO 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 from Setaria italica (93%), Zea mays (92%) and Sorghum 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 of EcACO between 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 susceptible E. crus-galli. Compared with the susceptible biotype, the EcACO 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.

Key words: Echinochloa crus-galli, 1-aminocyclopropane-1-carboxylate oxidase, gene cloning, site mutation, expression

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