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二氯喹啉酸诱导的稗草转录因子EcMYB1基因的克隆与表达
李岗,王强,蔡磊明,吴声敢,赵学平,吴长兴
浙江省农业科学院
Cloning and Expression of Quinclorac-induced Transcriptal Factor EcMYB1 in Echinochloa crus-galli
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摘要 稗草(Echinochloa crus-galli)是稻田恶性杂草,二氯喹啉酸(quinclorac)是防治稗草的特效药,但长期使用出现了抗药性稗草。为揭示稗草对二氯喹啉酸的抗药性机制,本研究采用cDNA末端快速扩增(rapid amplification of cDNA ends, RACE)技术从抗二氯喹啉酸的稗草中克隆了一个MYB转录因子基因,命名为EcMYB1 (GenBank登录号: JX518599), 其cDNA全长为1 844 bp,开放阅读框为 1 527 bp,编码508个氨基酸,理论分子量为55.4 kD,等电点为8.6。EcMYB1蛋白含有3个保守的MYB结构域,属于植物中不常见的R1R2R3-MYB类型。基于蛋白质序列的系统进化分析表明,EcMYB1与禾本科植物玉米(Zea mays)、高梁(Sorghum bicolor)的同源序列有较近的亲缘关系。采用整株量效实验,测定了二氯喹啉酸敏感和抗性的两种稗草近等基因系的抗药性水平,敏感生物型的GR50 (growth reduction by 50%,抑制生长50%时的浓度)值为158.3 g·AI(active ingredient)/hm2,抗性生物型的GR50值为858.0 g·AI/hm2,抗性指数为5.4。以稗草EcActin (GenBank登录号: HQ395760)作为内参的Real-time PCR实验,测定EcMYB1在抗、感稗草生物型的苗期叶、根和成株期根、茎、叶和种子中的表达,结果表明,EcMYB1在抗、感生物型的各组织中均有表达,相对表达量分别是26.22~52.50和6.85~34.62,其中在抗药性稗草苗期叶中的相对表达量最高为52.50,而在敏感稗草成株叶中的相对表达量最低为6.85,抗性稗草比敏感稗草高1.22~4.78倍。受二氯喹啉酸诱导后,其表达量在抗、感稗草中均升高,相对表达量分别为189.66~395.45和61.91~144.29,其中在抗性稗草苗期根中的相对表达量最高为395.45,而在敏感稗草的成株茎中的相对表达量最低为61.91,抗性稗草比敏感稗草高2.46~4.06倍。 抗、感稗草生物型的EcMYB1 mRNA水平差异表达暗示它可能参与了稗草对二氯喹啉酸的抗药性。这为后续该基因在稗草抗药性的功能研究提供了基础资料。
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李岗
王强
蔡磊明
吴声敢
赵学平
吴长兴
关键词 抗药性稗草二氯喹啉酸诱导基因EcMYB1基因克隆与表达    
Abstract:Echinochloa crus-galli is one of the most noxious grass weeds in Chinese rice paddies, which has evolved resistance to quinclorac after continuous application since 1990. But little is known about its resistance mechanism. In order to explore the mechanism involved, we described isolation and mRNA expression of a quinclorac-responsive gene MYB in E. crus-galli. Transcriptal factor MYBs played important roles in regulating plant growth and development and withstanding environmental stresses. But, little is known about expression profile of MYBs mRNA in herbicide-resistant barnyardgrass (E. crus-galli) under quinclorac stress. A homologue sequence of MYB was obtained from quinclora-resistant-identified E.crus-galli using RACE(rapid amplification of cDNA ends). The gene, designated EcMYB1 (GenBank accession number: JX518599) with whole length 1 844 bp, had a 1 527 bp open reading frame predicted to encode a protein of 508 amino acids with molecular weight 55.4 kD and isoelectric point 8.6. The predicted EcMYB1 contained three conserved MYB domains that make the EcMYB1 belong to the R1R2R3 gene family which was not common in plants. Based on phylogenic analysis of protein sequence between EcMYB1 and others MYB, EcMYB1 had the closest relationship with the MYBs of gramineous plants, such as Sorghum bicolor, Zea mays. The result of whole-plant dose-reponse experiments demonstrated that the near iso-genicline material used in this study was susceptible or resistant to quinclorac. The GR50 (for 50% growth reduction) was 158.3 g·AI/hm2 for quinclorac-susceptible (S) biotypes or 858.0 g·AI/hm2 for quinclorac-resistant (R) biotypes. Ratios (R/S) of GR50 value was 5.4. EcMYB1 transcriptional expression was monitored in seedlings (leaves and roots) and adult plants (leaves, roots, stems and seeds) of R and S biotypes of E.crus-galli through Real-time PCR experiment with EcActin (GenBank accession number: HQ395760) as the reference sequence. The relative expression of EcMYB1 in transcriptional level was 26.22~52.50 in R biotypes and 6.85~34.62 in S biotypes, such as 52.50 in leaves of seedling of R biotypes and 6.85 in leaves of S adult biotypes, and 1.22~4.78 times greater in the R plants than that in the S plants. After exposure to quinclorac, its increased relative expression was 189.66~395.45 in R biotypes and 61.91~144.29 in S biotypes,such as 395.45 in roots of seedling of R biotypes and 61.91 in stems of S adult biotypes, and 2.46~4.06 times greater in the R plants than that in the S plants. Its expression was higher in all tissues tested of R biotypes than in that of S plants before or after quinclorac treatment. The difference expression of EcMYB1 mRNA in R and S biotypes suggested that EcMYB1 maybe involve in resistance to quinclorac in E. crus-galli. The results of this study provide basic information for the further research of function of the EcMYB1 in resistance to quinclorac in barnyardgrass.
Key wordsQuinclorac-resistant barnyardgrass    Quinclorac-induced gene    Cloning and expression of EcMYB1
收稿日期: 2013-03-29     
通讯作者: 吴长兴   
引用本文:   
李岗,王强,蔡磊明,吴声敢,赵学平,吴长兴. 二氯喹啉酸诱导的稗草转录因子EcMYB1基因的克隆与表达[J]. , 2013, 21(10): 1166-1175.
链接本文:  
http://journal05.magtech.org.cn/Jwk_ny/CN/     或     http://journal05.magtech.org.cn/Jwk_ny/CN/Y2013/V21/I10/1166
 
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