INT J MOL SCI | 强胜团队揭示稗草突变体对二氯喹啉酸抗性机制

稗草很少有除草剂抗性报道，且目前为止还没有一例是关于二氯喹啉酸的抗性的报道。二氯喹啉酸(Quinclorac)是合成生长素类除草剂，被广泛用于稻田除草。中国稻田长期施用二氯喹啉酸提升了稗草对该除草剂的抗性。基于培养皿和小盆生测，我们揭示了来自国内3个省份的4个生物型的抗性排序：JS01-R>AH01-R>JS02-R>JX01-R。在这些生物型中，乙烯产量与抗性水平呈负相关，与生长抑制效果呈正相关。通过测定这些生物型的乙烯合成反应相关通路，发现1-氨基环丙烷-1-羧酸(ACC)含量、ACC合酶和氧化酶活性下调，以及对比敏感型来看，ACS与ACO基因具有更低的诱导上调水平，表明二氯喹啉酸抗性与乙烯的生物合成存在正相关性。乙烯生物合成反应通路中产生的氰化物主要被β-氰基丙氨酸合酶(β-CAS)降解。对比敏感型植株，抗性生物型具有更高的β-CAS活性。另外，在抗性个体中的EcCAS基因中还发现了部分核苷酸位点突变，导致了内部3个氨基酸残基替换(Asn-105-Lys, Gln-195-Glu, Gly-298-Val)，引起酶结构发生改变，增加了活性位点与辅因子(Cofactor)的结合残基(Binding Residues)数量，降低了结合自由能(BindingFree Energy)；因此，它的酶活性在抗性生物型中相对较高。而且，这些突变还增加了酶的结构稳定性。鉴于乙烯生物合成抑制、氰化物降解与抗性水平的正相关性，可以总结出乙烯反应通路的改变或因ACS和ACO基因的相对表达量降低导致ACC合酶、ACC氧化酶的酶活性变化，以及β-CAS活性增强，包括EcCAS基因相对表达量上调，可以认为是导致稗草产生二氯喹啉酸抗性的潜在机制。

作者：
MuhammadZiaUl Haq 农业系，费萨拉巴德农业大学 (巴基斯坦）
ZhengZhang杂草研究实验室，生命科学学院，南京农业大学
JiajiaWei杂草研究实验室，生命科学学院，南京农业大学
Qiangsheng*杂草研究实验室，生命科学学院，南京农业大学
发布期刊：INTERNATIONALJOURNAL OF MOLECULAR SCIENCES（IF:4.556）
中科院2区 JCRQ1
DOI：10.3390/ijms21051573
Abstract
Echinochloa crus-galli var. mitis has rarely been reported for herbicide resistance, and no case of quinclorac resistance has been reported so far. Synthetic auxin-type herbicide quinclorac is used extensively to control rice weeds worldwide. A long history of using quinclorac in Chinese rice fields escalated the resistance in E. crus-galli var. mitis against this herbicide. Bioassays in Petri plates and pots exhibited four biotypes that evolved into resistance to quinclorac ranking as JS01-R > AH01-R > JS02-R > JX01-R from three provinces of China. Ethylene production in these biotypes was negatively correlated with resistance level and positively correlated with growth inhibition. Determination of the related ethylene response pathway exhibited resistance in biotypes that recorded a decline in 1-aminocyclopropane-1-carboxylic acid (ACC) content, ACC synthase oxidase activities, and less inducible ACS and ACO genes expressions than the susceptible biotype, suggesting that there was a positive correlation between quinclorac resistance and ethylene biosynthesis inhibition. Cyanides produced during the ethylene biosynthesis pathway mainly degraded by the activity of β-cyanoalanine synthase (β-CAS). Resistant biotypes exhibited higher β-CAS activity than the susceptible ones. Nucleotide changes were found in the EcCAS gene of resistant biotypes as compared to sensitive ones that caused three amino acid substitutions (Asn-105-Lys, Gln-195-Glu, and Gly-298-Val), resulting in alteration of enzyme structure, increased binding residues in the active site with its cofactor, and decreased binding free energy; hence, its activity was higher in resistant biotypes. Moreover, these mutations increased the structural stability of the enzyme. In view of the positive correlation between ethylene biosynthesis inhibition and cyanide degradation with resistance level, it is concluded that the alteration in ethylene response pathway or at least variation in ACC synthase and ACC oxidase enzyme activities-due to less relative expression of ACS and ACO genes and enhanced β-CAS activity, as well as mutation and increased relative expression of EcCAS gene-can be considered as a probable mechanism of quinclorac resistance in E. crus-galli var. mitis.
Keywords: ACC synthase and ACC oxidase; ACS and ACO genes; Echinochloa crus-galli var. mitis; ethylene biosynthesis inhibition; quinclorac resistance; β-cyanoalanine synthase.

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