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1、PCR 联用 HPLC 快速筛选野生冠菌素生产菌 王春燕 胡堂路 姜峰 谭伟明 李召虎 段留生 中国农业大学农学院/植物生理生化重点实验室/教育部植物生长调节剂工程研究中心 中国农业大学园艺学院 摘 要： 冠菌素 (coronatine, COR) 是由丁香假单胞菌 (Pseudomonas syringae) 某些致病变种产生的一种新型植物生长调节剂。建立冠菌素生产野生菌快速检测方法对于丰富其菌种资源、推进冠菌素的研究和应用具有重要意义。本研究根据冠菌素合成相关功能基因 cor S、cor R 设计 2 对特异性引物, 利用 PCR 技术及高效液相色谱 (high-performance l
2、iquid chromatography, HPLC) 法从 32 株野生菌中筛选冠菌素产素菌。结果表明, 设计的特异性引物在以冠菌素产生菌模式菌为模板时能特异性扩增出目的片段, 分别为 850bp (cor S) 和 450 bp (cor R) , 以非产素参考菌株为模板时不能扩出此片段, 具有较好的特异性;用该 PCR 方法成功从 32 株野生菌中筛选获得 3 株冠菌素产素候选菌, 经 HPLC 复筛后, 其中 2 株发酵产物检出冠菌素, 其产量分别为 6.5 和 2.1 mg/L。与传统鉴定方法相比, 特异 PCR 与 HPLC 联用的方法在野生冠菌素产素菌的检测筛选中更快捷、更高效,
3、 可用于大批量野生菌的快速筛选, 发现产率更高的菌种资源并进行进一步的工程菌构建, 为生物调节剂冠菌素的工业化生产和规模化的农业应用提供支持。关键词： 冠菌素生产菌; 特异 PCR; 高效液相色谱 (HPLC) ; 快速筛选; 作者简介：段留生, 收稿日期：2017-04-29基金：国家高技术研究发展计划 (No.2011AA10A206) Rapid Screening of the Wild Coronatine-producing Stains Based on PCR and HPLCWANG Chun-Yan HU Tang-Lu JIANG Feng TAN Wei-Ming LI
4、 Zhao-Hu DUAN Liu-Sheng College of Agronomy, China Agricultural University, State Key Laboratory of Plant Physiology and Biochemistry/Engineering Research Center of Plant Growth Regulators, Ministry of Education; College of Horticulture, China Agricultural University; Abstract： Coronatine (COR) is a
5、 chlorosis-inducing non-host-specific phytotoxin that is produced by several pathovars of Pseudomonas syringae including P.syringae pv.tomato, P.syringae pv.glycinea, P.syringae pv.maculicola and P.syringae pv.atropurpurea.COR is a structural and functional mimic of jasmonates but it is more active
6、than jasmonates.COR is involved in a wide array of effects on plant development and defence responses including inhibition of root elongation, hypertrophy, senescence, accumulation of defense-related protease inhibitors, secondary metabolite production, ethylene emission and resistance to abiotic st
7、resses, and can function as a novel plant growth regulator.The objective of this study is to establish a rapid method for screening coronatine producing strains, which plays an important role in enriching its strain resources and promoting the research and application of COR.According to the key gen
8、es in COR biosynthesis gene clusters, cor S and cor R, two sets of PCR primers were designed.Polymerase chain reaction (PCR) and agarose gel electrophoresis gel electrophoresis were conducted for initial screening of wild COR-producing strains.The yield of COR for wild strains was determined by high
9、 performance liquid chromatography (HPLC) .As a result, the target bands cor S (850 bp) and cor R (450 bp) were amplified when we used the genomic DNA of a typical COR-producing strain P.syringae pv.glycinea MW123 as a template.And it was consistent with the result of colony PCR of P.syringae pv.gly
10、cinea MW123.The PCR method was verified with the typical CORproducing strains and non-COR-producing strains.The results showed that the cor S (850 bp) and cor R (450 bp) bands were amplified only from the typical COR-producing strains, including P.syringae pv.glycinea MW123, P.syringae pv.glycinea M
11、FB1 and P.syringae pv.tomato DC3000.The 2 target bands were not be obtained when using the non-COR-producing strains as templates.This showed that the designed primers had a fairly good specificity for COR-producing strains.The above PCR method was applied to preliminary screening of 32 wild stains
12、isolated from diseased plants in the field.The specific PCR assay displayed that a850 bp (cor S) band could be obtained from the strains numbered 7, 18, 19 and a 450 bp (cor R) band could be obtained only from the strains of 7 and 18.The 2 target bands, cor S (850 bp) and cor R (450 bp) , werent be
13、observed in the PCR products of the rest wild strains.Among 32 wild strains, 3 wild strains (7, 18 and 19) were picked out as potential COR-producing strains.The selected strains were inoculated and cultivated in HSC culture medium for COR fermentation.Subsequent HPLC analysis showed that the strain
14、s of 7 and 18 could synthesize COR and the yields of COR were 6.5 mg/L and 2.1 mg/L, respectively.Two novel producingCOR strains were successfully screened out from 32 wild strains by specific PCR and HPLC.In comparison, the specific PCR is more efficient for COR-producing strains and it is suitable
15、 for screening rapidly CORproducing strains which could help to found higher yield of COR-producing strain and provide support for the industrial production and large-scale agricultural applications of COR.Keyword： Coronatine-producing strains; Specific PCR; High-performance liquid chromatography (H
16、PLC) Rapid screening; Received： 2017-04-29冠菌素 (coronatine, COR) 分子式为 C18H25NO4, 是由冠烷酸 (coronamic acid, CMA) 和一个聚酮结构的冠菌酸 (coronafacic acid, CFA) 以酰胺键连结而成, 化学结构如下 (图 1) (Ichihara et al., 1977) 。冠菌素主要由丁香假单胞菌 (Pseudomonas syringae) 的一些致病变种产生, 包括丁香假单胞菌大豆致病变种 (P.syringae pv.glycinea) 、丁香假单胞菌番茄致病变种 (P.syringae pv.tomato) 、丁香假单胞菌麦叶赫斑致病变种 (P.syringae pv.atropurpurea) 、丁香假单胞菌李死致病变种 (P.syringae pv.morsprunorum) 、丁香假单胞菌斑生致病变种 (P.syringae pv.maculicola) 、丁香假单胞菌猕猴桃致病变种 (
