关键词: 甜瓜细菌性叶斑病, 锁式探针, 斑点杂交, 种子

Abstract: 【Objective】 Bacterial spot of melon leaves caused by Pseudomonas syringae pv. lachrymans is distributed widely in the world and inflicts different degrees of damage. P. syringae pv. lachrymans is a typical seed-borne pathogen.The objective of this study is to build effective, commercially viable and convenient detecting technologies and to prevent the spread of this pathogen.【Method】The house-keeping gene of DNA glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was selected as the target gene, the specific Padlock probe was designed for bacterial leaf spot of melon, and a bacterial detection system based on Padlock probe combined with dot-blot hybridization was developed. The target strain was selected as experimental materials, and DNA was extracted as a template for ligation reaction, enzyme-cleavage reaction and amplification reaction. The reaction temperature and reaction time of the ligation reaction, enzyme-cleavage reaction and amplification reaction were optimized. The optimized reactions were tested for healthy melon seeds, melon seeds with bacterial spot of melon leaves, sterile water and 25 experimental strains. In order to determine the sensitivity of padlock probe, DNA of the target strain was diluted to 1 ng·μL^-1, 100 pg·μL^-1, 10 pg·μL^-1, 1 pg·μL^-1, 100 fg·μL^-1, 10 fg·μL^-1 and 1 fg·μL^-1 as the templates. Sensitivity was determined by optimized ligation reaction, enzyme-cleavage reaction and amplification reaction. Padlock probe combined with dot-blot hybridization was developed, the amplification product in the course of the reaction was fixed on the nylon membrane, the reverse complementary sequence of the Zipcode sequence was synthesized into cZipcode (detection probe), the detection probe (cZipcode) was labeled with digoxigenin and hybridized with the amplified product. Padlock probe combined with dot-blot hybridization techniques were used for specific detection and sensitivity. Artificial infestation seeds were detected to further verify the reliability of the system. A total of 205 commercially melon seeds with suspected disease were detected by high throughput detection method.【Result】The specificity of the Padlock probe showed that a specific band of 105 bp was got from 26 strains of bacterial spot of melon leaves, while the remaining 25 strains and sterile water were not amplified products. the results of sensitivity showed that the target strain was diluted to 1 pg·μL^-1 and got a specific band of 105 bp, so the detection sensitivity of the Padlock probe was 1 pg·μL^-1. Padlock probe could distinguish bacterial spot of melon leaves from all other experimental strains and its sensitivity could be up to 1 pg·μL^-1. Twenty-six strains of bacterial spot of melon leaves had color reaction, the remaining 25 strains and sterile water did not have color reaction. The sensitivity of Padlock probe combined with dot-blot hybridization also could be up to 1 pg·μL^-1. Padlock probe combined with dot-blot hybridization could detect one bacterial seed from 1 000 healthy seeds, and the detection rate reached 0.1% (1/1 000). Seven commercially seed-borne bacteria were successfully detected from 205 commercially melon seeds. The seven seed-borne bacteria, respectively, were soaked in sterile water for 4 h, DNA was extracted for PCR amplification and sequencing, the results of DNA sequencing were compared with NCBI and verified the bacterial spot of melon leaves. 【Conclusion】Detecting system based on padlock probe combined with dot-blot hybridization could detect P. syringae pv. lachrymans from sweet melon fast and accurately.

Key words: bacterial spot of melon leaves, Padlock probe, dot-blot hybridization, seed