Ice nucleation active (INA) bacteria are the most potent heterogeneous ice nuclei in nature, which have become an important biological resource for diverse applica-tions. Many researches have proved that INA bacteria can raise the supercooling points (SCPs) of insect pests, then reduce their cold hardiness. However, INA bacteria抯 ineffi-cient colonization on the surface or in the guts of insects, and the high incidence of frost injury induced by their release hampered the application of INA bacteria in controlling in-sect pests in agricultural fields. In this study, we constructed a recombinant plasmid mob-Tn5-iceA with the ability of broad-host-range conjugal mobilization and integration of the ina gene of iceA into chromosomal DNA of many gram-negative bacteria by Tn5 transposition. In addition, Ent. cloacae strains stably carrying iceA and expressing high ice nucleation activity (INA), even in the absence of antibi-otic pressure, were constructed through conjugal mobiliza-tion and Tn5 transposition. Ent. cloacae strains have been reported to be able to efficiently colonize in the guts of in-sects, but have weak plant epiphytic ability. Therefore, these transgenic Ent. cloacae may be promising candidates for control of insect pests in agricultural fields.
Using the minitransposon pMini-Tn5 and the ice-nucleation active (INA) gene of iceA, a suicide recombinant plasmid pTnice1 was constructed, which has the ability of broad-host-range conjugal mobilization and integration of iceA into chromosomal DNA of many gram-negative bacteria by Tn5 transposition. We used this plasmid to integrate the iceA into chromosomal DNA of Ent. cloacae and obtained the transgentic strain Enc1.2022 ina. In this transgenic Ent. cloacae, iceA would never be transferred elsewhere through transposition, and constantly expressed high ice nucleation activity even in the absence of antibiotic pressure. The transgenic strain was ingested by corn borer larvae. Over the 7 d after ingestion, the mean supercooling points (SCPs) of the larvae was about 10℃ higher than those of larvae treated with distilled water (control). The maintenance of these high SCPs was related to the stable gut colonization of transgenic strain. At 6th day post ingestion, the larva was exposed at 5 or 7℃ for 12 h, the percentages of larvae frozen to death were 85 and 100%, respectively. In contrast, none or a small proportion of control larvae was frozen to death under the same conditions. Further studies demonstrated that this transgenic strain bore weak epiphytic ability. Therefore, this genetically engineered strain may be a promising candidate for control of insect pests in agricultural fields.
