Cloning Of A New Ice Nucleation Active Gene And Construction Of Ice Nucleation Active Enterobacter Cloacae For Insect Pest Control

The application research of bacterial ice nucleation active (ma) genes has beeninvolved in bacterial cell surface display, reporter gene system, detection ofpathogenic bacteria contaminating foods, breeding of cold resistant varieties, and soon.Before this study, no ma genes were available for research in China.Furthermore, ineffective gut-colonization in insect pests and the danger to incitefrost injury to crops hampered the application of ice nucleation active bacteria topest insect control in fields. In this study, the research to isolate an ma gene and toutilize the cloned ma gene to construct gene-engineered bacteria for insect pestcontrol in fields has been performed. The results go as follows.An ma gene was cloned from a Chinese ice nucleation active bacterial strainErwinia ananas 110 by PCR amplification. The primers were designed based on thepublished homologous ma genes. The cloned gene was sequenced. Its coding regioncontains 3921 nucleotides, which encodes 1306 amino acids consisting repetitiveregion (1104 an) with 16-residue as repetitive unit, flanked by non-repetitiveaminoterminal region (l6laa) and carboxyterminal region (4laa).The cloned gene was compared with other published ma genes and wasidentified to be a new ma gene, and registered in GenBank with an accessionnumber of AF3 87802. The new ma gene, named as iceA, has the greatest homologywith published ma gene inaA, with 94% homology both in amino acids andnucleotides. The prokaryotic expression vector prNA2O I for iceA was constructedand SDS-PAGE detection showed that specific 187 KDa translational product wasexpressed in Escherichia coil BL21(DE3)pLysS.The ma gene iceA was inserted into the region between restriction sites of Sailand BamHI of Tn5 of plamid pSZ2 1 and a recombinant plasmid mob-Tn5-iceA wasfirst constructed with the ability of broad-host-range conjugative mobilization andintegrating iceA into host chromosomal DNA of many gram-negative bacteria byTn5 transposition. The mob-Tn5-iceA was conjugatively mobilized intoEnterobacter cloacae 1.181 and 1.2022, and the ma gene iceA was further integratedinto the host cbromosomal DNA by Tn5 transposition, and genetically modifiedEnterobacter cloacae strains with stable ice nucleation activity came out. These twotransgenic strains were designated as Enc 181 `~ and Enc2O22ice, respectively.These transgenic bacteria (Enc 181 and Enc2022cc) were ingested by corn3borer (Pyrausta nubialis) and bollworm (Heliothis armigera) larvae through feedingon detached young corn stems and cotton leaves, respectively. The supercoolingcapacity of these larvae was examined. The mean supercooiing points (SCPs) ofcorn borer larvae ingesting transgenic bacteria were about -3.--40C, &?2C higherthan that of untreated larvae, which showed mean SCPs ofl ~- -17 C. The SCPs ofbollworm larvae ingesting transgenic bacteria were almost the same as that oftreated corn borer, 5?0C higher than that of untreated bollworm with mean SCPsof about 10'C. The ability of transgenic bacteria to raise SCPs of larvae remainedstably during the 7-day period of post-ingestion. After 9-day treatment, the treatedlarvae showed lowered SCPs, but still remained above ? C. These transgenicbacteria showed significant killing effects to insect pests. More than 90% of thelarvae ingesting transgenic bacteria froze and died when they were exposed at -5 Cfor 6h, while no check larvae died under the same condition. Placed at -7C for 6h,all the treated larvae froze and died, while only 7.4% of check corn borer and 14.3%of check bollworm died.These transgenic bacteria had stable and efficient gut colonization in theselarvae. After ingested by these larvae, large quantities of transgenic bacteria( 1 0~?1 O5cfu per larva) could be re-isolated durin...