Detection, Functional Evaluation And Application In Transgenic Of Antifreeze Proteins From Insects

Producing antifreeze proteins (AFPs) is one of the most important mechanismsunderlying insect cold tolerance. Depending on its high antifreeze activity, antifreeze proteinshave a broad application prospect, especially in the cultivation of transgenic crops. Today, asthe technology of transgenic plants is increasingly mature, the acquiring of high-activity AFPsgene resources has clearly become the key to the success of transgenic breeding of cropswhich have high cold tolerance. Aiming at the transgenic breeding of cold-tolerance crops, weattemped to look for high-activity AFPs in insects from cold regions, established theprokaryotic expression system for functional evaluation of objective genes and attemptedtransgenic operations. All of these studies are of great significance to getting more highquality AFPs genes of insects and their application in transgenic. The main research contentsand results are as follows:1. Detecting the AFPs in insects collected from cold regions: Firstly, we demonstratedthat the differential scanning calorimetry is feasible to identify whether one insect speciescontains antifreeze proteins through detecting thermal hysteresis activity of the total protein.Then, we collected seven insect species (Odontosia arnoldiana pupa, Ips subelongatus adults,Monochamus urussovi larvae, Coleophora dahurica larvae, Yponomeuta evonymallus larvae,Lymantria dispar eggs and Ambrostoma quadriimpressum adults) in the Daxing’anling ForestRegion in Inner Mongolia, and then prepared their hemolymph or total protein. Usingdifferential scanning calorimetry (DSC), the thermal hysteresis activity (THA) of the seveninsect species were respectively analyzed. Among the seven insect species, we had notdetected thermal hysteresis activity in the hemolymph of A. quadriimpressum adults, but theother six insect species all had THA. The results suggested that A. quadriimpressum cannotproduce antifreeze proteins but the other six insect species can. In the six insect speciesproduced AFPs, the THA of I. subelongatus (1.51-3.02℃) and M. urussovi (1.28-2.60℃) washigher, that of O. arnoldiana (0.36-1.39℃) and C. dahurica (1.18-1.57℃) was placed in themiddle and that of Y. evonymallus (0.34-0.43℃) and L. dispar (0.35-0.42℃) was lower.Compared with the THA of Tenebrio molitor larvae, all the THA detected is lower than that of hemolymph (2.54-4.34℃) and the THA of Y. evonymallus and L. dispar is at the samelevel with the total protein (0.49-0.98℃).2. Prokaryotic expression and activity determination of antifreeze proteins from T.molitor: In the study, we constructed a prokaryotic expression vector for T. molitor AFPgenes, induced the expression of the fusion protein through groping for conditions anddetected the corresponding activity successfully. Although the THA of the fusion protein islow (the THA is0.47-0.62℃at concentration of0.1mg/mL), the results sufficientlyindicated that the functional evaluation of objective genes through prokaryotic expression isavailable before transgenic.3. Identification of transgenic rice: We transferred4AFP genes cloned from T. molitorinto Oryza sativa ssp.japonica by gene marksmanship and obtained well grown transgenicplants. We identified quite a number of positive plants by using the PCR (the probability ofpositive plants were all above60%). The realization of expected results showed that thetransgenic of AFP genes from T. molitor won initial success and laid a good foundation forthe cold hardiness experiment of transgenic rice.