The Expression Analysis Of TPS And Trehlgenes Under Cold Induction In Harmonia Axvridis

Trehalose which is the blood sugar in insects is synthetized by Trehalose-6-phosphat Synthase (TPS) and degraded by Trehlase Trehalase in the spermatophore from the bean-shape accessory gland of the male mealworm beetle, Tenebrio molitor:purification, kinetic propertie and localization of the enzyme (Treh) into two molecules of glucose used by insects. Trehalose i not only the basis of maintaining insect individual and population life activities but also can b activated under stress conditions to resist undesirable environment and enhance their vitality an activity.In this experiment which used Harmonia axyridis as the object, the whole cDNA sequence c Treh1was cloned with homology cloning and Rapid Amplification of cDNA Ends (RACE), th both the expression of TPS as well as Trehl in different developmental stages of Harmonin axyridis and the prokary expression were conducted, the expression of TPS, Treh1and cold-resistant ability under short-term low temperature induction were studied. The main finding are as follows:Our experiment got the full cDNA of Trehl (GenBank accession number:HM05638) in h axyridis. The gene consists of1890nucleotides including75or150nucleotides fo5'-or3'-non-coding region respectively and an ORF of1665nucleotides that encodes555amon acid residues with a predicted mass of approximate63.7kDa and a p1of4.71. Comparation o homology reveals that insect Treh1has a high conservation including many conserved domains.The expression of TPS and Treh1at different developmental stages of H, axyridis wer detected by real time fluorescence quantitative PCR. The results showed that TPS and Trehl an expressed in every instar and changed with different developmental stages. The expression leve of TPS in pupal stage is the highest which is2.26times as high as control but Trehl is lower. The two types of Treh1have different expression patterns; the expression level of Trehl-2is relativel; stable with little changes.The prokaryotic expression of TPS and Treh was conducted. The conserved fragments o TPS and Treh gene got by cloning their conserved domains were transferred into BL21to perforn IPTG induction expression. The corresponding proteins including28kDa TPS protein,24kDa Treh1-1and Treh1-2protein were obtained.We detected the changes of supercooling point after short-term low temperature induction. The results showed that the supercooling point of insect rose under short-term low temperature induction treatment, but it decreased with the deepening of the induction. The induction may do harm to the insects. Under the treatment of the above induction, the regulative ability of TPS increased and the synergistic effect between Trehl and TPS can make insects resist to cold and maintain homeostasis. The injury caused by cold induction may be repaired within a certain time.