Functional Study Of Antifreeze Protein Gene From Tenebrio Molitor By Using RNAi

Antifreeze protein (AFP) is one of the important protective substances for insectto overwinter safely. However, recent studies have found that the afp genes from thedesert Tenebrionidae insects are also expressed in summer, and abiotic stresses, suchas low temperature, high temperature and drought could induce the expression of afpgenes. The results of in vitro experiments showed that the AFPs from a desert insectalso had thermal protection and strong water binding capacity. Therefore, it isnecessary to explore whether AFP also has the function of help insects to adapt to avariety of environmental stresses, except for its antifreeze function.Tenebrio molitor, a freeze avoiding insect, is a kind of storage pest inTenebrionidae. The larvae of T.molitor can tolerate low temperature. Its antifreezeproteins have high thermal hysteresis activity, and are the first well known AFPs intenebrionidae insect. Because the predicted structure of the desert insect AFP aresimilar to the AFP of T.molitor, and because it is convenient to obtain T.molitor forstudy from the market, we choose T.molitor as the research material to further verifythe potential protective effect of antifreeze proteins in insect resistance to abioticstress by using RNA interference (RNAi) technology. The RNAi was accomplishedby using bacterially expressed dsRNA which was injected into last instar larvae ofT.molitor to silence afp gene. After injection the T.molitors were treated with stressesof high temperature, low temperature and drought. Then the related physiologicalindicators, survival and gene expression level of the treated T.molitor insects weremeasured. The results preliminarily proved that antifreeze proteins confer T.molitor toresist abiotic stresses. The specific results are as follows:1. The mRNA levels of afp genes in each developmental stage of T.molitor undertemperature stresses was measured by using Real-time PCR. The results showed thatboth high temperature (38℃) and low temperature (4℃) could significantly inducethe expression of Tmafp genes. The extent of the small larvae induced by thermal stress was weaker than the middle larvae which was weaker than the older larvae andthe adult. The mRNA levels of Tmafp gene in the older larvae and the adult inresponse to high temperature was9.5~26.5-fold of the control; the expression levelsof the afp genes of them in response to low temperature was4.7~7.1-fold of thecontrol. Under normal conditions the basic expression levels of Tmafp gene wereregulated by developmental stages. Tmafp mRNA level in older larvae was the highest,therefore we selected the older larvae as the object for further RNAi experiments.2. The interference fragment of AF160497(Tmafp433) gene was designed, andconstructed to L4440interference vector and transformed into E.coli HT115. Thepositive clones could express double-strand RNA fragment targeting Tmafp gene byIPTG induction. After extraction and purification, the dsRNA was injected into theolder larvae, which influenced the survival of T.molitor, and the mortality of T.molitorafter injection showed a high dose-dependent manner. Changes in afp transcript levelsafter the RNAi were detected by Real-time quantitative PCR. The results showed thatall the different amount of the Tmafp-dsRNA could significantly inhibit theexpression of Tmafp gene, and the degree of the inhibition was positively correlatedwith the amounts. Furthermore, the inhibition to the expression of Tmafp genes by theTmafp-dsRNA varied with the time length after injection, and presented in a mannerof increase first and decline later. The strongest inhibition was reached on the thirdday of the RNAi, which was77.9%of the non-injected control, and the significantinhibition lasted for ten days.3. After afp gene silenced by RNAi in older larvae, the physiological indicatorsunder low temperature, high temperature and drought stress were tested. The resultsshowed that the survival of the non-injected T.molitor which were acclimated at4℃for5h increased significantly under extreme low temperature (-13℃for1h), but thesurvival of the T.molitor insects after RNAi decreased significantly. The averagesupercooling point (-10.4℃) of the injected insects was significantly higher than the control, and their hemolymph osmolality was significantly lower than the control,proving that the antifreeze protein could protect T.molitor from cold damage, andimprove their cold tolerance. After acclimated at38℃for5h, the survival rate of thenon-injected control insects at extreme high temperature (45℃for1h) was increaseddramatically, while after RNAi their survival rate dropped significantly. The mRNAlevels of the Tmafps in the RNAi T.molitor insects under high temperature were alsosignificantly decreased, while the Tmafps level of the control group was significantlyinduced by high temperature, suggesting that the antifreeze proteins play an importantrole in the thermal resistance for T.molitor. The water loss rate of the RNAi T.molitorinsects was much higher than that of the control group under drought stress, whichmight means that antifreeze proteins may function, to some extent, in the dehydrationtolerance of T.molitor.