| The western flower thrips, Frankliniella occidentalis is a worldwide insect pest of horticultural crops and economic crop. It has dispersed to many areas in China and the damage is becoming more and more serious since it was first reported in Beijing in 2003. During spread of F. occidentalis, they often encounter high temperature stress in summer. Unfortunately, this stress doesn’t limit its establishment in new regions. In this study, we uses the method of physiology and biochemistry and molecular biology to analysis the important roles of the antioxidant system under high temperature stress. As a result, understand the heat resistance mechanisms of western flower thrips could be illustrated, providing a theoretical basis for the spread of western flower thrips populations. The main findings are as follows:1. To study the effects of high temperature stress on antioxidant enzymes of the western flower thrips. The results show that, the western flower thrips under 0.5,1 and 2 hour high temperature stress, the activity of SOD, POD and CAT were significantly increased and then decreased. While, The activity of GSTs increased significantly under 0.5h high temperature treatment, but it didn’t change significantly under 1 and 2 hour. These shows that the antioxidant enzymes system of the western flower thrips can effectively resist the oxidative damage caused by high temperature stress.2. Seven reference genes (partial sequence) of 18S rRNA, EF-1, GAPDH, H3, TUB, ACT and RPL32, were cloned from F. occidentalis by RT-PCR. And these genes were evaluated for their suitability as normalization genes under different experimental conditions using the statistical software programs BestKeeper, geNorm, Normfinder and the comparative ACt method by qRT-PCR. Then, we chose a web-based comprehensive tool RefFinder to get the final ranking. The results indicated that the best sets of candidates as reference genes were as follows: Different developmental stages was EF-1 and RPL32; high temperature stress was RPL32 and GAPDH, low temperature stress was 18S and EF-1, high temperature stress gradient was TUB and GAPDH and the low temperature stress time gradient was EF-1 and TUB. Finally, we validated the suitable reference genes for gene expression profiling under different experimental conditions. So the choice of internal standard is necessary to normalize the target gene expression levels.3. CAT gene was cloned from F. occidentalis by RT-PCR and RACE, and was named FoCAT. The complete cDNA of FoCAT is 1870 bp, and their respective opening reading frames are 1521bp, which encode 506 amino acids with predicted molecular weights of 57.11 KDa. Sequence analysis suggested that the FoCAT is very conservative, and shared high similarity with those of other insects, and contained conserved motifs found in their respective family of other species. There are two signature sequence of the CAT family in FoCAT, it also shows that FoCAT is a member of CAT gene family. Expression patterns of FoCAT in response to development and different temperatures were examined by Real-time PCR. Results showed that high temperature stress could induce the expression of CAT, but there is no significantly influence of low temperature stress. The expression of CAT was significantly higher than other stages, and whether it is high or low temperature treatment in different time gradient treatments, the expression were significantly up regulation, it reached the maximum value at 2h.Taken together, relative strong tolerance to extreme temperature of the western flower thrips is a prerequisite for the rapid invasion, and the underlying mechanism may be related to antioxidant system against high temperature stress from oxidative damages. |
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