| Based on the results of high-throughput transcriptome sequencing of B. dorsalis, five trypsin genes (Trys), two aspartase genes (Asps) and two carboxypeptidase genes (CPs) were isolated. They were further investigated by using RT-PCR, as well asbioinformatic softwares. Furthermore, the expression profiles of these genes were analyzed in different developmental stages and conditions. The qPCR was also employed to evaluate the effects of beta-cypermethrin and proteases inhibitors treatment in molecular and proteases activity responses under stress. Additionally, a functional analysis of Trys in development was performed by RNA interference (RNAi). The results are helpful to explore the functions and regulating mechanism of proteases genes in B. dorsalis, and provide new ideas for controlling the pest. The main finding were as follows:1 Expression patterns of proteases genes of B. dorsalis1.1 Developmental expression patterns of proteases genesThe qPCR method was used to characterize the expression patterns of the nine proteases genes during different developmental stages:third-instar larvae, pupae and adults of different ages. The results showed that these genes were highly expressed during the larval stage, the expression peaks always appear at 3-5 day after hatch. Generally, the expression of nine proteases genes were highest in the larval stage, followed by the the expression in adult stage, and the expression of proteases genes in eggs are the lowest. We speculate that several genes play an important role in protein degradation in the larval and adult stages.1.2 Effect of trypsin proteases inhibitor on expression of proteases genesUnder the condition of TLCK treatment, nine proteases genes were up-regulated except for Aspl, while the effect of same concentration of SBTI on expression of proteases is not so obvious. Among the five trypsin genes, only genes Try3、Try4 are up-regulated. In other proteases genes, only CP1 and ASP2 are up regulated. The result shows that to relief the effect of proteases inhibitor, There were two types of compensatory response in insects, one is the up-regulation of those inhibitor-sensitive proteases, the other one is the change from predominantly sensitive to the inhibitor to predominantly insensitive.1.3 Expression Profiles of proteases genes in’hunger and feeding’experimentIn the’hunger and feeding’experiment, the larvae were normally reared for 3 days after hatching. When their growth state became stable, we stopped feeding. Twenty-four hours later, the expression level are quantified and used as control, then we resumed to feed the rest. We found that all five BdTrys were up regulated after refeeding and most of the BdTrys reached maximal expression levels at 12-18 hours post-meal (hpm). During this time the expression level of BdTryl, BdTry2, BdTry4, BdTry5 were up-regulated above 15 fold, and BdTryl and BdTry5 were especially increased nearly 23 fold. Interestingly, there was a sharp decline 24 h after refeeding but the expression level was still 5-10 times higher than the control. In the’pre-and post- meal’experiment, Try3 also performed irregularly. It seemed that Try3 was not affected by the hunger and feeding treatment and the expression level didn’t change over time but remained 3 times higher than control thro μ ugh the whole digestion process. Expression profiles of two Asps and two CPs in larvae midgut at 18 hpm were also measured. Compared with that in control, the expression levels of two CPs significantly increased. CP1 was up regulated 2 fold, and 5 fold for CP2. Conversely,2 Asps were not affected by feeding.1.4 Effect of beta-cypermethrin on expression of TrysThe transcriptional changes of all five Try genes transcripts in larvae exposed to β-Cypermethrin determined by qRT-PCR, which showed significant upregulation of BdTry3, BdTry5, BdTryl, BdTry2, and BdTry4 in larvae following the treatment with β-Cypermethrin, especially for BdTry3. When compared to the control, all five genes registered 10.9-,2.6-,1.2-,1.4-and 1.6-fold increase, respectively. The result showed that β-Cypermethrin can induce the expression of trypsin genes.2 Determination of trypsin proteases activity and total proteases activity of B. dorsalis2.1 Location of trypsin proteases activityTrypsin proteases activity in different development stages (larvae, pupa, dult) and tissues (midgut, fat body, cuticle) were measured, BApNA was used as the substrate. Through the determination, we found the activity in the larvae midgut is highest. We speculated that trypsin protease played an important role in larvae midgut.2.2 Effect of beta-cypermethrin on trypsin proteases activity.Activities of SPs were determined using BApNA and L-TAME as substrates for amidolytic and esterolytic activities, respectively. The results showed that in both processes, SP activities in the treatment group were significantly higher than those in the control group. For BApNA hydrolysis, the specific activity of SPs from the treatment group was 450.00 ± 32.97 nmol/mL/min/mg, which was significantly higher than that in the control group (302.67±79.93 nmol/mL/min/mg) (p<0.05). Similarly, for the L-TAME hydrolysis, the specific activity of SPs from the treatment group was 266.00±13.89 nmol/mL/min/mg, which was significantly higher than that in the control group (201.33±16.92 nmol/mL/min/mg) (p<0.05).2.3 Effect of trypsin proteases inhibitor on trypsin proteases activity and total proteases activity.Assay results showed that serine protease activity was severely inhibited by SBTI and TLCK, which decreased approximately 70% of activity towards BApNA, these data might indicate a larger proportion of trypsin-like protease in serine protease, B. dorsalis. However, total proteases activity (towards Azocasein) kept constant under the inhibitors’treatment, which may due to the compensatory response of other digestive proteases. The result indicated that the insect was relieving the effect of proteases inhibitor by up-regulation of those inhibitor-insensitive proteases. |
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