| The oriental fruit fly, Bactrocera dorsalis (Hendel) (Dipttera:Tephritidae), was widely found in tropical and subtropical areas of the world. Its strong adaptability to surroundings, migratory and highly fecundity makes it possess ability to make a lot of yield loss for fruits and vegetables every year. Typically, females oviposit in fruit, developing tunnel for larvae through the fleshy mesocarp that they feed on. This causes fruit rotting and drop and further directly leads to economical loss. The control of this pest is mainly depended on the insecticides applying in the field. However, the population of this pest has developed high level of resistance to many classes of insecticides. Therefore new methods and ideas should be exploited to control the pest. Heat shock protein involves in many important physiological processes such as growth and development, resilience and cell differentiation. It is necessary to investigate the molecular mechanism of small heat shock protein genes in regulating metamorphosis and lay the foundation of novel insecticides development.In this thesis, based on the information from Bactrocera dorsalis transcriptome database, nine cDNA sequences of heat shock protein gene we cloned from the Bactrocera dorsalis by using RT-PCR and their characteristic sequence were annotated. qPCR was conducted to analyze the nine genes expression patterns at different developmental stages, tissues, heat stress and exogenous 20E treatment in Bactrocera dorsalis. Finally, the function of Bdhsp23.8 and 3315 in ovarian development and another two genes’(Bdhsp23.8, Bdhsp20.6) in thermal stresses was revealed by using RNAi technology. The conclusions in this study will help us to understand the function and regulation mechanism of oriental fruit fly small heat shock protein genes, providing new ideas and methods for the Bactrocera dorsalis prevention and control. The main findings are as follows.1 Cloning and sequencing of nine sHSP genes in Bactrocera dorsalisNine sHSP genes fragments were cloned hrough the RT-PCR technology according to the unigene sequence from Bactrocera dorsalis transcriptome database. Eight of them were analyzed to obtain their open reading frame and amino acid sequence. They are Bdhsp20.4, Bdhsp21.6, Bdhsp23.8, Bdhsp20.6, Bdhsp23.0, Bdhsp17.6, Bdhsp18.4, Bdhsp11.1 and 3315.2 The expression pattern of sHSP genes2.1 Nine sHSP genes expression patterns in different developmental stagesThe mRNA expressions patterns in egg, larvae, pupae and adult stage of nine small heat shock protein genes were determined by qPCR technology. The results showed that the expression patterns of these nine genes varied among developmental stages, which Bdhsp20.4 and Bdhsp18.4 had a low expression in egg, larvae and early pupae but very high level in the late pupae and adult stage. The relative expression of Bdhsp23.8 and Bdhsp23.0 in egg, pre-larva and pre-pupa was significantly higher than the post-larvae and post-pupae. The relative expression of Bdhsp21.6 was extremely low in the egg, larva and pupa stage. Bdhsp17.6, which had almost no expression in the egg stage compared to the larvae stage (expression higher in prophase than late stage), had a gradually increased expression in pupae stage. The qPCR results indicated that these small heat shock protein genes had function difference among different developmental stages.2.2 Tissue-specific expression patterns of nine sHSP genes in adultTissue-specific expression patterns of the nine sHSP genes were analyzed by qPCR in seven tissues of adults including head, thorax, midgut, malpighian tubules, fat body, testis and ovary. The results showed that the relative expression of Bdhsp23.8 and 3315 in the ovary was significantly higher than that of other tissues, indicated that Bdhsp23.8 and 3315 were essential in the ovary development. The relative expression ofdhsp21.6, Bdhsp23.0, Bdhsp20.6 and Bdhsp11.1 also showed a significant difference in all the tissues,but the levels were not as much as Bdhsp23.8 and 3315’s. Bdhsp20.4 expression in the fat body was significantly higher than other tissues, which can be inferred that this gene related to energy metabolism pathways. Bdhspl7.6’s relative expression in the head, chest and fat body was significantly higher than that of other tissues, no expression in testis and ovary. Bdhsp18.4 had high expression in the chest, but low expression in other tissues. The relative expression results indicated that these nine sHSP genes in different tissues, obviously, had different functions.3 Effects of exogenous 20E and thermal stress on the expression of nine sHSP genes3.1 Effects of exogenous 20E on the expression of sHSP genesAfter 12h 100 ng 20E treatment, the relative expression level of Bdhsp20.4, Bdhsp21.6, Bdhsp23.8, Bdhsp23.0, Bdhsp20.6, Bdhsp18.4 and 3315 had a sharp increase and of Bdhsp11.1 significantly reduced but of Bdhspl7.6 had no significant change. It is reasoned that, under exogenous 20E stimulation, sHSP gene may adapt to environmental stimuli through its gene expression regulation.3.2 Effects of thermal stress on the expression of nine sHSP genesThe relative expression of Bdhsp20.4 and Bdhsp21.6 significantly increased only under-5℃ treatment. The expression level of Bdhsp23.8, Bdhsp20.6 and Bdhsp 18.4 were significantly upregulated under-5℃ and 40℃. The relative expression of Bdhsp17.6 was not affected by temperature. After-5℃ and 0℃ stress treatment, the relative expression of Bdhsp11.1 significantly reduced. Therefore we estimated that small heat shock proteins play an important role as the molecular chaperone during thermal stress, easing the pressure from the outside environment.4 Functional study of Bdhsp23.8, Bdhsp20.6 and 3315 in B.dorsalisThe microinjection method was used to deliver the synthesized dsRNA of target Bdhsp23.8 and 3315 genes to 4-day-old virgin female Bactrocera dorsalis. After 72 h, the ovaries were dissected and observed under stereoscope. qPCR was conducted to determine gene silencing efficiency. The results showed that the relative mRNA expression was significantly reduced and ovary development delay occurred by comparison with the control. The function of Bdhsp20.6 and Bdhsp23.8 was studied as the same methods stated above using B.dorsalis treated at -5℃ and 40℃. The results showed that mortality was significantly higher than control after 12h treatment and the relative expression of Bdhsp20.6 was significantly decreased. However the mortality and silencing efficiency are showing a significant reduction in mRNA after 48hs. By injection dsBdhsp23.8, almost no deaths and no significantly gene expression reduction were observed. The relative expression of the other eight small heat shock protein genes also had no significant decrease. |
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