| The English grain aphid, Sitobion avenae, is of wing dimorphism, namely, winged and wingless aphid. The aphid's wing differentiation is manipulated by a complicate factors system, and the ratio of the wing dimorphism depends on the intrinsic and external environment factors. So in order to uncover the mechanism of aphid's wing differentiation, through the related genes expression level by molecular biology approach, which will be very important to enrich the basical biology of aphid, and also benefit to further develop new method to control the aphid based on the knowledge of the manipulation of aphid's wing differentiation.In this experiment thesis, Cdc42 gene tissue location and accurate relative expression levels of S. avenae were determined by using in situ hybridization (ISH) and real-time fluorescence quantitative PCR method to determine the Cdc42 gene in the location and relative expression levels of the organization in Sitobion avenae. Then the change dynamic of Cdc42 gene expression, and population and the ratio of alatae and apterae aphid were detected with dose variation of juvenile hormone (JH) and its analogues by smearing 2 instar nymphae aphid. The aim is to understand preliminarily for the interaction of JH in vitro, the Cdc42 gene expression, and the function of manipulation of wheat aphid's wing dimorphism. The major results are as follows:First, ISH results showed that drip liquid hybridization specificity of tissue biopsies were specific stained in brown with nucleic acid probe of Cdc42 gene, while without the same staining characteristic on the negative control, which indicated that ISH could be sensitive to identify the tissue locaton of the gene in the aphid. ISH results showed that Cdc42 expressed in the head and abdomen parts of the aphid, and focused between the two complicated eyes by aphid's head.In the abdomen the gene expressions were almost concentrated the pseudo-embryos. According to the brown staining. The Cdc42 gene specific stainging spread all over on the alate thorax part, and it was deduced by the number staining dot, that the highest expression level occurred on the thorax part of alate aphids, but hardly not expressed on the thorax part of apterae aphid.Tissue and stage specific transcriptive levels were determined by real-time fluorescence quantitative PCR and showed that Cdc42 was transcripted in multiples tissues and at different stages. There was higher transcriptive level in the nymphae stages than that in adult stage, and higher in alate aphid than that of apterous one, and significantly higher on the thorax part of alate than that in apterae aphid. The gene's expression start up at the aphid's pseudo-embryo stage, and even with very high expressed level in the stage.Application juvenile hormone (JH) and its analogues ZR-515 on aphid's thorax in vitro by three concentrations, to study the impact of JH dose on the expression of Cdc42 gene and wing differentiation, the results showed that with the dose increasing of JHâ…¢and its analogues ZR-515, the expression level of Cdc42 gene in the whole part of alate and apterae aphid decreasing gradually, which were significant lower in the three treated concentration than that in control of alate aphid, by real-time fluorescence quantitative PCR detection, and the expression level of Cdc42 in the treated apterae aphid was only one thousandth of the level compared with aphid in control.There were also significant difference among the different treatments. The number of apterae adults increasing, but the number of alate aphids decreasing under the JH and ZR-515 treatments.In this thesis, a micro-insect gene tissue localization and quantitative analysis, method has been established, and the results provide a primary data for the interaction of JH, Cdc42 gene and wing differentiation in aphid. |
PDF Full Text Request