| In recent years, with the improvement of people s living condition, the risk of suffering a cardiovascular disease and myocardial infarction was not only greatly increased, and it showed that the age onset to be younger, which has became an important problem to be solved urgently for modern medicine. Studies have shown that endurance exercise can be used as a beneficial intervention to maintain the function of the key organ and systems in our body. Thus, as non invasive intervention, exercise has become an important research project. Anything else, due to the lack of the concept of overall ecological and extensive use of pesticides for a long time, it directly leads to the enhancement of undesirable tendency of agricultural development. At the same time, the broad-spectrum pesticides which used by people can cause damage to the pets, infants and young children as well as the harmful ones. Therefore, many countries have begun to study the new specific pesticides for reducing and avoiding more serious damage to our habitat and other organisms, which has an important significance in agriculture.CAMRs family was evolutionarily highly conserved proteins, which is the major intracellular receptor of the immunosuppressive drugs RAPA and FK506. It widely distributed in the cytoplasm of mammals, birds, and insects, and involved in a variety of cellular physiological processes. Ca2+ is one of the most important intracellular messenger, and the balance of calcium homeostasis plays an important role in the development and function of musculature and reproductive system in fruit flies. CAMRs regulated the concentration of Ca2+ in cytoplasm and sarcoplasmic reticulum by combining with RyR receptors, thus caused a series of physiological and biochemical process. In addition, CAMRs and extracellular matrix also have a close relationship, which regulate the process of protein folding, signal regulation inside and outside cells.Drosophila melanogaster is one of the significant model organisms for the research of human disease and played a more and more extensive role in modern life sciences which is an useful genetic tools for the study of insect metamorphosis and diseases associated with human muscle. As a powerful genetics tool, fruit fly has a certain representativeness of insecta. By studying the expression patterns of CAMR1 and CAMR2 proteins in Drosophila adult skeletal muscle, cardiac muscle and the reproductive system, it can provide valuable molecular basis for the biological control of pests in agricultural applications. At the same times, it can also offer a reference and biomolecular evidence for the treatment of disease which related human cardiovascular and muscle development.In this study we selected Drosophila melanogaster Canton-S strains as the experiment materials. This project was divided into five parts to study the spatial and temporal expression patterns of CAMR1 and CAMR2 protein in fruit flies. Firstly, we carried out histological and image analysis to learn the structure of muscle and reproductive system in adult. Secondly, using in situ Hybridization in Routine Paraffin section method we observed the expression and localization of CAMR1 and CAMR2 mRNA in adult flies. Thirdly, we observed the distribution and expression of CAMR1 and CAMR2 proteins in muscle and reproductive system of adult by immunohistochemical staining. At last, following a regular exercise, we investegated the expression changes of CAMRs proteins before and after exercise by Western blot. Consequently, we discussed the role of CAMR1 and CAMR2 in the metamorphosis development of Drosophila. The results on the above criteria are as follows:Firstly, we found that CAMR1 and CAMR2 are both expressed in adult flies with the method of RT-PCR at the level of mRNA, which indicated that CAMR1 and CAMR2 may be involved in the growth and development process.Secondly, we analyzed the histological structure of system in fruit fly. HE staining results showed the nuclear mass distribution in mature ovaries, undifferentiated eggs, heart tube and skeletal muscle groups; then we studied the complex extracellular matrix, skeletal muscle and collagen structure and distribution in the body with Masson staining in adult tissue section. The results show that the presence of fibers in insect visceral muscle; Through the analysis of PASM staining in exoskeleton cuticle, cardiac muscle and gonad structure, we discovered a large number of proteoglycans existed in basement membrane, which will provided a basis for the preliminary localization of CAMRs protein in Drosophila.Thirdly, we detected the expression and localization of CAMR1 and CAMR2 mRNA in adult using in situ hybridization in paraffin-embedded sections. Our results showed that CAMR1 and CAMR2 were both detected in fruit flies and the expression patterns have similarities and differences. CAMR1 mRNA was mainly expressed in the reproductive system, heart, brain cytoplasm and cell membrane, and CAMR2 mRNA was just weakly detected in the dorsal muscle of Drosophila. In addition, both of them were detected in the digestive system and stomach muscles.Fourthly, we identified the expression and localization of CAMR1 and CAMR2 proteins by the method of Immunohistochemical Staining on Paraffin-Embedded tissue slide. The results showed that CAMR1 protein was expressed widely in imaginal tissues, especially in the mature ovary and undifferentiated yolk as well as the coated vitelline membrane. At the same time, the heart tube and brain also have strong expression. However, the expression in the digestive system such as stomach and midgut was relatively low. CAMR2 protein expression mainly located in dorsal longitudinal muscle and dorsal ventral muscle, but it just had a lower expression.Then, through a planned exercise to some conditioned flies, we detected the change of expression of CAMRs proteins by western blot before and after exercise. We aimed to explore the relationship between the CAMRs proteins and myocardial development. The results showed that CAMR1 had a higher expression in the group of medium-dose cordyceps non-exercise fruit flies after 30 days. After 2-3 weeks excrise, the expression of CAMR1 was highest in the male, and CAMR2 protein had a high expression level in low-dose concentration groupConclusion:CAMR1 and CAMR2 have different expression level among different tissues in Drosophila, and highly expressed specificity in skeletal muscle and smooth muscle, myocardial and gonads. Through a 2-3 weeks exercise with different concentration of cordyceps sinensis, the expression of CAMR1 and CAMR2 protein was dramatic up-regulated. Also, there are big differences among the groups. If we just feed the cordyceps sinensis, both of them had a high expression level in medium-dose concentration group.However, if we give the same intensity exercise further. Both of the proteins had a high expression level in low-dose concentration group All the results in this study would provide valuable information for the research and development of specific pesticides and the treatment of muscle related diseases of human beings, and give some references for the treatment of cardiovascular and cerebrovascular diseases. |
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