Study On The Expression Pattern Of RyR Protein And Its Regulatory Protein During Drosophila Development

In organisms,calcium ion plays an important role in regulating the metabolism as a common chemical substances and a second messenger.The mainly effect of calcium ion is to regulate the excitability of nerve and muscle.In addition,calcium ion also participated in a variety of important activities such as the fertilization,embryo development,cell differentiation and cell apoptosis.Intracellular calcium ion is mostly stored in the endoplasmic reticulum cells and sarcoplasmic reticulum,so there has is a lower concentration of calcium ion in the cytoplasm.The calcium pool regulates the physiological activities based on the release and uptake of calcium ion and the change of concentration of calcium ion in the cytoplasm.The release of intracellular Ca²⁺ ion is regulated by two types of channel protein receptor.One is the three phosphoinositide receptor?IP3 receptors?;another is the ryanodine receptors?RyR?which can specifically binding with alkaloid ryanodine.RyR receptors which are made up of four identical subunits and are located on the endoplasmic reticulum cells.The relative molecular mass of each subunit is about 560 KDa and total molecular weight is greater than 2000 KDa.Each subunit has the N terminus of N in the cytoplasm area and the C terminus of C is in the transmembrane area.The N terminus in which the terminus of N cytoplasm area interacts with and the membrane receptor of dihydropyridine DHPR and L type voltage-gated Ca²⁺ ion channels.In mammals,there are three types of RyR receptors,namely the skeletal muscle type?RyRl?,myocardial type?RyR2?and cerebral?RyR3?.Currently,researches on the functions of RyR1 and RyR2 are relatively clear.They are play an important parts in the process of skeletal muscle and myocardial excitation-contraction coupling?excitation contraction coupling E-C coupling?respectively.In skeletal muscle,the effect of DHPR and RyR1 E-C coupling on the regulation of the release of calcium is induced by depolarization,while the effect of DHPR and RyR2 E-C coupling on the regulation of the release of calcium is induced by calcium in myocardium.However,in most studies to date,only in the vertebrate RyR receptor has been studied.Especially The RyR receptor in insect research has been remained at the initial stage.Thus,it is essential to investigate RyR receptor in invertebrates.The currently research on fruit fly RyR receptor are is focused on investigating the function of RyR and the expression of RyR in the age of 2nd,3rd instar larval and adult body using in vitro system.And there are no patterns for exploration exploring the expression of RyR in drosophila embryo RyR in different periods.Drosophila melanogaster with a short life cycle and high reproductive rate is widely used in the process of as a biological model.And the Drosophila embryo is one of classic models for early embryonic developmental because of the ease to of collection and observation.Thus,using Drosophila embryo model,the major aims of the current study was to illustrate the RNA and protein expression of RyR in fruit fly embryonic in different development periods,using the mature fruit fly embryo paraffin slice preparation technology as the foundation.And also to illustrate the RNA and protein expression of RyR in the adult fruit fly.The main results include the following points.?1?In the early stage of drosophila embryo,we detect the mRNA expression of RyR was found using RT-PCR technique.These resultes demonstrated that RyR are expressed in Stage blastoderm and Satge gastrula corresponding to the data obtained in the following result.?2?Based on the above basis,we combined with situ hybridization and immunohistochemical technology to explore the patterns of the change of expression of RyR in the fruit fly embryonic in different development periods.The results showed that the RyR gene was expressed in the fruit fly in all the embryonic development periods.On the longitudinal section,the gene expression of RyR in syncytial blastocyst?0-2h?was uniform.As the process of the blastula cells is completed,the mRNA and protein expression of RyR in the blastocyst?2?3 h?were mainly distributed in the blastula cells layer and the distribution showed symmetrical between the left and right sides and uniform.On the longitudinal section and cross section,the protein expression of RyR in blastocyst abdominal cells presented specificity.With the start of the process of gastrula?3?24 h?,blastocyst abdominal inward concave then appeared in the mesoderm cells.The RyR was expressed in abdominal mesoderm in the early period,therewith the expression of RyR in the ventral and also was expressed in the intestinal lumen at the same time.As the specificity process of differentiation of embryonic organ,the gene expression of RyR in different organs showed differently.There was had a highly RNA and protein expression of RyR in the digestive tract epithelium and chest dorsal flight muscle in adult fruit fly by using situ hybridization and immunohistochemical technique.?3?The results of immunoblotting showed that the protein expression of CAMR2protein was increased gradually during the blastocyst stage,cytoplasmic blastocyst stage and early gastrocnemius development,and the expression of protein in advanced gastrula was decreased.?4?In situ hybridization and immunohistochemistry showed that the expression pattern of CAMRs gene in blastocyst was significantly different from that in syncytial blastocysts.CAMR1 mRNA was mainly expressed in cytoplasm,while CAMR2 mRNA was expressed in cytoplasmic membrane.The expression of CAMR1 mRNA in blastocysts was expressed in blastocysts.The expression of CAMR1 mRNA in blastocysts was different from that of RyR gene in different organs trending to be synchronized.?5?RyR and its regulatory protein CAMR play a key role in regulating the systolic function of muscle cells,so this topic also explores the Drosophila movement method.According to our experimental method,the Drosophila Drosophila melanogaster had a shorter diastolic and diastolic cycle than that of the untreated Drosophila,and the heart rate increased and the ejection volume decreased,indicating that the exercise stress had an effect on the fruit fly.To further analyze the RyR complex.The role of the process laid a good foundation.The results showed that RyR gene was involved in the regulation of dorsal ventral axis in the early stage of embryonic development of Drosophila and involved in the regulation of mesenchymal organ development and differentiation.The RyR gene is also involved in the differentiation and development of the gastrula.The CAMRs gene is involved in the regulation of the anteroposterior axis and is involved in the specific tissues and organs of the Drosophila embryo.Differentiation and development regulation of Drosophila melanogaster can be used as an indicator of changes in the physiological state of Drosophila melanogaster after stress.