| Teleogryllus emma (Gryllusmitratus Burmeister) bleongs to the genus Telegryllus (Orthoptera:Gryllidae). T.emma species is widely distributed in our country. As one kind of experimental materials, T.emma used in gene expression regulation, embryonic developmentl, entomological physiology as will as toxicology aspects because of the shorter life cycle, the bigger individual and easyly to be dissected. The most of multicellular organisms development process are regulated by steroid hormone, The insect development mainly is controled by ecdysteroid and juvenile hormone. Usually ecdysteroid combines with ecdysone receptor(EcR) and regulates the insect growth and development.Estrogen-related receptor(ERR), as an orphan nuclear receptor, play some improtant roles in biosynthesis of mitochondria, oxidative phosphorylation and lipid metabolism. Previous studies found that ecdysone receptor response element is similar to estrogen-related receptor response element and they all possess "GGTCA" mode, which may imply that EcR and ERR have correlation, togethor regulate physiological function of insects.In this study, we have investigated the expression location and expression level of EcR protein in T. emma by florescent real-time quantitative RT-PCR (qRT-PCR), Western blot and immunohistochemical methods. The relationship in protein level between EcR with ERR was also explored by RNA interference (RNAi) technique. The results are as follows.1.TeEcR gene expression in the head and thorax of T.emma(1).Real-time quantitative RT-PCR analysis was performed to quantify the relative expression levels of TeEcR transcripts at the different development stage of head and thorax. The results displayed that TeEcR was highly expressed in all samples at different levels in head and thorax. Compared with embryo stage, in the early larvae period (1 to 3 larvae) mRNA expression level is lower, in the later larvae period (4 to 6 larvae) and adult mRNA expression level is higher,4 instar larvae reached the highest. According to the mRNA expression level of adult, we found that the male transcription level slightly higher than the female. These results are highly consistent with TeEcR mRNA expression in the male and female individual of T.emma by previous studies results in our lab. Based on that we hypothesized that TeEcR participate in the learning, memory, development of the nervous system and other life activities in T.emma. Our study found TeEcR in the thorax transcription level is slightly higher than the head's, this implys TeEcR may regulate T.emma's wing and foot development as well as correlated activation.(2)The expression quantity of TeEcR protein in the different developmental stages head and thorax was investigated by western blot. The results show that TeEcR protein is expressed throughout all developmental stages, and presents a specific expression pattern in different stages. In the early larvae period (1 to 3 larvae) TeEcR protein expression level is lower. In the embryo, later larvae period (4 to 6 larvae) and adult TeEcR protein expression level is higher. This implies that TeEcR plays a more important role in regulation on late development in T. emma. The 4th larvae period began to distinguish between male and female, the yolk protein synthesis of 4th and 5th larvae have reached a peak period. We speculate, that TeEcR protein expression increasing rapidly in this period may be closely related with the testes and ovarian developent in T.emma.(3)Immunohistochemistry method was used to anlysis the expression location and quantity of TeEcR protein in 4th larvae and adults of T.emma. The results indicate that TeEcR proteins distribute widely in many different tissues, and the distrbution pattern of male and female brain is similar. There are some strong positive reaction granules in the forebrain mushroom bodies, optic lobe, central complex and midbrain olfactory lobe but lower positive reaction granules in the corpora allata and wai pharynx nerve cord fibrous tissue. Among them, the strongest positive reaction granules exist in Kenyon cells of the mushroom bodies and the optic fibre tissues of optic lobe. TeEcR proteins spreading in the brain organizations widely have proved that TeEcR participate in regulating the nerve development and corresponding complex behaviors process in T.emma. Especially TeEcR may play an extremely important role in regulating the development of nervous system,learning, memory and behaviour activities in T.emma. 2.TeEcR gene effects in regulating the growth and development of T.emma.(1)After TeEcR-RNAi, real-time quantitative RT-PCR analysis was performed to quantify the relative expression levels of TeEcR transcripts at the different development stage of head and thorax. The mRNA level of TeEcR gene in the experimental groups has decreased significantly (P< 0.05) reached total of 37.8%-65% after RNAi, this explain that injecting 3?g TeEcR-dsRNA can be effectively silence TeEcR gene in the body after 48h.(2)After TeEcR-RNAi, using Western blot and immunohistochemistry techniques to detect the TeEcR protein level change, the results show that TeEcR proteins reduced in the different development stages of the head and thorax in T.emma. The immunohistochemical results show that the TeEcR positive reaction granules in the brain frontal mushroom bodies, the midbrain olfactory lobe and hindrain corpora allata are significantly lower, the positive particles distribution in the central complex decrease in a certain degree, the optic lobe and wai pharynx nerve cord are not obvious change. These suggests that the forebrain mushroom bodies and the midbrain olfactory lobe may be the center of TeEcR regulation for the ant molt, growth, development and the sugar uptake.(3)Observing the phenotype of T.emma after TeEcR-RN Ai, we found the experimental groups ants molted slowly even didn't shed their exoskeleton and finally didn't finish their development, particularly appeared on the wing inextensible and abdominal swell. These may explain that TeEcR play very important roles in regulating the T.emma wing and foot development and corresponding activities. Compared with the control groups, the ant body size of experimental groups significantly is smaller, females spawning rods shorter, and eating less. These implies that TeEcR associated with energy storage in T. emma. The mortality and malformation rate from the investigation shown that the insect mortality has obvious enhancement, organization development imperfect, and malformation rate is high as 75%. These variation also demonstrates TeEcR belong to a very important gene in regulating the embryogenesis process, cell proliferation and differentiation in T.emma. The high mortality rates also shows the ecdysone mediated by TeEcR participateing in the regulation of the lifetime, and when TeEcR silented, the ants lifetime will be reduced.Above the results show that TeEcR may play some important roles for regulating the ant development and physiological activities. When the TeEcR gene is silenced, the T.emma is imperfect development, malformation and even death.3.The correlation between TeEcR and TeERR(1)After TeEcR-RNAi, real-time quantitative RT-PCR analysis and Western blot were performed to quantify the relative expression levels of TeERR mRNA and protein transcripts level changs. The results shown that compared with the control groups, TeERR in the experimental groups changes in the expression of mRNA and protein levels. No matter in the head or thorax, in the early larvae period (1 to 3 larvae) TeERR expression level is higher, in the 4th larvae and adult TeERR expression level is lower. However, There are the changes of TeERR expression level in T.emma 5th and 6th larvae period. We suggest that TeEcR and TeERR may have correlation, and the trend of changes may be associated with their function characteristic in regulating T.emma development. In 1-3 larvae instar stage may be mutual synergy, and in the 4th larvae and adult may be mutual antagonism effect, but these mechanisms are not clear, and need to be further studied.(2)After TeEcR-RNAi, using immunohistochemistry techniques to detect the TeERR proteins level change. The results shown that TeERR proteins in T.emma obviously decreased in the head of 4th larvae, compared with the control groups, the mushroom bodies not change, however, in the frontal lobe, the midbrain and hindbrain, the positive particles decreased significantly. TeERR protein positive particles reduced in the female head tissue, but in the middle of the mushroom bodies, central complex and optic lobe were not evident changes. We suggest that the relationship between TeEcR and TeERR varies from development period of T.emma, exhibiting tissue specificity. TeERR expression level decreased in the forebrain optic lobe, the midbrain olfactory lobe of 4th larvae and the female head tissue imply that the optic lobe, olfactory lobe and the female head tissue may be the interacting site between TeEcR and TeERR, and they may mutual participate in the olfactory system and visual system development regulation. |
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