| Nuclear receptor belong to one type of important transcription factors, which involved in the regulation of various physiologic functions by integrating internal and external signal of organisms to maintain homeostasis. Nuclear receptor superfamilies are widespreaded in multicellular organisms, one class of nuclear receptors is known as orphan receptor which is short of the ligand. Estrogen-related receptor (ERR) as the first discovered orphan receptor owns highly homologous with estrogen receptors (ERs). ERR regulates the transcription of target gene by some cofactors or forming monomers and dimmers, which then bind to specific DNA sequences of target gene, namely hormones response element. ERR possesses various biological functions in regulating the growth and development of the mammals. However, ERR had been found in most of insects. ERR is involved in the glycolytic pathway of Drosophila melanogaster Meigen and also has effects in the castes differentiation of Polyrhachis vicina Roger according to some reports. About ERR physiological function in regulating Teleogryllus emma Ohmachi & Matsumura has not been reported by now. Ecdysteroid receptor (EcR) is a regulatory factor of signaling transduction pathway for ecdysone in insects, EcR usually combine with ultraspiracle protein (USP) into heterogenous dimers to mediate the transmission of ecdysone signal in insect body, and then affect diverse physiological processes of insects. Research has been demonstrated that the response elements of ERR and EcR were found to be similar, but whether they are functionally related has not been reported.Teleogryllus emma Ohmachi & Matsumura (T.emma) belong to insects of Orthoptera, Gryllidae. There are three stages during the development of T.emma, including the egg stage, 1th to 6th instar nymph stages and adult stages. The male and female can be distinguished during 4th instar, and covered with 80 days developmenting to adult. T.emma as an ideal experiment material is used to study the economic insect exploitation and utilization as well as the pest prevention because of its shorter developmental cycle and distinct instar division.In this study, the T.emma was used as experimental material to analyze the localization and quantization of TeERR mRNA and protein in the head and thorax of T.emma in different development stage by qRT-PCR, Western-blot and SABC immunohistochemistry. Besides that, RNAi technique was used to interfere TeERR mRNA expression, the change of T.emma phenotypic and behavior were observed, and the effects and possible molecular mechanism of ERR were analyzed. Finally, according to the expression variations of TeEcR mRNA and protein that were tested by the experiments as described above, the correlation between TeERR and TeEcR in regulating the growth and development of T.emma was put forward. The results are as follows:1. qRT-PCR was used to detect TeERR mRNA expression level in the thorax of T.emma in different development stage, combining our lab previous research that TeERR mRNA expression levels in head, integrated analysis imply that TeERR is found to be expressed in both head and thorax, and the expression level is different in various developmental stages and tissues.In the head, the expression level of TeERR mRNA was decreased with the development of individual, and falled to the lowest level in the 4th instar then raised until adult. From the 6th instar nymph to adult, TeERR mRNA expression level was higher in females than in males. The expression level of TeERR mRNA in the thorax is similar to the head, the difference extis in the 5th instar, TeERR mRNA expression level was higher in the head than in the thorax, but appeared an opposite situation in the 6th instar and adult. The expression level of TeERR mRNA was exhibited differentiation in the difference development stages, tissue, male and female. These results show that TeERR plays specific roles and is very important in regulating the growth and development of T.emma. The difference tissue of TeERR mRNA expression may be related to its regulation on the physiological functions. The TeERR mRNA expression in the females was higher than in the males, which probably show that TeERR may exhibit more impotant functions in regulating the females development. 2. Western-blot was used to detect TeERR protein expression level in the head andthorax of T.emma in different development stage. The results show that the protein expression of TeERR is similar to the transcription level, which indicate that the role of TeERR protein in regulating different development stages and tissues is almost the same as TeERR mRNA. These illustrates that the regulatory roles of TeERR for T.emma growth and development are essentially no difference at transcription level or translation level. 3. Immunohistochemistry was used to detect the location expression of TeERR protein in the head of 4th instar nymph and adult. The results indicate that TeERR protein is widely expressed in the head of T.emma, and presents diversity in different tissues.In the forebrain, strong positive particles of TeERR protein were widely distributed in the mushroom bodies, central complex body and protocerebral bridge, especially in the mushroom bodies crown and Kenyon cells, followed by the central complex body and protocerebral bridge. In the olfactory lobe of midbrain and tritocerebrum, positive particles were still obvious, however, in the corpora allatum, positive particles were undetectable. The positioning results of TeERR protein in the T.emma head indicate that TeERR may play some important roles in regulating the development of brain's nerve and the behaviors of T.emma.4. RNAi technique was used. TeERR-dsRNA which synthesized in vitro were delivered into the individuals through appropriate ways to interfere the expression of TeERR, then the expression variations of TeERR and TeEcR were tested at mRNA and protein level by the experiments as described above. Besides that, the change of T.emma phenotypic and behavior were observed. The results indicate that the mortality and abnormality rate increased after the expression of TeERR was disturbed. The change mainly represents as dying of the eggs, expending of the abdomen, shrinking of the wings, and molting time increased but which most couldn't complete this behavior. These reflect that ERR is a key gene in regulating growth and development of T.emma. The abnormal expression of TeERR can effect the normal expression of TeEcR, mainly reflected in the TeEcR expression level reduced in the 4th instar and increased in the 5th and 6th instar after the expression of TeERR was inhibited. In the other stages of the T.emma development, the decrease of TeERR expression seemed to have no obvious effect on TeEcR expression. These show that a closely relevance may exist in these two genes, they could promote each other in the 4th instar and inhibit each other in the 5th and 6th instar. Synergistic effects and inhibitive effects were observed between TeERR and TeEcR, which means that they may interact with each other to regulate the growth and development of T.emma.This is the first study that the expression of ERR protein is systematically studied throughout all stages of development in the incomplete metamorphosis insects, which further demonstrated the roles of ERR on regulating the growth and development of insects, enriched research concent in the entomology field. RNAi on the expression of TeERR was successfully performed in this study, and the interaction between TeERR and TeEcR in regulating the growth and development of T.emma was explored. These may provide theoretical basis for studying the genes level regulation related with the economic insect exploitation and utilization as well as the pest prevention. |
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