| Polyrhachis vicina Roger(P.vicina),belongs to Formicidae and Polyrhachis.It is mainly distributed in Yunnan,Guangxi and Zhejiang in China and in Burma,India and Australia.It is a typical social insect that lives in groups.P.vicina is an economic insect which has medicinal and therapeutic value.It has formic acid,28 kinds of amino acids,many kinds of vitamins and many kinds of trace elements,especially for the content of zinc is 180 mg/kg.Many research showed that P.vicina can assist in the treatment of hepatitis B,rheumatic arthritis,rheumatoid arthritis,neurasthenia and other diseases.In 1993,the China Ministry of Health identified it as an excellent ant species with both food and medicine harmless.In recent years,many scholars have carried out researches on the development process of P.vicina,in order to establish a systematic development biology research system of social insect,and lay a solid foundation for the theoretical research,species protection and industrial application of P.vicina.Ecdysteroid hormones(EH)is an important hormone regulating molting and metamorphosis of P.vicina and other insects.Its active form is 20-hydroxyecdysone(20E).Meanwhile,the juvenile hormone(JH)is also a very important endogenous hormone besides ecdysone in insect body.JH is secreted by corpora allata,its main function is to keep the characteristics of ant in its infancy,prevent the differentiation and metamorphosis of internal organs,that is to prevent the appearance of adult characters.As soon as pupation,the secretion of JH will be reduced or stopped.The treatment of adult insects with JH can cause sterility.The secretion and function of JH are mainly regulated by some key genes and proteins,among which methoprene tolerant(Met),as a potential receptor of insect JH,plays an important role in the process of growth and development.At the same time,there is another receptor,estrogen related receptor(ERR),which is closely related to the growth,development and reproduction of insects.The regulation of gene transcription and expression mediated by ligand-receptor binding has become one of the hotpots in molecular biology.The proteins encoded by ERR and Met,two major genes of P.vicina involved in this experiment,are very important transcription factors and participate in a variety of physiological activities in insects.Early studies on ERR mainly focused on its role in mammals.Although it has been reported that there is also ERR in insects,but there is no estrogen in insects,so how ERR plays a role in insects deserves further attention.In the early research,our laboratory used P.vicina as experimental materials,using molecular biology,histochemistry,physiology,behavior training and other related biological means,systematically studied the characteristics and some molecular mechanisms of P.vicina in the individual reproduction,growth and development,physiological activities,etc.,which laid a solid foundation for the smooth development of the experiment in this paper.For example,previous studies in our lab showed that there was a correlation between ERR and Ecdysteroid receptor(EcR)gene,suggesting that ERR may be involved in the regulation of gonadal hormones.Besides Met,there are also Kruppel-homologl(kr-h1)Steroid Receptor Coactivator(SRC)and other hormone participate in the signal pathway of JH in insects.In this study,RNAinterference(RNAi),real-time quantitative PCR and Western blotting were used to explore the functional relationship among ERR,Met,Kr-h1 and SRC.The purpose is to explore the functional relationship between ERR and JH signaling pathway,so as to lay a theoretical foundation for better understanding the growth and development process of P.vicina.At the same time,it is of great significance for the further study of ERR function in insects,the understanding of the growth and development mode of insects,the regulation of beneficial insect reproduction ability and the control of harmful insects.The results are as follows:1.The partial cDNA sequences of Met,Kr-h1 and SRC were successfully cloned and identified in P.vicina.The PCR products were detected by agarose gel electrophoresis,and the size was consistent with the expectation.Sequencing results were compared by blast sequence homology and bio-informatics analysis,showed that the partial cDNA sequences of Met,Kr-h1 and SRC cloned from P.vicina were highly homologous with the corresponding genes of known model insects.The accuracy of the cloned gene was confirmed,so it was named PvMet,PvKr-h1 andPvSRC.2.In this chapter,after cloning partial cDNA sequences of PvMet,PvKr-h1 andPvSRC,and the known cDNA sequences of PvERR,the mRNA expression patterns of the four genes were analyzed in the five different development stages:2nd instar larvae,4th instar larvae,pupa stage,female adults and male adults,as well as the changes of the expression levels after applying exogenous juvenile hormone JH?.The results showed that:(1)From the 2nd instar larvae to the adults,the expression of PvERR increased gradually,showing significant difference.(2)The expression of,PvMet,PvKr-h1 and PvSRC decreased gradually from the 2nd instar larvae to the pupa stage,and there was a significant difference.(3)There was no significant difference in the expression of PvMet between 2nd instar larvae and female adults,but between female adults and male adults,there was significant difference.(4)There was also no significant difference in the expression of PvKr-h1 between male adults,female adults and the 2nd instar larvae.(5)Meanwhile,no significant difference in the expression of PvSRC between male adults,female adults and the 4th instar larvae.PvERR can participate in the physiological regulation of 20E signal pathway during the growth and development of P.vicina.Previous studies in our laboratory have confirmed this result.However,20E is closely related to JH signaling pathway,which can mediate the some identical gene expression to complete the metamorphosis and development of insects.In this chapter,the study shows there was a correlation between PvERR and JH signaling pathway in P.vicina.The expression of PvERR is negatively correlated with.the expression of genes related to JH signaling pathway,which coincides with the known 20E regulation of insect metamorphosis,JH control of molting and larva morphology.Therefore,PvERR and JH signal pathway are considered to be related.The expression of key genes of JH signaling pathway in 4th instar larvae and pupae was relatively low in 5 different developmental stages,so the two stages were chosen.Then,the expression of PvERR,PvMet,PvKr-h1 and PvSRC was studied after 1,6,12 and 24 hours of JH ? treatment.The results showed that after 1 hour of JH ?treatment,there was no significant change in the expression of the four genes in the 4th instar larvae and pupa stage.After 6 hours of treatment,the expression of PvERR decreased significantly,and with the extension of treatment time,the expression decreased gradually,especially in the 4th instar larvae stage.After 6 hours of treatment,the expression of PvMet in 4th instar larvae and pupae increased significantly,which was positively correlated with the treatment time.After 6,12 and 24 hours of treatment,the expression of PvKr-h1 in 4th instar larvae and pupa stage increased significantly,but there was no significant difference among the three time treatment groups.After 6 hours of treatment,the expression ofPvSRC in 4th instar larvae and pupae increased significantly,and showed a positive correlation with the treatment time.These results indicate that the expression levels of PvMet,PvKr-h1 andPvSRC genes in JH signaling pathway are significantly increased under the induction of exogenous JH?.It is preliminary confirmed that these three genes are indeed involved in JH signaling pathway in P.vicina.PvERR was also able to respond to exogenous JH?,but the expression of PvERR was contrary to PvMet,PvKr-h1 andPvSRC,that indicating a negative correlation between PvERR and JH.3.The study further confirmed the correlation between PvERR and JH signaling pathway by RNA interference technology.The 2nd instar larvae and female adults were used as the experimental materials.Double-stranded RNA of PvERR was designed by PvERR gene sequence,named as dsPvERR.RNA interferencewas treated with different doses and times by feeding.After selecting the appropriate interference dose and time,the changes of mRNA and proteinexpression of key genes PvMet,PvKr-h1 andPvSRC in JH signaling pathway were detected when PvERR expression was inhibited,and the further correlation betweenPvERR and JH signaling pathway was analyzed.The results showed that compared with the control group and GFP-RNAi group,after PvERR-RNAi,the mRNA and proteinexpression levels of PvMet,PvKr-h1 and PvSRC in the 2nd instar larvae and female adults were significantly increased,which indicated that down regulating the mRNA expression of PvERR would enhance the expression of key genes PvMet,PvKr-h1,PvSRCin JH signaling pathway.4.This study found that PvMet,PvKr-h1 and PvSRC were involved in JH signaling pathway in the P.vicina,and the expression of PvKr-h1 and PvSRC would be regulated byPvMet,which might be related to the upper and lower levels.PvERR and PvMet can interact with each other through 20E and JH signaling pathways in insects.The 2nd instar larvae and female adults were used as the experimental materials,design dsPvMet,which was treated with RNAi in different doses and at different times by feeding method to determining the best doses and treat time.The changes of mRNA and proteinexpression levels of PvERR,PvKr-h1 andPvSRC were detected when the expression of PvMet was inhibited.Compared with the control group and GFP-RNAi group,the expression of PvKr-h1 and PvSRC in the 2nd instar larvae and female adults decreased significantly after PvMet-RNAi.After PvMet RNAi,the expression of PvERR was enhanced,which was consistent with that of PvMet-RNAi.5.The results showed that after PvMet-RNAi,it could impair the PvKr-h1,PvSRC and PvERR respond to exogenous JH?.After PvKr-h1-RNAi,it could impair thePvSRCrespond to exogenous JH?.There may be one-way regulation of PvMet to PvKr-h1.PvMet and PvKr-h1 were directly related to the response of PvSRC to exogenous JH?.PvERR response to exogenous JH?wass dependent on PvMet expression,but does not require PvKr-h1 participation.In this chapter,the 4th instar larvae were used as experimental materials,design dsPvKr-h1,different doses of PvMet-RNAi and PvKr-h1-RNAi were respectively treated for 6 days by feeding method.The mRNA expression levels of PvMet and PvKr-h1 after interference were detected respectively to determine the best dose.The 4th instar larvae were divided into eight groups,the four were PvMet-RNAi group,and the other were PvKr-h1-RNAi group.Every four groups were treated with acetone,exogenous JH ?,dsPvMet or dsPvKr-h1 for RNAi,ds PvMet or dsPvKr-h1 for 6 days,then treated with exogenous JH? for 24 hours.At the end of the experiment,the mRNA expression of related genes was detected.PvMet-RNAi group:after adding exogenous JH?,the expression level of PvKr-h1 and PvSRC increased significantly,and the mRNA expression level of PvERR was inhibited;after PvMet-RNAi,the mRNA expression level of PvKr-h1 and PvSRC decreased significantly,and the mRNA expression level of PvERR increased significantly.After PvMet-RNAi treatment for 6 days,then treated with exogenous JH?for 24 hours,the expression ofPvKr-h1 and PvSRC could not be up regulated,and the expression of PvERR was no longer inhibited.It suggested that PvMet is required for these three genes to respond to exogenous JH?.PvKr-h1-RNAi group:after adding exogenous JH?,the mRNA expression level of PvMet and PvSRC increased significantly,mRNA expression level of PvERR decreased significantly.After PvKr-h1-RNAi,the expression of PvMet and PvERR did not change significantly,and the expression level of PvSRC decreased significantly.After PvKr-h1-RNAi treatment for 6 days,then treated with exogenous JH? for 24 hours,it was found that the expression of PvMet was still significantly up-regulated,and the expression of PvERR was still significantly reduced,but the expression of PvSRC could not be up-regulated.To summarize,the partial cDNA sequences of Met,Kr-h1 and SRC were successfully cloned and identified in P.vicina,the four genes PvERR,PvMet,PvKr-hl and PvSRC were analyzed in the five different development stages:2nd instar larvae,4th instar larvae,pupa stage,female adults and male adults,preliminary confirmed that there was a correlation between PvERR and JH signaling pathway.The relationship between PvERR,PvMet,PvKr-hl and PvSRC and their roles in the JH signaling cascade were discussed by RNAi and JH? induction technology,and it was further clear that PvERR was involved in JH signaling pathway.These results provide valuable experimental data for further understanding of the growth,development and reproduction of social insects and the complex physiological mechanism of higher animals. |
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