Functional Study On PSMA1 Gene Of Nilaparavata Lugens(St?l)

Nilaparvata lugens(St?l)is one of the main pests on rice in China and many parts of Asia.The growth and yield of rice are seriously harmed and reduced by its behaviors such as sap-sucking on rice,oviposition and transmission of rice virus disease.N.lugens has characteristics of migration,gregariousness and explosibility,which make it really difficult to control.The N.lugens,which eats only rice juice and has an unbalanced feeding structure,can still grow and reproduce normally,which is closely related to its internal yeast-like symbionts(YLSs).YLSs could provide amino acids,sterols and other essential nutrients for N.lugens,as well as improve the pesticide resistance and immunity of N.lugens,thus enhance the adaptability of N.lugens.YLSs are mainly distributed in the abdomen of N.lugens and transmitted between N.lugens generations by vertical transmission,but the specific mechanism of the transmission is still unclear.In this study,the PSMA1(Proteasome subunit,alpha type 1)is a subunit in the 20 S core particle ? ring of the proteasome complex,which plays an important role in the process of proteasome degradation or catalysis.It has been confirmed that YLSs are first released from fat bodies into hemolymph when the vertical transmission is started,and then enter the ovary through the epithelial plug of the ovariole,and finally YLSs enter the oocyte to complete the transmission.After transcriptome sequencing of ovariole petiole,epithelium piug(EP)and so on,we found that the expression of PSMA1 in the EP follicular cells of N.lugens ovariole is more than twice as large as its neighbor cells.Therefore,in this study we designed a series of experiments to study the function of PSMA1 in the vertical transmission process of YLSs.In the study,we also found that the expression of PSMA1 interfered with RNA interference technology leaded to the delay of N.lugens ovary development.The followings are the results of this study.1.Sequence analysis and expression characters of PSMA1 in N.lugensThe PSMA1 gene of N.lugens was cloned with the length of 1897 bp and the open reading frame(ORF)length of 846 bp,which encodes 281 amino acids and the predicted protein size is 30.94 kDa with the pI value of 6.03.It was found that PSMA1 gene was relatively conserved in insects through amino acid comparison and evolutionary tree analysis.The expression of PSMA1 at different developmentalstages of N.lugens was detected and analyzed via fluorescence quantitative polymerase chain reaction(qPCR),and we found that PSMA1 was expressed in every stage.In nymph stage,the expression of PSMA1 increased gradually with the development of nymph.During 1~5 day-age of adults,the expression of PSMA1 in macropterous females increased gradually during 1~3 day-age and reached the highest value on 3 day-age,and then gradually decreased.When analyzing the expression level of PSMA1 in different tissues of N.lugens,it was discovered that the expression level of PSMA1 was the highest in the ovary,followed by the fat body,and the lowest in the gut.2.RNA interference and functional analysis of PSMA1 in N.lugensDouble-stranded RNA(dsRNA)was synthesized for interfering PSMA1 by in-vitro transcription to interfere with the newly-emerged macropterous females of N.lugens.Compared with the control group,the expression of PSMA1 in the experimental group was significantly decreased.After RNA interference,compared with the control group,the ovary development of N.lugens in the experimental group was delayed for 7~8 days,and the number of eggs was reduced by about 35%~40%,and the hatching rate of eggs was also significantly reduced.In addition,compared with the control group,starting from the second day after RNA interference,the experimental group had a large amount of free abdominal fat bodies with loose structure,and a large number of symbiotic bacteriocytes were released.Later,in the YLSs counting experiment of mature ovary eggs,the result showed that the number of YLSs in the experimental group was significantly higher than that in the control group.In the YLS counting experiment of 1 day-age egg after oviposition,the result also showed that the number of YLSs in the experimental group was significantly higher than that in the control group.Thus,it is speculated that PSMA1 plays important roles in the ovary development of N.lugens and the transmission of YLSs.