The Preliminary Analysis Of Flightin And TnC4Genes In The Brown Planthopper(Nilaparvata Lugens)

Brown planthopper (BPH, Nilaparvata lugens), a wing dimorphism species, is one of the most important pests of rice. Flightin and TroponinC4(TnC4) are both muscle related proteins. Flightin was originally found in indirect flight muscle (IFM) of Drosophila melanogaster, bingding with myosin. It guides the correct assembly of thick filaments and plays an important role in muscle stretching activity. TnC4is one of TroponinC (TnC) isoforms and promotes actin-myosin interaction by specifically combing with Ca2+, so that muscle contraction occurs. The two proteins are very important for structural integrity and sarcomere function, but their expressional and functional differences between macropterous and brachypterous of BPH are unknown. We amplified flightin from BPH cDNA by PCR and3’RACE. The result showed that flightin open reading frame (ORF) is630bp in length, which is potential to encode a predicted25.49kDa protein containing209amino acid residues. At the same time, we cloned TnC4from female BPH. The TnC4ORF is459bp in length, which is potential to encode a predicted17.42kDa protein containing152amino acid residues. The alignment of amino acid sequence showed that flightin is relatively conservative, while TnC4is highly conservative with a Ca2+bingding site in the N terminal conservative regions. Both flightin and TnC4were successfully expressed in Escherichia coli and the expressed proteins were used to prepared polyclonal antibodies. RT-PCR and western blot analysis showed that flightin and TnC4were expressed much higher in macropterous females than those in brachypterous female adults. They both start to express from the first day of the adult stage and mainly located in the thoracic tissue. Immunofluorescence localization experiments further confirmed that flightin and TnC4distrubuted only in IFM of BPH adults. After microinjecting to the third instar BPH with dsRNA of flightin and TnC4, both the two gene expression levels were significantly down-regulated, and the BPHs lost their ability to fly. In this study, we verified the differential expression of flightin and TnC4between macropterous and brachypterous adults. Our results laid a basis for the further study of flight muscle development, wing dimorphism, migration and control of this important rice pest.