| Under the influence of different external environmental conditions,the same organisms may develop into individuals with different morphologies and habits,namely development plasticity.Development plasticity is an extremely important survival strategy for organisms to adapt to the changing ecological environment under the pressure of natural selection.Many insects,such as bees,ants,locusts,and rice planthoppers,have this capacity of development plasticity.Rice planthopper is an important insect pest,which is divided into two wing form: Long-and short-winged morphs.The long winged individual mainly comes from poor host nutrition.The long wing individual can migratory flight,the fore-wing is longer than the hind-wing,and both of them are membranous and the area is big,the motor ability is strong but the feeding and the reproduction ability is weak;The short winged individual cannot fly,the length of the fore-wing is very short,and the hind-wing is degenerate into wing-buds.The motor ability is weaker but the feeding and reproduction ability is stronger.With the Wing polymorphism,the rice planthopper is highly competitive under the natural rule of the fittest.Over the years,the wing dimorphism of rice planthopper has attracted the interest of many insect scholars,and has made a lot of research progress.The switch gene of the brown planthopper for wing morph transformation has been identified: insulin receptor Insulin Receptor 1/2(In R1/2).However,In R1/2 is a upstream regulator,while wing development genes usually act as the specific executors in the process of wing development.However,up to now,how the wing development genes participate in or respond to the process that the switch gene insulin receptor 1/2 determine wing differentiation is still unclear.Ultrabithorax(Ubx),Decapentaplegi(Dpp),Vestigial(Vg),Apterous-a(ap A)were selected as our research objects,and we used homology,molecular cloning,microinjection RNAi,quantification PCR,in-situ hybridization,Gal4-UAS transgenic fly and some other biological techniques to study these four genes' function in wing development with wild-type and our lab-maintained strain of long-winged and short-winged strains of rice planthoppers,respectively.The main results achieved are as follows: The regulation of Ubx(Nl Ubx)on the wing development and the wing differentiation.Through quantitative PCR and In-situ hybridization,it was found that the Ubx expression pattern of rice planthoppers was different from that of the known insecta Ubx,which was more similar to that of crustaceans.Ubx of rice planthoppers were expressed in both fore-and hind-wing,and the expression levels of the short winged strain was higher than that of long winged strain in the 5th instar nymph wingpad.In both long-winged and short-winged strains,the Ubx expression of hindwing pad of the 5th instar nymph was higher than that of forewing pad.However,the known Ubx of insecta is only expressed in the hindwing,and the fore-wing is not expressed.The Ubx protein of rice planthoppers is also structurally different from the known insecta Ubx,which is more similar to that of crustaceans.The MXSXFE motif sequence of Nl Ubx is MMNSYFE,QA motif sequence is QAQAAK,and Ploy-Ala motif sequence is AAALAASQAQ.The known Ubx of insecta,the sequences of these three structural domains are: MNSYFE,QAQAQK and AAAAAAAAAA.Ubx of rice planthoppers is not only expressed in the forewing,but also plays an important role in the development of the forewing and regulates the size of the forewing and the hindwing.By RNAi interference,reducing the Ubx expression of rice planthopper,produced the following phenotype: both long winged and short winged strains of adult forewing and hindwing were deformity,short wings adult forewing and the hindwing bristles increased,The changed size of the hindwing of the short winged adult are particularly obvious.Ubx of rice planthopper has different regulatory modes for its target genes in long-winged and short-winged strain.According to previous studies,Nl Dpp,Nl Vg,Nlap A and Nl ASH genes are the target genes of Ubx.By q PCR,the expression of these target genes were detected after the interference of Ubx in the long-and short-wing strains of brown planthopper.The results showed that Nl Ubx had different regulatory effects on the target genes in the fore-and hindwing of the long-winged and short-winged strains.Considering that Nl Ubx has different expression levels of the wingpad of nymphs in the short-winged and long-winged strain,and that Nl Ubx has a regulatory effect on the size of the wings,it is speculated that Nl Ubx may regulate the size of the wings of brown planthopper by dose effect.To test this speculation,in this study we used Gal4-UAS transgenic flies strains to drive BPH Ubx(Nl Ubx),fruit flies(Drosophila melanogaster)Ubx(Dm Ubx Ia),brine shrimp(Acanthokara kaputensis)of Ubx(Ak Ubx)in the fruit fly wing(Ubx empty in this tissue)ectopic expression,and through the three different intensity of promoter(Nub-Gal4(strong),Sd-Gal4(in);C765-gal4(weak)allows transgenic fruit flies to express different doses of Ubx in their forewings.Results showed that the Dm Ubx Ia expressed in forewing with high,medium and low doses,flies forewing is still haltere,and Nl Ubx and Ak Ubx in medium doses,fly wing into a smaller wing;In low doses,flies forewing is transformed into a relatively completely wing.Therefore,the dose effect of Dm Ubx Ia is not obviously,while the dose effect of Nl Ubx and Ak Ubx is obviously,and the size of wings can be flexibly changed through the change of dose.It also indicates that Nl Ubx and Ak Ubx have a weaker inhibitory effect on thewing develoment genes than Dm Ubx Ia.By point mutation technology makes drosophila forewing express mutation Dm Ubx Ia,Nl Ubx,Ak Ubx,proving the Nl Ubx motif for inhibiting wing development genes significantly weaker than Dm Ubx Ia,similar to Ak Ubx suppression ability.Therefore,the Ubx protein of rice planthoppers retains or partially retains the ancestral Ubx structure,which is the molecular basis for its sensitive dose effect.On the basis of this,we also found that the expression of rice planthopper In R2 became higher by feeding nutrient-rich tillering stage rice,and the expression of rice planthopper In R1 became higher by feeding nutrient-poor ripening stage tillering stage rice.Ubx also has a response to host nutrition,that is,the expression of rice planthopper Ubx increased by feeding the nutrient-rich tillering stage rice and decreased by feeding the nutrient-poor rice seedling stage rice.Regulatory effects of ap A(Nlap A)on wing development of brown planthopperAp A was significantly higher in the 2rd and 4th instar nymphs of short winged strain brown planthoppers than that of long-winged strain.After Nlap A is disturbed,the forelegal wing bristles disappear,the wings curl,and the wings length became shorter.Further studies found that ap A RNAi resulted in less wing bristles of fore wing of rice planthoppers because of the decreasing expression of ap A resulted in decreased expression of the key gene achaete-scute homolog(Nl ASH)controlling the development of bristles.Nlap A is the upstream regulator of Nl ASH.Regulation of wing development by Dpp(Nl Dpp)and Vg(Nl Vg)Expression levels of Dpp and Vg were significantly different in nymphs of long-winged and short-winged stable,and were significantly higher in long-winged nymphs than in short-winged strain.The expression of Dpp and Vg in the 3rd instar nymphs of the long and short wings strain were interfered.After Vg was interfered,blisters appeared in the top corner of the forewing and hindwing of long-winged adult.However,there are no blisters in the top corner of the forewing gluteal region of the adult short-winged adult(the hind wing is a fin-bud shape,and there is no top corner of the buttock region).In this study,four wing development genes,Ubx,Dpp,Vg,and ap A were selected as our research objects.The difference of expression profiles were compared between long-winged and short-winged BPH strains for revealing the regulation mechanism of wing development and differentiation.Our results will enrich the theory of the molecular mechanism of the wing morph differentiation of incomplete metamorphosis insects.It is of great significance to reveal the internal causes of the rice planthoppers outbreak,to realize the fine prediction of the migration time and to control the source rice planthoppers moving out. |
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