Identification And Functional Characterization Of Homeobox Genes In The Brown Planthopper,Nilaparvata Lugens

Homeobox genes are highly conserved and perform an analogous function in metazoans.Homeobox genes encode about 60 amino acids called homeodomain（HD）.The HD is a DNA binding domain and hence homeodomain proteins are essentially transcription factors.The brown planthopper,Nilaparvata lugens（St（?）l,1854）（Hemiptera:Delphacidae）,an incomplete metamorphosis,is one of the most notorious pests in China and Southeast Asian countries.Moreover,the homeobox genes have rarely been studied in N.lugens.In this study,we identified homeobox genes in N.lugens based on genomic and transcriptomic database and analyzed the gene functions through RNAi,q RT-PCR,western blot,Immunohistochemistry（IHC）,etc.The main results are as follows:1.This study identified amount and classification of homeobox genes in N.lugens.We identified 94 homeobox genes and 75 gene families through analysis of BPH genomic and transcriptomic database.Based on the analysis of sequence of amino acid,conserved domain and HD,all homeobox genes could be divided into 9 classes,including ANTP,PRD,LIM,POU,SINE,TALE,CUT,ZF,and CERS,and the ANTP class could be divided into HOXL and NKL subclass.With the exception of Pknox,93BPH homeobox genes were found to be Drosophila orthologues.Orthologues for the Pknox gene are present in insects such as the honeybee,red flour beetle although none has been found in Drosophila.In addition,11 homeobox genes（cad,Dfd,btn,Dll,Dr,al,oc,prd,repo,ap,and Lim3）had been duplicated during the evolution of the BPH.2.This study analyzed spatio-temporal patterns of BPH homeobox genes.The spatio-temporal expression of BPH homeobox genes was examined by RNA-seq data derived from different BPH developmental stages,including embryonic,nymphal and adult stages at 15 time points,and 8 different tissues from adult females.The developmental profile showed that the majority of homeobox genes（n=81,88%）had expression that was biased to the egg stage indicating that these genes played important roles during embryonic development.Tissue distribution analysis showed that 54homeobox genes（59%）were extremely tissue-specific.A relatively higher expression of 10 genes,18 genes,10 genes,16 genes genes was detected in the ovipositor,antenna,head,and ovary,respectively.3.This study revealed the function of homeobox genes in N.lugens.Totally 89genes were PCR amplified and then used as a template for double-stranded RNA（ds RNA）synthesis.The RNAi-mediated gene knockdown assay in 4^st-instar nymph showed that depletion of 8 homeobox genes（pb,abd-A,vnd,ap-1,otp,Lim3＿2,vvl and ct）caused complete lethality in nymphs indicating the importance of these genes in nymphal development.Knockdown of additional 14 homeobox genes resulted in four major morphological defects.Adults treated with ds RNA targeting Dll＿1 and Dll＿2failed to establish distal limb structures.Knockdown of eyg and Scr led to adults with a shrunken,thin body.Knockdown of al＿2 and Awh led to a malformed ovipositor and the first valvifers were widely extended.Knockdown of Ubx,nub,Dr＿1,ap＿1,mirr,zfh2,exd and hth led to abnormal wings after eclosion.RNAi-mediated gene knockdown assay in 5^st-instar nymph showed that knockdown of 9 homeobox genes（schlank,Lim3＿2,ap＿1,Pknox,vis,abd-A,C15,nub and prd＿2）affected reproduction.The parental RNAi for 8 Hox genes（lab,pb,Dfd＿1,Scr,Antp,Ubx,abd-A and Abd-B）showed that depletion of 4 homeobox genes（lab,pb,Dfd＿1 and Scr）led to the offspring with malformed head and appendages.Silence of Ubx led to additional leg-like appendages in A1 segment.Knockdown of Abd-B resulted in an additional abdomen segment.RNAi for abd-A led to additional leg-like appendages in A2-A8 and resulted in lethal embryo.The parental RNAi indicated that the BPH Hox genes also took part in the determining some morphological traits and played some different roles between the holometabolous and hemimetabolous insects.4.This study revealed the function of Ubx gene in N.lugens.RNAi-mediated silencing of Ubx in short-winged BPH nymphs significantly induced the development of wing-like appendages from wingbuds,and this effect likely depends on the activity of Akt,a key component of the insulin/insulin-like signaling pathway.RNAi knockdown of Ubx in long-winged BPH nymphs led to a transformation from hindwings to forewings.The q RT-PCR,western blot and IHC assay showed that Ubx was easily detected in T3 wingbuds and maybe a trace amount in T2 wingbuds.Additionally,silencing of Ubx not only dramatically changed the T3 morphology,but also led to jumping defect of T3 legs.First-instar nymphs derived from parental RNAi had an additional leg-like appendages on A1.