Fuctional Analysis Of AQP Genes In Rice Stem Borer,Chilo Suppressalis Walker (Lepodoptera:Pyralidae)

The rice stem borer,Chilo suppressalis(Walker),is one of the key pests of rice,which distributes widely and distributes from north to south in China and it has strong adaptability to environment.The stable and high yield of rice is faced with a big challenge because of occurrence of C.suppressalis.Stusdies have shown that there are many kinds of AQPs in insects and were associated with water channel proteins(AQPs),which involved in transportation of water and regulation of osmotic pressure.In present study,two cDNAs are cloned from C.suppressalis by RACE,then their expression patterns over different life stages,distribution in tissues from 5th larvae and in different relative humidity and temperature treatment were monitored by real-time PCR.In addition,we used Xenopus oocytes to analyse the potential role of transport water and solutes of three AQPs in C.suppressalis.The main results are as following:Two full length of CsAQP12 CDNAs(CsAQP12L1 and CsAQP12L2)were cloned from C.suppressalis larvae by RT-PCR and RACE.The full length of CsAQP12L1 consists of 1604 bp with a 873 bp of opening reading fragment(ORF),which encodes 269 amino acids residues.The 5'UTR and 3'UTR are 219 bp and 512 bp,respectively.The full length of CsAQP12L2 is 1698 bp encoding 324 amino acids residues,with a 975 bp of ORF.The 5'UTR and 3'UTR are 210 bp and 513 bp,respectively.A comparison of deduced amino acid sequences showed that the CsAQP12L exhibited a high level of amino acid identity with other insects,including Amyelois transitella(89%),Plutella xylostella(87%),Papilio machaon(82%),and Papilio Xuthus(82%).Both CsAQP12L1 and-2 have nonconserved CPY(Cys-Pro-Tyr)and NPV(Asn-Pro-Val)motifs associated with unorthodox members of the aquaporin superfamily,which is same to Bemisia tabaci.The ar/R constriction site is not conserved with orthodox AQPs consisting of Tyr88,Val214,Gly(CsAQP12L1:223;-2:257),Leu229(CsAQP12L1:229;-2:263).TM topology predictions for both CsAQP12L1 and-2 varied:four(TMHMM),five(RHYTHM,HHMTOP),or seven(Pbobius,TOPCONS).We used different life stages and sexes in C.suppressalis to find expression changes of CsAQP12L1 and-2.CsAQP12L1 and-2 mRNAs were detected in all stages and sexes.Real-time quantitative PCR verified that CsAQP12Ll and-2 mRNAs were expressed in C.suppressalis foregut,midgut,hindgut,heads,epidermis and not in hemocytes,malpighian tubules and fat body.The highest expression of CsAQP12L1 was in heads,followed by hindgut and epidermis.Although expression of CsAQP12L1 and-2 in 1st instar larvae and male adults are high,there are no difference with male pupae,5th larvae,4th larvae,3rd larvae and 2nd larvae.The lowest experession of CsAQP12Ll and-2 were both in eggs.Our showed that there is no significant difference between different relative humidity and temperature,different stages and sexes(data not shown).Our water and solutes permeability assays in Xenopus oocytes showed that oocytes experessing CsAQP12Ll and-2 showed no significant permeability compared to control.Then we tested glycerol,trehalose and urea permeability of CsAQP12L1 and-2 in swelling rate.Compared to control,expression of CsAQP12L1 and-2 cannot significantly induce the rate of water loss.And another two kinds AQPs CsAQP1 and CsAQP3 can significantly increase the water permeability of the oocytes and these two AQPs cannot significantly induce the rate of water loss either.