Cloning And Functional Analysis On Genes Involving In Arginine Biosynthesis In Nilaparvata Lugens

During the long evolutionary history,there is an intimate symbiosis between insects and their microbes.In this symbiosis,the insects offer a stable inche to the latter and benifite from microbes to adapt to the environmental pressure.Nilaparvata lugens(Stal)is a serious phloem-feeding pest on rice.However,phloem sap in rice is riched with simple sugars but lowed with nitrogenous chemicals,such as essential amino acids.As phloem feeder,N.lugens harbours yeast-like symbionts(YLS)in mycetocytes formed by abdominal fat body cells.Previous studies have proposed that N.lugens lacks the ability to de novo synthesize 10 essential amino acids(arginine,histidine,isoleucine,leucine,lysine,methionine,phenylalanine,threonine,tryptophan and valine).However,YLS is speculated to involve in the process of arginine biosynthesis for N.lugens,avoiding this disablity.To our limitied knowledge,the speculation is obtained from bioinformatics analysis.In this paper,we cloned the genes involved in arginine synthesis pathway by RT-PCR and RACE,investigated the phylogenetic relationship of these genes,respecitively.Moreover,we evaluated the contributions of YLS orgined gene argininosuccinate lyase(EdArg4)to the growth and development of N.lugens by RNA interference(RNAi).The main results were listed as follows:1.Cloning and analysis of the arginine biosynthetic genes in N.lugens and YLSWe cloned the arginine biosynthetic genes in N.lugens and YLS.In summary,16 genes were cloned.Of these genes,5 members are N.Augens origined,encoding 3 enzymes(amino-acid N-acetyltransferase,EC 2.3· 1 · 1,NAGS;ornithine cyclodeaminase,EC 4.3.1.12,OCD;ornithine aminotransferase,EC 2.6.1.13,OAT).The remaining genes were YLS origined genes,encoding 11 enzymes(amino-acid N-acetyltransferase,EC 2.3.1.1,Arg2;acetylglutamate kinase,EC 2.7.2.8,AGK or Arg5,6;N-acetyl-gamma-glutamyl-phosphate reductase,EC 1.2.1.38;acetylornithine transaminase,EC 2.6.1.11,Arg8;acetylornithine deacetylase,EC 3.5.1.16,AODA;ornithine transcarbamylase,EC 2.1.3.3,Arg3;argininosuccinate synthetase,EC 6.3.4.5,Arg1;argininosuccinate lyase,EC 4.3.2.1,Arg4;glutamate 5-kinase,EC 2.7.2.11,Prol;glutamate-5-semialdehyde dehydrogenase,EC 1.2.1.41,Pro2;OCD).According to that,arginine biosynthesis pathway is consisted of 8 reactions that were catalyzed by EC 2.3.1.1,EC 2.7.2.8,EC 1.2.1.38,EC 2.6.1.11.EC 3.5.1.16,EC 2.1.3.3,EC 6.3.4.5 and EC 4.3.2.1 respectively.It is worth mentioning that N.lugens genome lacks most of arginine biosynthetic genes,except for the gene encoding EC 2.3.1.1.In contrast,YLS genome contained the complete functional gene set for arginine arginine biosynthetic pathway.Furthermore,the phylogenetic relationship analysis indicates that NAGS-like protein(amino-acid N-acetyltransferase,EC 2.3.1.1)derived from N.lugens f-ormed a large cluster with that of insects,and the orther genes derived from YLS formed a cluster with that of fungi species,respectively.This result suggests YLS might supply arginie to N.lugens.2.Expreesion pattern and RNA interference of EdArg4 in N.lugensIn present paper,we cloned a complete cDNA of an YLS gene EdArg4 that encodes arginineininosuccinate lyase.EdArg4 consisted of 1687 bp,with an ORF of 1401 bp and encoding 466 amino acids.The molecular weight of deduced EdArg4 is 52.3 kDa.EdArg4 possesses twenty four a-helices and five β-sheets,contributing to three distinct structural domains(Domain 1,2 and 3).Domains 1(residues 23-119)and 3(residues 368-437)have similar structure and topology with two helix-tun-helix motifs in perpendicular arrangement.Domain 2(residues 120-367)has nine helices and five of them form the central bundle with up-down-up-down-up topology.Noticeably,the three domains(C1,C2 and C3),the substrate-binding residues and the catalytic residue were conserved in EdArg4.Multiple sequence alignment revealed that EdArg4 shares 96%similarity with that from Cer at aphis brasiliensis YLS.The phylogenetic analysis indicated that the EdArg4 clustered together with that from C.brasiliensis YLS and Tolypocladium inflatum,supported by 100%bootstrap value.The tissue expression patterns of EdArg4 showed that the mRNA level was the highest in fat body,compared with midgut,leg,ovary and integument.The temporal expression profiles suggested that EdArg4 was expressed through all developmental stages,with the highest level in the first day of fourth nymphal instars,followed by the third day of third nymphal instars,and the lowest level is detected in the second day of second nymphal instars.In addition,we found that the target EdArg4 transcript and content of free arginine in hymolymph were significantly downregulated with 20%-60%and 50%after dsRNA injection,respectively.Furthermore,knockdown of EdArg4 caused higher nymphal lethality(15%to 25%)and longer nymphal development period(0.5 to 1 day),compared to the control.We also found that the expression levels of the other seven genes involved in arginine biosynthesis pathway were disturbed,comparing with control group.3.The impact of knocking down EdArg4 on NO signaling pathway,ILP signaling pathway,20E signaling pathway and TOR signaling pathwayqPCR was used to determine the expression of the genes in NO,ILP,20E and TOR signaling pathways at day 2,4 and 6 after dsRNA application,respecitvely.In case of NO signaling pathway,6 days after injection,the expression level of NlNOS-1 increased with 50%-100%,compared to the control.The mRNA levels of three downstream elements of the NO/cGMP signaling pathway,NlcGKs,NlvATPase-D and NlvATPase-E were down(or up)regulated.For example,at 4 and 6 days of dsRNA injection,the expression level of NlcGK-1 increased significantly with 20%-30%,respectively.In contast,the expression levels of NlNATPase-D and NlvATPase-E were decreased significantly with 20%-30%,respectively.In case of 20E signaling pathway,four days after injection,the expression level of NlE75 in dsArg4-2 groups increased significantly with 100%,compared to the control.Similarly,the expression levels of NIFTZ-F1 and NIPHM in dsArg4-1 groups were increased significantly by 70%.However,NlHR3 mRNA levels were significantly decreased with 70%-90%,respectively.In case of TOR signaling pathway,the expression levels of NlTor,NlelF4B and NlS6-2 in dsArg4-treated groups were decreased significantly at the second day,except for the up-regulateion for NIS6-2 in dsArg4-1 group.In case of ILP signaling pathtway,the expression levels of NlILP1,NlILP3,NlInR1 and NlInR2 were significantly decreased with 20%-90%at 2 days after injection,compared to the control.In conclusion,knockdown of EdArg4 inhibited NO and ILP signaling pathways,disturbed TOR signaling pathways and downstream genes of 20E in N.lugens.Our results provided the fundamental materials for studying the mechanisms of amino acids nutrition to the metamorphosis.