Functional Studies Of Growth And Development Related Genes In Chilo Suppressalis By RNAi

The rice stem borer,Chilo suppressalis Walker,(Lepidoptera:Pyralidae)is one of the most important rice pests in Asia,north Africa and southern Europe.Currently chemical control strategy is used to manage this pest.The extensive and long term use of pesticides do not only pose serious dangers to the environment and human health but has also resulted in the development of resistance by Chilo suppresalis to a wide variety of pesticides including chlorantraniliprole.Thus,there is an urgent need for an alternative effective and environmental friendly strategy to be developed against this pest.RNA interference(RNAi)is a technology that has become a potential robust tool for crop protection against insect pests.It is a specific gene silencing mechanism mediated by double-stranded RNA(dsRNA),which has been harnessed as a useful reverse genetics tool in insects.The selection of target genes is a key step in achieving pest control with RNAi.Juvenile Hormone(JH)and Molting Hormone(MH)are two important insect hormones that play key roles in the insect’s development and reproduction.During larval growth and development,alterations in any of these hormones results in developmental delays and sometimes death.Therefore,the key genes involved in the biosynthesis of these two hormones could be used as potential targets for pest control using the RNAi technology.In this study,a transcriptome of C.suppressalis was carried out,based on this data,the molecular characteristics and functions of some important genes involved in JH biosynthesis,its metabolism,and 20E biosynthesis in C.suppressalis were analyzed comprehensively.The main results are as follows;1.Transcriptional analysis of C.suppressalisIn this project,after low quality reads were screened,100,748,860 clean reads were obtained via Illumina Hiseq 2000 sequencing.Then all the reads were assembled which tallied up to t 46,603 Unigenes.The average length and N50 of Unigenes were 966 bp and 1,904 bp,respectively.There were 19,990(42.89%)Unigenes annotated by 7 functional databases,and 17,647 Unigenes annotated in NR database.Among these Unigenes,14,383,8,048 and 5,106 Unigenes were annotated in GO,KOG and KEGG databases,respectively.2.The gene functional study of JH biosynthesis and metabolic relative genes in C.suppressalisIn this study,13 selected genes involved in JH biosynthesis and metabolism and the receptor of JH were cloned and characterized.Among these genes,the open reading frames(ORF)of Csaact、Cshmgs、Cshmgr、Csmevk、Csmevpk、Csmevppd、Csippi、Csfpps.Csjheh and Csjhdk were obtained.The putative amino acids sequences,physicochemical properties,transmembrane regions and signal peptide were predicted and annotated in details.The gene phylogenetics indicated that these genes are clustered together in the lepidopterans i.e.,Bombyx mori,Helicoverpa armigera and Cnaphalocrocis medinalis.While this relationship was far when compared with other insects such as Dipterans and Coleopterans.These results were matched with the traditional taxonomic position they have.The expression patterns of these genes(JH biosynthesis relative genes:Csaact,Cshmgr,Csmevk,Csmevpk,Csippi and Csfpps;JH metabolic relative genes:Csjhe,Csjheh and Csjhdk;JH receptor gene:Csmet)during different developmental stages(early and late 1st to 3rd instar larvae)and different tissues(head,midgut,epidermis,fat body and trachea)were analyzed by q-PCR.Results revealed that the expression patterns of the 6 genes involved JH biosynthesis were similar,with the highest expression level on the first day of each instar which significantly reduced when the larva developed into the next instar.All of the 6 genes were highly expressed in the head,and there was a significant difference between head and other tissue.This may be because the corpus allatum(CA)is located in the head of larva.The expression patterns of JH metabolism genes were more complex.Results revealed different expression patterns for these 3 genes with at least one of them being significantly increased during ecdysis.Furthermore,,the expression proportions of these 3 genes were different in different instars.These observed differences may have come about as a result of differences in the action mode and time of the 3 metabolizing enzymes.Spatial patterns showed that both Csjhe and Cshjheh were highly expressed in the midgut and fat body,however,Csjhdk was highest only in the midgut.The expression pattern of Csmet in different development stages was similar to the JH biosynthesis related genes,and it was not only high expression in head but also in other tissues which are the target tissues of JH such as the fat body and midgut.After silencing the expression of these genes by feeding bacterially expressed dsRNA,the mRNA levels of these genes in treated larvae were significantly reduced compared with the control(dsegfp)except in Csaact which was significantly increased in dsaact treatment.Furthermore,knocked down of these genes resulted in high larval mortality and extended development durations.For example in the dshmgr,dsmevk,dsmevpk and dsfpps groups,15 days of ceaseless feeding resulted in significantly reduced survival rates of 29.8%,62.5%,59.2%,and 45.5%respectively,compared with control.In addition,almost all dead larvae had abnormal phenotypes,such as "half-ecdysis"during the molting process and ’melanism’ in head or thorax.However,there were no effects on larval growth and developmental rates after the JH metabolism-related gene were suppressed.This might be due to the involvement of 3 kinds of metabolic enzymes in JH metabolism.Suppressing only one of them will not have a great JH degradation effect.3.Gene functional studies of 20E biosynthesis relative genes in C.suppressalis20-hydroxyecdysone(20E)is the most active molting hormone in insect.In this study,9 selected genes involved with 20E biosynthesis and it’s receptor Csecr were cloned and characterized.Through sequence analysis,six of them i.e.,Csspook,Csphantom,Csdib,Csshade,Csnm-g and Csecr encoding 20E biosynthesis enzymes comprised the complete open reading frame.We performed the bioinformatics analysis of these genes,including the sequence of amino acids,characteristics,composition of amino acids,trans-membrane motif and signaling peptide.Phylogenetic analysis based on the amino acid sequence showed that,these genes are clustered in the branch of Lepidoptera including B.mori,H.armigera and M.sexta,showing a closer relationship than Diptera and Coleoptera.Using RT-PCR,we identified the gene expression mode of these genes in the 1st,2nd and 3rd instar larvae.The results showed 20E biosynthesis related genes had a similar gene expression tendency,that is,these genes were significantly up-regulated on the last day of each instar and down-regulated on the first day of the next instar.This tendency is identical with the changes of the 20E titers during larval development.All genes except Csptth and Csshade showed significantly high transcriptional levels in the head than the other tissues.There was no significant difference amongst the tissues for Csptth expression.Csshade and Csecr showed high abundance in all the tissues.Through feeding bacterially expressed dsRNA targeting 20E biosysthesis genes and their receptor,C.suppresalis larvae significantly down-regulated all the corresponding genes except for dsshade.dsRNA treatments resulted in a reduced survival rate and extended developmental duration,e.g.,continuous feeding of dsspook,dsnm-g,dsptth and dstorso for 15 days resulted in reduced survival rates of 28.8%,64.4%,35.6%and 53.6%respectively,compared with the control group.Phenotypic changes indicated that dsRNA treatments severely inhibited larval development and molting during the instar change or ecdysis..In summary,we interfered the growth and development related genes’ expression in C.suppressalis by using oral administration of dsRNAs in the present study.The results revealed that silencing of JH and 20E biosynthesis related gene expression negatively affected its development,leading to a decreasing larval survival.We screened four crucial genes,i.e.,Csfpps,Csmevk,Cstorso and Csnm-g,playing an important roles in larval growth regulation.The silencing of these genes in transcriptional level caused to extended larval duration and block of molting,along with a significantly decreased larval survival.Our results provided important targets for pest management by using molecular methods,and shed light on exploring novel control strategies.