Using RNAi Technology To Silence CmUAP And CmGFAT Genes Of Rice Leaf Roller

C.medinalis,one of the most destructive of all rice pests,is a serious threat to the development of the rice industry as it causes massive losses to rice production every year.Insect chitin synthesis begins with algin and endswith chitin,with a total of eight enzymes involved in the reaction.Insect UDP-N-acetylglucosamine pyrophosphorylase(UAP)and glutamine:fructose-6-phosphate transaminase(GFAT)are the fourth and seventh enzymes in the insect chitin synthesis pathway,respectively.These two enzymes regulate two key sub-reactions of chitin synthesis,which play an important role in insect chitin synthesis and are ideal targets for pest management.With the continuous development of RNA interference technology,it has been widely applied to the study of insect gene function and pest control in recent years.In this study,two genes,CmUAP and CmGFAT,were cloned and comprehensively characterized and expressed.The two genes were comprehensively characterized and their expression patterns were analyzed.The two genes were used as targets to synthesize double-stranded RNA in vitro,and the inhibitory effect of dsRNA on C.medinalis target genes was detected by RNAi injection to explore the important role of these two genes in the growth and development of C.medinalis.1.Cloning and expression analysis of the CmGFAT geneGFAT plays an important regulatory role in the biosynthetic pathway of aminohexose in insects.It combines fructose 6-phosphate with glutamine in the glycolytic pathway to produce glucosamine 6-phosphate,and is a very important rate-limiting enzyme in the insect titin synthesis pathway.In this study,the cDNA sequence of CmGFAT gene was cloned by RT-PCR combined with RACE-PCR,and a series of bioinformatics analyses of the gene and its encoded protein were carried out using various software.The results showed that the cDNA of CmGFAT is 2117 bp long,contains an open reading frame of 2025 bp and encodes 674 amino acids.Homology and phylogenetic analyses showed that CmGFAT had high amino acid similarity with H.vitessoides,O.furnacalis and A.transitella,reaching 97%,95% and 95%,respectively.The sequence identity of CmGFAT with the published glutamine 6-phosphofructose transaminase genes of various lepidopteran insects was over 70%.Homology modelling showed that the three-dimensional molecular structure of CmGFAT contained 18 ?-helices,19 ?-folded sheets and 40 random coils.dd PCR results showed that CmGFAT was expressed in all developmental stages of C.medinalis and in 12 tissues of adult worms,with a progressive increase in expression in the lower larval stages and significantly higher expression in the adult worms than in the The highest expression was found in the midgut and body wall of adult C.medinalis,and lower expression was found in the head,wings and other tissues.2.Cloning and expression analysis of CmUAP geneIn this study,the CmUAP gene of C.medinalis was cloned using a combination of RT-PCR and RACE-PCR,and its bioinformatics and spatio-temporal expression patterns were determined.The results showed that the cDNA of CmUAP was 1788 bp long,containing an open reading frame of 1464 bp and encoding 487 amino acids.Homology and clustering analyses showed that CmUAP was similar to G.pyloalis,O.furnacalis and H.vitessoides with up to 91.79%,87.89% and 82.75%homology,respectively.Homology modelling showed that the three-dimensional molecular structure of CmUAP formed a dimeric structure containing 37 ?-helices,37 ?-folded sheets and 71 random coils.The expression pattern of the CmUAP gene was determined using microtitre digital PCR,and the results showed that CmUAP was expressed in all eight developmental stages of the life history of C.medinalis and in 12 tissues of the adult,with significantly higher expression levels in the adult stage than in other developmental stages,and higher expression levels in the midgut,epidermis and body wall of the adult.3.Injection of dsRNA to silence the CmGFAT gene of C.medinalisIn vitro dsRNA was synthesized and microinjected to silence the CmGFAT gene of C.medinalis,which significantly reduced the expression of the gene by up to 59%,resulting in abnormal moult,affected body colour,increased mortality and reduced feeding and activity.The CmGFAT gene was confirmed to be essential for the growth and development of C.medinalis,laying a theoretical foundation for targeting the CmGFAT gene by RNAi technology to control this insect.4.Injection of dsRNA to silence the CmUAP gene of C.medinalisThe dsRNA was synthesized in vitro and microinjected to silence the CmUAP gene of C.medinalis to investigate the function and role of the CmUAP gene in the growth and development of C.medinalis.A high concentration of high quality dsRNA was synthesized and successfully injected into C.medinalis 3rd instar larvae,targeting and silencing the expression of CmUAP gene with an RNAi efficiency of 68.6%,resulting in a series of serious developmental disorders such as death,slow growth,reduced activity and excretion,weight loss and developmental deformities in C.medinalis.The CmUAP gene is essential for the growth and development of C.medinalis,and targeted silencing of the CmUAP gene leadsto developmental disorders and death of C.medinalis,laying a theoretical foundation for targeting silencing of the CmUAP gene by RNAi technology to control this insect.