Genome And Silencing Of CmHK And CmGNA Genes In Cnaphalocrocos Medinalis

Rice is an indispensable and major food crop in the world.Nearly half of the world's population feeds on rice.Insect pests have severely reduced rice production.The rice leaf folder,(Cnaphalocrocis medinalis)(Lepidoptera:Pyralidae)is one of the most serious insect pest of rice.Synthetic chemical insecticides are being used to control of this crop damage.However,C.medinalis have produced resistance against insecticide.In the present study,we tried to sequence,assemble,and annotate the genome of C.medinalis.We cloned two chitin biosynthesis gene such as,CmHK and CmGNA and analyzed the bioinformatics of CmHK and CmGNA.We observed the spatio-temporal expression pattern of CmHK and CmGNA during developmental and life stages.We also examined theRNAi and pRNAi effects of CmHK and CmGNA in C.medinalis.Our findings provide scientific data to analyze the effects ofRNAi and pRNAi,and to develop an effective control of C.medinalis.Results of this study given below.1.Genome sequencing of C.medinalis A genome sequence contained effective data of 136.8 Gb,including Pacbio sequencing of 130.34 Gb.Thirty two chromosomes were identified Hi-C technology.The heterozygosity and repetition rate were 2.39% and 51.2%,respectively.The total length of Contig was 475.1 Mb contained N50 of 3.1 Mb.The GC content was 38.45.Unigenes assessed the integrity of the gene region was 81%.2.Cloning,Characterization,Expression analysis,andRNA interference of CmHKThe cDNA cloned length of CmHK was 1581 bp,consisting of an ORF of1359 nucleotides(nt)that encodes 452 aa,respectively.The 5? untranslated region(UTR)of CmHK contained 88 bp,while,3? untranslated region(UTR)have 134 bp.The CmHK protein has a molecular weight of 50.06 k Da and theoretical isoelectric point was 5.99.CmHK have five phosphorylated sites and no glycosylation sites and signal peptides or transmembrane structures.In case of CmHK expression analysis,highest expression level was analyzed in fifth instar larvae followed by the first and fourth instar larvae of C.medinalis.Furthermore,expression of CmHK was analyzed in the all body tissues with the highest expression level in the ovary.3.Knockdown of CmHK gene using ParentalRNAi(pRNAi)To analyze the effects of pRNAi,daRNA was synthesized and introduced into the 4th instar larvae of C.medinalis using a micro-syringe.pRNAi caused significant phenotypic deformities,significant weight loss,significant reduction of laid eggs and hatched eggs per female,higher larval and pupal mortality,and less male and female emergence rate in G1,G2,and G3 progenies.RT-q PCR results showed the significant reduction of mRNA expression level was observed in G1,G2,and G3 progenies.4.Cloning,Characterization,Expression analysis,andRNA interference of CmGNAThe length of cDNA sequence of CmGNA contained 859 bp with ORF of600 bp,encodes a 199 aa protein.The cDNA sequence contained 76 nt(UTR)located at 5? stand upstream,while 3? UTR consisted of 83 nt The molecular weight(Mw)and isoelectric point was predicted 22.299 k Da and 8.65,respectively.CmGNA have twenty four O-glycosylation sites with five Nglycosylation sites and have no signal peptide or transmembrane structures.The tissue expression levels of CmGNA revealed that CmGNA was highly expressed in integument followed by the head.While,at developmental stages,the expression levels of CmGNA was highest in second larval instar followed by fourth larval-instar.5.Knockdown of CmGNA gene using ParentalRNAi(pRNAi)The pRNAi results showed significant less laid eggs and hatched eggs per female,higher larval and pupal mortality as compared to control were observed in G1,G2,and G3 progenies.Phenotypic deformities was higher as compared to control.In addition,male and female emergence,and mRNA transcriptional level was significantly reduced in G1,G2,and G3 progenies.