Molecular Cloning and Sequence Analysis of Chitin Synthase cDNA from Mamestra brassicae (L.) (Lepidoptera: Noctuidae) Cuticle

Chitin is the most widespread amino polysaccharide in nature. Chitin synthase （CHS） plays an important role in chitin formation in the cuticle and the peritrophic membrane （PM） lining the midgut. Total RNA was isolated from the cuticle of Mamestra brassicae （L.） fourth instar larva, cDNA sequence was cloned by RT-PCR and Rapid Amplification of cDNA Ends （RACE）. cDNA 5 220 bp in length, contained an open reading frame of 4 704 bp coding for a polypeptide of 1 567 amino acid residues with a predicted molecular weight of 178.3 ku and its pI was 6.42. The deduced amino acid sequence from Mi brassicae （L.） shared the high level of identity with chitin synthase sequences from other insects, especially lepidopteran insects, cDNA sequence has been deposited with GenBank under accession No. GQ281761

Chitin synthase:A potential agricultural and therapeutic target

Chitin synthase(CHS)is a potential agricultural and therapeutic target.Recently,the crystal structures of CHS from Phytophthora sojae(PsChs1)and Candida albicans(CaChs2)were reported.Here,we highlight the achievements of PsChs1 and CaChs2,which would give some clues for design new inhibitor targeted upon CHS.

Chitin synthase 1 and five cuticle protein genes are involved in serosal cuticle formation during early embryogenesis to enhance eggshells in Nilaparvata lugens

Many holo-and hemimetabolous insects enhance their eggshells during embryogenesis by forming a serosal cuticle(SC).To date,scholarly understanding of the SC composition and SC-related gene functions has been limited,especially for hemimetabolous insects.In this study,we initially performed transmission electron microscopic(TEM)observation and chitin staining of the SC in Nilaparvata lugens,a hemimetabolous rice pest known as the brown planthopper(BPH).We confirmed that the SC was a chitin-rich lamellar structure deposited gradually during the early embryogenesis.Parental RNA interference(RNAi)against Nilaparvata lugens chitin synthase 1(NlCHS1)in newly emerged and matured females resulted in decreases of egg hatchability by 100%and 76%,respectively.Ultrastructural analyses revealed loss of the lamellar structure of the SC in dsNlCHS1-treated eggs.According to temporal expression profiles,five cuticle protein coding genes,NlugCpr1/2/3/8/90,were specifically or highly expressed during the SC formation period,and NlugCpr1/2/3/90 were further detected in 72 h eggshells extract by ultra-performance liquid chromatography-tandem mass spectrometry/mass spectrometry.NlugCpr2/3/90 were likely three SC-specific cuticle proteins.TEM observations of the SC following parental RNAi against NlugCpr1/2/3/8/90 demonstrated that NlugCpr3/8/90 were essential for SC formation.The study provided an understanding of the SC formation process and SC-related cuticle proteins in BPHs,which offer potential targets for pest control in the egg stage as well.

Cloning and sequence analysis of chitin synthase gene fragments of Demodex mites

To our knowledge,few reports on Demodex studied at the molecular level are available at present.In this study our group,for the first time,cloned,sequenced and analyzed the chitin synthase(CHS) gene fragments of Demodex folliculorum,Demodex brevis,and Demodex canis(three isolates from each species) from Xi'an China,by designing specific primers based on the only partial sequence of the CHS gene of D.canis from Japan,retrieved from GenBank.Results show that amplification was successful only in three D.canis isolates and one D.brevis isolate out of the nine Demodex isolates.The obtained fragments were sequenced to be 339 bp for D.canis and 338 bp for D.brevis.The CHS gene sequence similarities between the three Xi'an D.canis isolates and one Japanese D.canis isolate ranged from 99.7% to 100.0%,and those between four D.canis isolates and one D.brevis isolate were 99.1%-99.4%.Phylogenetic trees based on maximum parsimony(MP) and maximum likelihood(ML) methods shared the same clusters,according with the traditional classification.Two open reading frames(ORFs) were identified in each CHS gene sequenced,and their corresponding amino acid sequences were located at the catalytic domain.The relatively conserved sequences could be deduced to be a CHS class A gene,which is associated with chitin synthesis in the integument of Demodex mites.

Molecular and functional analysis of chitin synthase genes in Chilo suppressalis(Lepidoptera:Crambidae)

The rice stem borer,Chilo suppressalis,has developed a high level of resistance to many of the compounds currently used for control.There is therefore an urgent need to develop novel control methods for C.suppressalis.Insect chitin synthases(CHS)have attracted interest as a potential target for insect pest management.However,to date,CHS have not been characterized in C.suppressalis.Two CHS genes(CsCHSI and CsCHS2)were identified and cloned from C.suppressalis.Two transcript variants were identified for CsCHS1,CsCHSla and CsCHS1b.Spatiotemporal expression profiling showed that both transcripts of CsCHS1 are most highly expressed on the last day of each larval instar stage and show the highest expression levels in the integument.In contrast,CsCHS2 is predominantly expressed during the larval feeding stages and shows the highest expression levels in the midgut.Knockdown of CsCHSI by RNA interference significantly inhibited the molting and pupation of C.suppressalis,and knockdown of CsCHS2 significantly affected growth during the larval stage,but had no significant effect on the pupation.Moreover,knockout of CsCHS1 by CRISPR/Cas9 genome editing severely lowered the hatching rate,larval survivorship,pupation rate,and eclosion rate,but only larval survivorship at the GO generation was lowered after the knockout of CsCHS2.These results demonstrate that CsCHS1 and CsCHS2 play vital roles in the growth and development of C.suppressalis,and so have potential as insecticidal targets for the control of this highly damaging pest.

Biochemical characterization of chitin synthase activity and inhibition in the African malaria mosquito, Anopheles gambiae

Chitin synthase （CHS） is an important enzyme catalyzing the formation of chitin polymers in all chitin containing organisms and a potential target site for insect pest control. However, our understanding of biochemical properties of insect CHSs has been very limited. We here report enzymatic and inhibitory properties of CHS prepared from the African malaria mosquito, Anopheles gambiae. Our study, which represents the first time to use a nonradioactive method to assay CHS activity in an insect species, determined the optimal conditions for measuring the enzyme activity, including pH, temperature, and concentrations of the substrate uridine diphosphate N-acetyl-D-glucosamine （UDP- GlcNAc） and Mg＋＋. The optimal pH was about 6.5-7.0, and the highest activity was detected at temperatures between 37℃ and 44℃. Dithithreitol is required to prevent melanization of the enzyme extract. CHS activity was enhanced at low concentration of GIcNAc, but inhibited at high concentrations. Proteolytic activation of the activity is significant both in the 500×g supernatant and the 40 000×g pellet. Our study revealed only slight in vitro inhibition ofA. gambiae CHS activity by diflubenzuron and nikkomycin Z at the highest concentration （2.5μmol/L） examined. There was no in vitro inhibition by polyoxin D at any concentration examined. Furthermore, we did not observe any in vivo inhibition of CHS activity by any of these chemicals at any concentration examined. Our results suggest that the inhibition of chitin synthesis by these chemicals is not due to direct inhibition of CHS in A. gambiae.

Design and synthesis of chitin synthase inhibitors as potent fungicides

Chitin is a structural component of fungal cell walls but is absent in vertebrates,mammals,and humans.Chitin synthase is thus an attractive molecular target for developing fungicides.Based on the structure of its donor substrate,UDP-N-acetyl-glucosamine,as well as the modelled structure of the bacterial chitin synthase NodC,we designed a novel scaffold which was then further optimized into a series of chitin synthase inhibitors.The most potent inhibitor,compound 13,exhibited high chitin synthase inhibitory activity with an IC（50） value of 64.5 μmol/L All of the inhibitors exhibited antifungal activities against the growth of agriculturally-destructive fungi,Fusarium graminearum,Botrytis cinerea.and Colletotrichum lagenarium.This work presents a new scaffold which can be used for the development of novel fungicides.

Putative Chitin Synthases from Branchiostoma floridae Show Extracellular Matrix-related Domains and Mosaic Structures

The transition from unicellular to multicellular life forms requires the development of a specialized structural component, the extracel- lular matrix （ECM）. In Metazoans, there are two main supportive systems, which are based on chitin and collagen/hyaluronan, respec- tively. Chitin is the major constituent of fungal cell walls and arthropod exoskeleton. However, presence of chitin/chitooligosaccharides has been reported in lower chordates and during specific stages of vertebrate development. In this study, the occurrence of chitin syn- thases （CHSs） was investigated with a bioinformatics approach in the cephalochordate Branchiostomafloridae, in which the presence of chitin was initially reported in the skeletal rods of the pharyngeal gill basket. Twelve genes coding for proteins containing conserved amino acid residues of processive glycosyltransferases from GT2 family were found and 10 of them display mosaic structures with novel domains never reported previously in a chitin synthase. In particular, the presence of a discoidin （DS） and a sterile alpha motif （SAM） domain was found in nine identified proteins. Sequence analyses and homology modelling suggest that these domains might interact with the extracellular matrix and mediate protein-protein interaction. The multi-domain putative chitin synthases from B. floridae constitute an emblematic example of the explosion of domain innovation and shuffling which predate Metazoans.