The Functions Of Chitinase Genes From Locusta Migratoria

There are multiple genes encoding chitinases and chitinase-like proteins in all insect species. These chitinases have different functions. In order to provide important basis for effective pest control based on RNAi, the temporal and spatial expression characterizations and the biological functions of chitinase family genes from L. migratoria were investigated and chitinase gene that can cause locusts death during development was screened. Transgenic maize with locust chitinase gene dsRNA were obtained for further study on RNAi-mediated crop protection against insects. The main contents are as below:First of all, seven chitinase-like gene fragments were obtained from Locust Database and the sequences of these genes were analyzed by bioinformatics methods. The first-strand cDNAs were synthesized using RNA isolated from the fourth day of locust at different developmental stages and various tissues of the 5th-instar nymphs, RT-PCR was carried out to analyze the gene expression patterns. The results showed that temporal and spatial expression patterns among seven chitinase genes were significantly different and LmCht10 was highly expressed in the integument.Secondly, cDNA of LmCht10 were cloned by degenerate primer and RACE-PCR amplication. cDNA fragment of 9815 bp was obtained and contained an open reading frame (ORF) of 8658 bp encoded 2886 amino acids and a non-coding region of 1157 bp at the 3'end, but about 600 bp cDNA from the 5'end was failed to amplify. The coding region of 2886 amino acids contained five catalytic domains and six chitin-binding domains (CBDs). The first-strand cDNAs were synthesized using RNA isolated from different days of the 5th-instar nymphs, Real-time results showed that LmCht10 was highly expressed in the sixth day of the 5th-instar nymphs. The nymphs from the sixth day of the 5th-instar nymphs were dissected to get different tissues for RNA isolation. The highest expression of LmCht10 was detected in the foregut of Locust by real-time PCR, followed in integument or hindgut, foregut or fatbody, the lowest expression was found in gastric caeca or Malpighian tubules.Thirdly, biological functions of LmCht10 were studied by RNA interference, the results showed that the corresponding transcript level was reduced in the nymphs after LmCht10 dsRNA injection compared with the control insects; the nymphs injected with dsLmCht10 displayed slowly development, failed to detach from the old cuticle during molting process and died eventually; After adults were injected with dsLmCht10, the survival days of females shortened significantly and fecundity decreased simultaneously compared with control insects injected with dsGFP, about 30% eggs laid by females injected with dsLmCht10 appeared malformation or could not hatch. No difference were observed for males between control and treatment.Based on the important role of LmCht10 during the development of L. migratoria, RNAi-mediated transgenic plant against insects was further studied. The recombinant plasmid pBI 121 expressed dsRNA of LmCht10 was constructed; the transgenic maize plants were obtained by pollen-mediated transformation method. The screening of positive transgenic maize plants and bioassay are being conducted.In conclusion, multiple chitinase genes were found in locust and the temporal and spatial expression profiles of these genes were analyzed; LmCht10 is a large chitinase that has five catalytic domains and six CBDs, the RNAi results suggested that LmCht10 plays an important role in the molting process of L. migratoria, and gene silencing results in the failure of locust ecdysis and even death. RNAi-mediated crop protection against insects will be promising in the future.