| Chitin is an important component of insect cuticle. Chitinases (EC 3.2.1.14), which belong to endonucleases, are involved in chitin degradation of insect cuticle. Thus, chitinases play key roles in insect molting and development. Insect chitinases genes (Chts) are family genes, essential for insect development, digestion and defense. Because chitin is absent in humans and other higher animals, it is relatively safe for insect pest control by attacking chitin synthesis degradation pathway. In recent years, with rapid developing, RNA interference (RNAi) is more and more widely used for insect gene function analysis and pest control. Locusta migratoria is sensitive for RNAi. In this study, we first silenced expression of chitinase genes to influence the chitin metabolism of nymphs, thus disrupted the normal molting process which led to insect death. We also screen the target dsRNA based on chitin metabolism pathway for RNAi-mediated pest control. The results had important theoretical and practical values for developing innovative molecular targets and new plant protection technology.In this study, we systemically analysized chitinase family genes from the transcriptome and genome of L. migratoria, an important agriculture pest in China. Sixteen chitinase genes were identified and their cDNA sequences were cloned; 2 chitinase genes were involved in molting by using RNAi. dsRNAs with high specificity and insecticidal efficiently were designed and used for generating dsRNA-mediated pest resistant transgenic corn. The main results are as follows:1. Sixteen chitinase family genes were identified from L. migratoria.The full length sequences of 16 chitinase and chitinase-like genes were identified by searching locust transcriptome- and RACE-PCR amplification. The phylogenetic analysis showed that the 15 genes could be classified into 9 groups of chitinase genes except LmCht9. The homologous genes of LmChtl and LmCht7 could be found in vertebrate. There were significantlly different among cDNA squences of the 16 genes. Analysis of genomic structures of these genes showed common characteristics, which are numerous large introns, except LmChtl (only one exon).2. Molecular expression characteristics of chitinase family genes in L. migratoriaThe expression profiles of 16 chitinase genes were analyzed in different tissues and developmental stags by RT-qPCR. The results showed that the genes express differently in locust life cycle. The high transcripts of LmCht5-1, LmCht5-2 and LmCht10 were detectable in the integument, foregut and hindgut, suggested that they were involved in degradation of old cuticle before molting. The diverse expression patterns of the above 3 chitinase genes might reflect their various functions during egg development. The expression profiles of the above 3 chitinase genes in integument during the fifth-instar were similar with that of ecdysone titre. The transcription of the above 3 genes responses to 20-hydroecdysone (20E) treatment and RNAi-mediated suppression of EcR (the 20E receptor gene) suggested that the expressions of LmCht5-1, LmCht5-2 and LmCht10 are regulated by 20E.3. The duplication of Cht5 genes (LmCht5-1 and LmCht5-2) with different functions were detected in L. migratoria.The duplication of Cht5 has been reported only in mosquito species. In this study, we first found two Cht5 genes possibly originated for a duplication event in L. migratoria, one of incomplete metamorphosis of insects. Phylogenetic analysis of the domain structures suggested that LmCht5-1 and LmCht5-2 were orthologous to other insect Cht5 genes, which is different with mosquito duplicated Cht5s. Amino acid sequence alignments showed that both LmCht5-1 and LmCht5-2 had the catalytically critical glutamate (E) residue in conserved motif "DWEYP" of the catalytic domain, suggesting that both of them still had the chitinase catalytic activity. RNAi-mediated suppression of LmCht5-1 transcript led to severe molting defects and lethality from 5th instar nymph to adult, but such effects were not sfound in LmCht5-2 RNAi, suggesting that LmCht5-1 was essential for development and survivorship of the locust.4. LmCht10 played an important role in molting of L. migratoria during development.RNAi was used to explore functions of LmCht10. Results showed that specific knockdown transcripts for LmCht10 prevented molting of nymph of all instars. When adult locusts were treated with dsRNA, the reproductivity of females were significantly down-regulated, and the transcript of LmCht10 during egg development were suppressed. dsLmCht10-injected nymphs could break the egg shell, but could not release their out layer membranes, which lead to their death. Besides, samples were collected from the abdominal integument of each day insects after dsRNA injection (N5D2-N5D7). Then, these samples were used for analyzing silencing efficiency of LmCht10, total chitinase activity assay, integument tissue section and chitin content. The results indicated that LmCht10 was essential for old cuticle degradation during molting.5. The efficient and specific target dsRNA molecules for pest control were screened from L. migratoria.The results of RNAi test of the chitinase family genes and P-N-acetylglucosaminidase gene (NAG) suggested that only dsLmCht5-1, dsLmCht10, and dsLmNAG were lethal for locust nymphs, and dsLmChl10 was the most efficient. Further study showed supression expression of Cht10 in L. migratoria, Oxya chinensis and Tribolium castaneum, chould cause the failure of molting. This results indicated that insect group ? genes had the similar function. Two dsRNAs of LmCht10 designed from different region had different mortality effects to the three insect species. The dsCCD located on the conserved chitinase catalytic domains (CCD) had highest mortality (above 90%) in all the three insects. The dsCBD located in the L. migratoria and O. chinensis chitin binding domain (CBD) had the specific lethal effects in only the two grasshoppers. These results implied that dsCCD could be used for wide spectrum pest control, and dsCBD could be specifically used for locust control.6. Transgenic maiz with dsLmCht10 were obtainedThe dsCCD fragment used for constructing the recombinant plasmid for plant transformation, and transgenic maize plants were generated by pollen-mediated transformation method and following self-pollination and molecular verification. We obtained four positive lines (dsLmCht10-11 to dsLmCht10-14) in the T3 generation. The feeding bioassay showed that molting difficulty and even death appeared in the first-instar locust nymphs after feeding with leaves of all four transgenic lines. The transgenic line with dsLmCht10-11 caused the highest locust mortality were used for northern blot analysis. The results suggested that siRNA of dsLmCht 10 was the major expressing in transgenic plant. The first-instar locust shown the lethal phenotype (53%) after feeding the leaves of transgenic corn. The mRNA expression of LmCht 10 was suppressed after locust feeding transgenic maize with dsLmCht10. These results indicated dsRNA-mediated transgenic maize has the insect resistant effects.In summary,16 chitinase family genes were identified from L. migratoria by bioinformatics and RACE-PCR; RT-qPCR analysis revealed the distribution and expression variation of Chts in different tissues and developmental stages; and LmCht 10 was identified as the key gene during molting by RNAi test; the two dsRNAs of LmCht10 showed different mortality effects in three insect species; dsCCD, which showed wide spectrum lethality for pests, were used for generating trangenic maize; four transgenic maize lines showed the resistantce to locust in bioassay. Taken together, we obtained the effective target dsRNA molecular sequences in insect chitin metabolic pathways and established RNAi-mediated pest control technology system, which will promote the plant protection technology and had important theoretical significance and practical values. |
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