| The brown planthopper (BPH), Nilaparvata lugens, a kind of monophagous insect, only feeding on rice and having a high reproductive rate. BPH is an important rice pest causing huge yield in China and many Southeast Asia or Northeast Asia countries. Epigenetic modifications are indispensable for development and are crucial in many biological processes without altering the genomic DNA. In this work, the full-length cDNAs of DNA (cytosine-5)-methyltransferase 1(Dnmtl), N6-adenine-specific DNA methyltransferase 2 (N6amt2) and DNA methyltransferase 1-associated protein (DM4P1) were cloned by rapid amplification of cDNA ends (RACE). Then Dnmtl and N6amt2 genes were analyzed. The existence of different DNA methyltransferase genes in diverse insect taxa were investigated. RNAi-mediated lethal genes were cloned in Spodoptera exigua, the pest which had caused huge yield in agricultural production. The main results are summarized as follows:1. Molecular cloning of Dnmtl, N6amt2 and DMAP1 in BPHThe full-length cDNAs of two DNA methyltransferases (Dnmtl and N6amt2) and one DNA methyltransferase-associated protein (DMAP1) were successfully cloned by RACE. Then amino acids of three genes were used to calculate molecular weights (Mw) and isoelectric points (pI). Proteins secondary structures were also predicted. All three cDNA sequences were submitted to GenBank and got the accession numbers with KF294265, KF294266, KF294267, respectively.2. Gene structure and evolution analysis of BPH Dnmtl and N6amt2Sequence analysis of BPH Dnmtl and N6amt2 were conducted, including domain analysis, sequence alignments, gene structures and phylogenetic analysis. Domain analysis showed that BPH Dnmtl has the same domain-pattern with H.sapiens, M.musculus, X.tropicalis and D.rerio. As for N6amt2, there is only one domain in all species. Sequences alignment of Dnmtl catalytic domain showed a high similarity among various species. Amino acid sequence of this domain in BPH had 75% identities to those from X.tropicalis and D.rerio,74% to H.sapiens and A.mellifera. all N6amt2 in diverse taxa contain a highly conserved QFW motif and an DPPFmotif, and sequence in BPH shares the greatest identity with A.mellifera (74%), B.mori (69%) and A.pisum (65%), respectively. Gene lengths of Dnmt1 and N6amt2 in BPH have apparent differences compared with their counterparts in other species. Among various species, the number and the length of exons and introns in Dnmtl genes are significantly different but in N6amt2 are not. Phylogenetic trees showed that both Dnmt1 and N6amt2 of BPH have closely evolutionary relationships with other insects. The investigation of DNA methylation tool kit in diverse taxa showed that BPH own three Dnmts, which is the same as mammals and hymenopterous insects. N6amt2 is present in most insects.3. Expression patterns of Dnmt1 and N6amt2 in different BPH developmental stages and wing-type populationsDevelopmental expression pattern of Dnmt1 and N6amt2 were analyzed in BPH, including the larvae from the first to fifth instars, brachypterous and macropterous females within 24h of final molt and ones at egg-laying stage, brachypterous and macropterous males. The results indicated that the aboundance of Dnmtl and N6amt2 mRNA were not different at larvae stage, but change a lot at adult stage. The expression levels of Dnmtl and N6amt2 were low in females within 24h of final molting than at egg-laying stage, no matter of brachypterous or macropterous. Relative Dnmtl and N6amt2 mRNA levels in the female were higher than the male. These results showed that DNA methylation might have important functions in reproductive development and ovary maturation in BPH.The aboundance of Dnmt1 and N6amt2 mRNA in differernt days of adult stage had a similar tendency, increasing at first several days and decreasing later. Expression pattern of Dnmt1 and N6amt2 at larvae stage of brachypterous and macropterous populations showed gradually decreasing.We also found that the aboundance of Dnmtl in brachypterous larvea was higher than macropterous larvea, but the abundance of N6amt2 is similar beteween brachypterous and macropterous populations.4. RNA interference of Dnmt1 and N6amt2 in BPHFeeding-based RNA interference was conducted with dsRNAs designed on the sequences of Dnmt1 and N6amt2. Second instar larvae were selected for this experiment. The levels of Dnmtl and N6amt2 mRNA on the first, second, fifth, seventh, tenth and fifth day after dsRNA feeding were detected by qRT-PCR. Unexpectedly, the results showed Dnmt1 were increased compared with the control groups, especially in the first, second, tenth and fifth day. As for N6amt2, the levels of mRNA at first day after feeding were highly elevated. However, the levels were continuous declining from the second day until the tenth day. The aboundence of N6amt2 decreased with 62% compared with the control level. Phenotypic changes were observed in the RNAi-treated groups.The functions of Dnmt 1 and N6amt2 requires more investigations.5. Cloning RNAi-mediated lethal genes in Spodoptera exiguaThe cDNAs of four RNAi-mediated lethal genes (arfl, arf2, tubulinl, tubulin2) were successfully cloned by RACE. All four cDNA sequences were submitted to GenBank and got the accession numbers with JF915770, JQ653045, JQ653042, JQ653043, respectively. Then amino acids of four genes were used to calculate molecular weights (Mw) and isoelectric points (pI). Proteins secondary structures were also predicted. |
PDF Full Text Request