| Mythimna separata (Walker), a typical seasonal migration pest in China, belongs to Noctuidae of Lepidoptera. Insect migration is an adaptive behaviour strategy in a long-term complicated and changeable environment, which cause heavy agricultural losses. So the mechanism of the formation and regulation of insect migratory behavior are always the popular issue in the behaviour research field. Insect migratory potential were assessed by the time length of preoviposition period and flighy duration, which subject to the nutrition formed in larval stage. The feeding behaviour of M. separate larvae is associated with the rearing density of larvae. Those who reared in the crowded conditions feed more actively and take in more food. So the development of molecular mechanism on feeding behaviour in M. separata is necessary supplement for the migratory mechanism research. The research on the molecular mechanism on feeding behaviour in M. separate has important academic significance and application value. The approach of candidate gene offers a shortcut for non-model insect at the molecular level.The cDNA of the PKG, NOS, OAR, QM andβ-actin from M.separata were cloned using RT-PCR and the RACE technique. These cDNAs and the deduced proteins are analyzed using bioinformatics. Real-time PCR assays of PKG, NOS, OAR and QM, RNA levels in the heads of the two phases at four stages were performed. The results are showed as follows:1. The full-length 2,065 base-pair (bp) cDNA of PKG (called MsPKG) was cloned from M. separata (Walker) containing a 5'-untranslated region (5'-UTR) of 93 bp, a 3'-UTR of 193 bp with a canonical polyadenylation signal sequence (AATAAA) and a 17 bp poly (A) tail,, and an open reading frame of 1,779 bp encoding a 592-amino acid protein. The initiating codon ATG is at positions94-96 and the stop codon TAA is at positions 1,870-1,872. The deduced MsPKG protein shares 94.43% identity with that from Bombyx mori. The functional motif analysis showed that four predicted function domains are in the PKG protein sequence. These motifs are characteristic of the PKG family of proteins. These results indicate the newly isolated cDNA is PKG homologue. The full-length cDNA sequence of PKG from M. separate was submitted to GenBAnk and GenBank accession number is GQ844298.2. The full-length 4,073 base-pair (bp) cDNAof NOS (called MsNOS) was cloned from M. separata (Walker) containing a 5'-untranslated region (5'-UTR) of 95 bp, a 3'-UTR of 642 bp with a canonical polyadenylation signal sequence (AATAAA) and a 16 bp poly (A) tail,, and an open reading frame of 3,336 bp encoding a 1,111 amino acid protein. The initiating codon ATG is at positions96-98 and the stop codon TAG is at positions 3,429-3,431. The deduced MsPKG protein shares 73% identity with that from Maduca sexta. The functional motif analysis showed that double predicted function domains are in the NOS protein sequence. These motifs are characteristic of the NOS family of proteins. These results indicate the newly isolated cDNA is NOS homologue. The full-length cDNA sequence of NOS from M. separate was submitted to GenBAnk and GenBank accession number is JF502067.3. The partical 1,397 base-pair (bp) cDNA of OAR was cloned from M. separata (Walker) The deduced ORA protein shares 88% identity with that from B. mori. These results indicate the newly isolated cDNA is ORA homologue. The cDNA sequence of OAR from M. separate was submitted to GenBAnk and GenBank accession number is JF502068.4. The full-length 832 base-pair (bp) cDNAof QM (called MsQM) was cloned from M. separata (Walker) containing a 5'-untranslated region (5'-UTR) of 115 bp, a 3'-UTR of 57 bp with a canonical polyadenylation signal sequence (AATAAA) and a 19 bp poly (A) tail,, and an open reading frame of 660 bp encoding a 219 amino acid protein. The initiating codon ATG is at positions116-118 and the stop codon TAA is at positions 773-775. The deduced MsQM protein shares 98% identity with that from B. mori. The functional motif analysis showed that a ribosomao protein L10signature in the deduced protein.These results indicate the newly isolated cDNA isQM homologue. The full-length cDNA sequence of QM from M. separate was submitted to GenBAnk and GenBank accession number is HM467199.5. The full-length 1,472 base-pair (bp) cDNA ofβ-actin was cloned from M. separata (Walker) containing a 5'-untranslated region (5'-UTR) of 28 bp, a 3'-UTR of 273 bp with a canonical polyadenylation signal sequence (AATAAA) and a 19 bp poly (A) tail,, and an open reading frame of 1,131 bp encoding a 376-amino acid protein. The initiating codon ATG is at positions69-71 and the stop codon TAA is at positions 1,197-1,199. The deducedβ-actin protein shares 98% identity with that from Bombyx mori. The functional motif analysis showed that three actin protein family signatur in theβ-actin protein sequence. These results indicate the newly isolated cDNA isβ-actin homologue. The full-length cDNA sequence ofβ-actin from M. separate was submitted to GenBAnk and GenBank accession number is GQ856238.6. Real-time PCR assays of the feeding behaviour candidate genes mRNA levels in the heads of the two different lifestyles at four different developmental stages were performed. The results showed that MsPKG is differentially expressed with higher expression levels in the solitary larvae relative to the gregarious ones. Interestingly, this trend reverses in the adult stage. Ther MsPKG expression levels are statistically significant higher in the solitary morph (L4 and L5) than the gregarious larvae (L4 and L5). The adults emerging from the gregarious larvae showed significantly higher expression than those from the solitary larvae; MsNOS is differentially expressed in the heads from two different lifestyles, and there is no significant deffence expression in these two different lifestyles. The OAR expression is significant higher in gregarious larvae (L4, L5 and L6) than those in solitary larvae. There is no statistically significant deffence expression of QM mRNA at four stages in two lifestyles. The results of MsPKG and MsNOS expression are similar to the findings reported in other animals. Taken together, the results of these studies suggest that MsPKG and MsNOS may be associated with the feeding behaviour in M. separate. However, more research is needed to verify this assumption.The cDNAs of MsPKG, MsNOS, OAR, MsQM andβ-actin were cloned successfully from M. separate for the first time, and submitted GenBank. Real-time quantitative PCR compared these genes expression in two different lifestyles. These results may provide the necessary data for further investigation on the molecular mechanism of feeding behaviour in M.separata...
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