Figure 1.
Schematic diagram of clones used to compile the complete CmRyR cDNA sequence.
The top line indicates the full-length CmRyR cDNA in kilobases. Thin lines (P1–P14) represent CmRyR cDNA clones obtained by RT-PCR. Solid lines (R5 and R3) represent CmRyR cDNA clones obtained by RACE.
Table 1.
Oligonucleotide primers used for RT-PCR, RACE and diagnostic PCR.
Figure 2.
Phylogenetic tree of the RyR family.
The CmRyR amino acid sequence was aligned to 9 representative RyR isoforms from 7 species and used for phylogenetic analysis. The Neighbor-joining tree was generated in MEGA5 with 1000 bootstrap replicates. RyR sequences are obtained from the following GenBank entries: DJ085056 for Bombyx mori (sRyR); AET09964 for Plutella xylostella (PxRyR); EFN78897 for Harpegnathos saltator (HsRyR); BAA41471 for Drosophila melanogaster (DmRyR); EAT44864 for Aedes aegypti (AaRyR); BAA08309 for Caenorhabditis elegans (CeRyR); CAA33279 for Oryctolagus cuniculus RyR1 (OcRyR1); NP_001076226 for Oryctolagus cuniculus RyR2 (OcRyR2); NP_001076231 for Oryctolagus cuniculus RyR3 (OcRyR3).
Table 2.
Nucleotide polymorphisms of the CmRyR cDNA.
Figure 3.
Schematic showing the location of putative transmembrane domains (TMDs) and conserved structural domains identified using the Conserved Domains Database (NCBI), including: MIR (Mannosyltransferase, IP3R and RyR) domains (pfam02815), RIH (RyR and IP3R Homology) domains (pfam01365), SPRY (SPla and the RyR) domains (pfam00622), RyR domains (RyR repeated domain) (pfam02026), RIH-associated domains (RyR and IP3R Homology associated) (pfam08454), EF hands, and suppressor-domain-like domains (SD) (pfam08709).
Figure 4.
Alignment of COOH-terminal region of the insect RyR isoforms.
Identical amino acids are shown in black boxes and similar amino acids are highlighted in gray boxes. Gaps have been introduced to permit alignment. Asterisks below the alignment indicate the position of residues discussed in the text (E4134, I4946, R4962, and D4966 of CmRyR). Triangles below the alignment indicate unique residues (N4922, N4924, N4935, L4950, L4981, N5013 and T5064 of CmRyR) in the lepidopteran homologues. Abbreviations and GenBank entries for the RyR isoforms are described in Figure 2.
Figure 5.
Nucleotide and inferred amino acid sequences of alternative exons in the CmRyR gene.
Figure 6.
Relative frequencies of individual CmRyR alternative exon usage in eggs (E), first instar larvae (L1), third instar larvae (L3), fifth instar larvae (L5), pupae (P), adult heads (AH), and adult bodies (AB).
Mutually exclusive exons are represented by hatched bars (exon a) and open bars (exon b). Optional exon c is indicated by black bars. Numbers in parentheses represent the total numbers of clones investigated.