Cloning, characterisation and expression profiling of the cDNA encoding the ryanodine receptor in diamondback moth, Plutella xylostella (L.) (Lepidoptera: Plutellidae)

Abstract

There are at least two alternative splices of PxRyR expressed in all developmental stages and tissues in P. xylostella at various levels. The results provided the basis for further understanding of the mechanisms of resistance to diamide insecticides in P. xylostella.

Ryanodine receptor genes of the rice stem borer, Chilo suppressalis: Molecular cloning, alternative splicing and expression profiling

“…As with other published data for insects, we found that the egg stage generally is the lowest expression level of RyR mRNA during the different developmental stages, just like in T. castaneum [48], B. dorsalis [32], H. armigera [42], O. furnacalis [17], Sogatella furcifera [47], and L. decemlineata [31]. In C. suppressalis, CsRyR expression level is highest in 3 rd instar larvae and much lower in eggs, 6 th instar larvae and adults, which is similar to but not the same as the RyR mRNA expression in P. xylostella [14,39] and D. melanogaster [49].…”

“…However, Guo et al [14] deduced that both P. xylostella RyRs may have a similar function and this phenomenon might be attributed to the alternative splicing of mRNA or the diversity of insect RyRs. To date, splicing variants have been observed in several insects, such as P. xylostella [39], Heliothis virescens [41], C. medinalis [15], Helicoverpa armigera [42], O. furnacalis [17], and D. melanogaster [18].…”

“…Due to difficulties in separation of the brain and muscle of C. suppressalis head, and the importance of RyR expression profile in other M A N U S C R I P T ACCEPTED MANUSCRIPT 4 insects [14,31,32], the whole head, including brain and muscle, was chosen for the detection of CsRyR mRNA. The qRT-PCR was performed on the ABI 7500 system (Applied Biosystems, CA, USA) in 20 μL reactions containing 1 μL cDNA, 10 μL SYBR ® Premix Ex Taq (Perfect Real Time) (Takara Co., Otsu, Japan), 0.4 μL forward GSP (qCsRyR-F and G3PDH-1-F) and reverse GSP (qCsRyR-R and G3PDH-1-R) (10 μM), 0.4 μL ROX Reference dye II and 7.8 μL ddH 2 O.…”

“…To investigate the relative transcription levels of CsRyR, different developmental stages (such as eggs, 1 st to 6 th instar larvae, pupae and adults), various tissues from the 5 th instar larvae (for example, integument, fat body, haemolymph, Malpighian tubules, midgut and silk gland), and the head were separated, harvested and ground for the total RNA purification following the procedure of Guo et al [14] and the cDNA was obtained after genomic DNA clearance and RT-PCR. Due to difficulties in separation of the brain and muscle of C. suppressalis head, and the importance of RyR expression profile in other M A N U S C R I P T ACCEPTED MANUSCRIPT 4 insects [14,31,32], the whole head, including brain and muscle, was chosen for the detection of CsRyR mRNA.…”

“…Two isoforms of RyRs (RyR A and RyR B ) are found in fish, amphibians and birds [12,13]. Although a variety of insect RyR variants has been reported and may affect the channel function properties due to alternative splicing [14,15], only one isoform of RyR gene has been detected in insects and nematodes so far [16,17].…”