Cloning And Expression Analysis Of Glycogen Phosphorylase Gene In Ostrinia Fumacalis (Guenee)(Lepidoptera: Crambidae)

Diapause, a type of hibernation, is a life feature of insect adapted to the natural evolution history inorder to survive adverse conditions, such as cold winter, hot summer, unavailable of host plants, etc.Research on the physiology and biochemistry of insect diapause, especially the cold hardiness ofdiapause insect are important components for understanding the mechanism of diapause, which willprovide scientific evidence for developing novel methods against agricultural insect pests. The researchefforts have been further turned to the molecular mechanism. In general, glycogen is a major source oflow molecular weight polyols and/or sugars as significant metabolites accumulated by many insectsduring hibernation. Glycogen phosphorylase (GP) is the key enzyme to degrade glycogen, which isimportant in understanding the mechanism of insect diapause. The Asian corn borer Ostrinia furnacalis(Guenée) is the most distructive insect pest on maize. During midsummer to autumn, day lengthsshorten and temperatures begin to cool, which leads to diapause of5thinstars for overwinter. It plays animportant role for cold hardiness of diapause larvae in survival through the fall and winter, which willdirectely determine the population dencity of the first generation next year. The objective of this study isto investigate the relationship between GP and diapause as well as cold hardiness of diapause larvaebased on the gene clone, cNDA sequence and protein structure characterization through RT-PCR, RACE,bioinformatics methods and expression pattern through Real-time quantitative PCR (qPCR) techniques.A cDNA encoding GP was cloned and sequenced from Asian corn borer. It had a3’ untranslatedregion (3’UTR) of222bp. A single Open reading frame（ORF）was2526bp nucleotides encoding841amino acids polypeptide which was estimated as96.49kD for molecular weight. The enzyme had aputative isoelectric point of6.04. The ORF sequence is most similar to GPs from Spodoptera exiguaand Bombyx mori with81%and79%sequence identity and the sequence also has high homology of76%with Danaus plexippus. The deduced amino acid sequence has the typical conservation reqions of GPs,including Ser-14phosphorylation site, AMP binding site, pyridoxal phosphate cofactor binding site,glucose binding site (active site), and glycogen storage site. The three dimentional structure showed33α-helixes and24β-sheets, variation happens much easier when the amino acid is exposed outside of thestructure. The sequence reported here has been deposited in GenBank database.The expression levels of OfurGP varied significantly in the2nd-5thinstars larvae and pupa with thehighest in the4thinstar larvae reared under longday photoperiod. The similar trend was showed for thelarvae reared under shortday photoperiod. However, the expression quantity maintained at a low levelfrom20d to31d, which was inferred as the pre-diapause phase and then followed with a sharp increaseat about36d, so we could conclude that the ACB larvae may enter diapause phase at about36d.Compared with longday treatment, the expression levels of OfurGP in the2nd-4thinstars reared undershorday photoperiod were higher, but it was significantly lower in the5thinstar. In conclusion, theincreasing expression level of OfurGP is the result for ACB larvae to enter diapause rather than reason. For the larvae reared under longday photoperiod, haemolymph ranked the highest in the expression ofOfurGP, then malpighian tubule, and there were no difference detected among head, fat body, andmdiguts, but they all were lower than haemolymph and malpighian tubule. The lowest expression was inthe body wall. In contrast, the expression was the highest in the fat body for the larvae reared undershortday photoperiod, but it was significalty declined in the other tissues, proved the importance of fatbody for diapause insects. For the larvae collected in the field from Oct2012to Feb2013, theexpression levels of OfurGP increased linearly from Oct to Jan, but decreased significantly in Feb. Thedynamics were negatively correlated with the changes of the environmental temperature. In conclusion,expression of OfurGP in Asian corn borer larvae was significantly influenced with the shortdayphotoperiod and lower temperature which lead to diapause of the5thinstar; the higher expression levelof OfurGP may enhance the cold hardiness of the diapause larvae.