Molecular Identification Of Ostrinia Furnacalis(Guenée) Thioredoxin Gene

Tioredoxin(Trx), a small, ubiquitous thiol [sulfydryl(-SH)] protein, is one of the most important regulators of cellular processes, including the control of cellular redox balance, the promotion of cell growth, the inhibition of apoptosis and the regulation of gene expression. In this experiment, the characterizations of thioredoxin from Ostrinia furnacalis were studied and these studies further provide foundation to understand the insect response to oxidative stress damage. The main results are as follows:(1) Ostrinia furnacalis tioredoxin(Ofur Trx) c DNA was isolated by using reverse transcription polymerase chain reaction(RT-PCR) and rapid amplification of c DNA 3’ ends(RACE). Sequence analysis revealed that the open reading frame of Ofur Trx consists of 321 nucleotides encoding 106 amino acid residues with a predicted molecular weight of 11.85 k Da. Homolog research revealed that Ofur Trx shares a common active site, CGPC, with other insect counterparts. Ofur Trx share 60%~ 96% identity with other insects Trx. Altogether, these results suggest that the gene obtained belongs to Trx family genes(Gen Bank accession number KJ744039.1).(2) The developmental analysis and tissue distribution of Ofur Trx was studied using Real-time PCR. The results showed that the Ofur Trx is expressed in all developmental stages, but the expression is different. Ofur Trx expression peaked at 24 h in the 4th-instar larvae; Its transcript peaked at 0 h in the 5th-instar larvae and decrease in the 5th instar in feeding stages with the growth. The expression levels of Ofur Trx was lower in 3-day pupae than 5-day; and it was relatively lower in adult stage. Tissues expression analysis revealed Ofur Trx transcript expressed abundantly in the epidermis, secondly in the fat body, compared with that in the head and midgut.(3) The effect of adverse stress factors(including high and low temperatures, starvation, ultraviolet light, mechanical injury, and hydrogen peroxide)on the Ofur Trx expression were analysis using q PCR. The results showed that these adverse stress factors dramatically induce Ofur Trx transcript expression, except ultraviolet light.(4) Larvae were injected with hormone to investigate the effect of molting hormone(20-hydroxyecdysone) and juvenile hormone analogues(methoprenen) on the expression of Ofur Trx. The results showed that methoprene and 20 E significantly induce Ofur Trx expression, it was showed a significantly promotion at 30 min, but decrease after 2 h-20 E treatment and 1 h methoprenen treatment.(5) To further clarify the regulation mechanism of molting hormone on Ofur Trx expression, the double-stranded RNA of the ecdysone receptor and ultraspiracle isoform were transcripted synthesis and injected into the fifth 24 h instar larvae in vitro. The expression of ecr and usp gene are decreased significantly after interference of ecdysone receptor by injecting the double-stranded RNA into the fifth instar 24 h larvaes. Meanwhile we have injected the hormone to the larvaes of which the ecdysone receptors have been interferenced, then we found that the expression of Ofur Trx is up regulation. These results futher demonstrate that the molting hormone inhibit the expression of Ofur Trx gene.