Identification of 20-hydroxyecdysone late-response genes in the chitin biosynthesis pathway.
BACKGROUND
20-hydroxyecdysone (20E) and its receptor complex ecdysone receptor (EcR) and ultraspiracle (USP) play a crucial role in controlling development, metamorphosis, reproduction and diapause. The ligand-receptor complex 20E-EcR/USP directly activates a small set of early-response genes and a much larger set of late-response genes. However, ecdysone-responsive genes have not been previously characterized in the context of insect chitin biosynthesis.


RESULTS
Here, we show that injection-based RNA interference (RNAi) directed towards a common region of the two isoforms of SeEcR in a lepidopteron insect Spodoptera exigua was effective, with phenotypes including a high mortality prior to pupation and developmental defects. After gene specific RNAi, chitin contents in the cuticle of an abnormal larva significantly decreased. The expression levels of five genes in the chitin biosynthesis pathway, SeTre-1, SeG6PI, SeUAP, SeCHSA and SeCHSB, were significantly reduced, while there was no difference in the expression of SeTre-2 prior to 72 hr after injection of EcR dsRNA. Meanwhile, injection of 20E in vivo induced the expression of the five genes mentioned above. Moreover, the SeTre-1, SeG6PI, SeUAP and SeCHSB genes showed late responses to the hormone and the induction of SeTre-1, SeG6PI, SeUAP and SeCHSB genes by 20E were able to be inhibited by the protein synthesis inhibitor cycloheximide in vitro indicating these genes are 20E late-response genes.


CONCLUSIONS
We conclude that SeTre-1, SeG6PI, SeUAP and SeCHSB in the chitin biosynthesis pathway are 20E late-response genes and 20E and its specific receptors plays a key role in the regulation of chitin biosynthesis via inducing their expression.
State Key Laboratory of Biocontrol, School of Life Sciences, Sun Yat-sen University, Guangzhou, China.
Qiong
Q
Yao
Daowei
D
Zhang
Bin
B
Tang
Jie
J
Chen
Jing
J
Chen
Liang
L
Lu
Wenqing
W
Zhang
D000818:Animals; D053898:Biosynthetic Pathways; D015153:Blotting, Western; D002686:Chitin / Q000096:biosynthesis*; D003513:Cycloheximide / Q000494:pharmacology; D004441:Ecdysterone / Q000494:pharmacology*; D005786:Gene Expression Regulation / Q000187:drug effects; D019476:Insect Proteins / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D010802:Phylogeny; D020033:Protein Isoforms / Q000145:classification / Q000235:genetics / Q000378:metabolism; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology; D034622:RNA Interference; D011987:Receptors, Steroid / Q000145:classification / Q000235:genetics / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D018411:Spodoptera / Q000235:genetics / Q000378:metabolism*; D013997:Time Factors
D019476:Insect Proteins; D020033:Protein Isoforms; D011500:Protein Synthesis Inhibitors; D011987:Receptors, Steroid; C055469:ecdysone receptor; D002686:Chitin; D004441:Ecdysterone; D003513:Cycloheximide