二化螟滞育生物钟蛋白TIME-EA4基因的克隆及时空和温度诱导表达分析

Cloning and spatiotemporal and temperature-induced expression profiling of diapause bioclock protein TIME-EA4 gene in the rice stem borer,Chilo suppressalis( Lepidoptera: Pyralidae)

【目的】生物测时蛋白TIME-EA4是昆虫特有的一种滞育生物钟蛋白。本文旨在克隆二化螟Chilo suppressalis TIME-EA4基因,研究其在二化螟不同发育时期和不同组织中的时空表达模式及在不同田间种群和不同温度诱导下的滞育和非滞育种群中的表达水平。【方法】通过RACE技术克隆二化螟TIME-EA4基因全长c DNA序列,利用荧光定量PCR的方法检测TIME-EA4基因在二化螟不同发育时期、6龄幼虫不同组织中的表达模式,及在不同季节采集的田间种群和不同温度处理1h后的滞育和非滞育种群6龄幼虫中的表达量变化。【结果】从二化螟中克隆获得TIME-EA4的c DNA序列(Gen Bank登录号:KU356855),全长821 bp,开放阅读框516 bp,编码172个氨基酸;TIME-EA4基因在二化螟不同发育时期和不同组织中均有表达,且在早期蛹、雌雄成虫以及6龄幼虫的头部、脂肪体和中肠中表达水平较高,同时在滞育幼虫组织中的表达高于非滞育幼虫。此外,TIME-EA4基因在滞育种群中的表达量是非滞育种群的3倍左右,同时在非滞育种群中,该基因的表达明显受10℃及以下低温的诱导,且在10℃时表达量最高,而在滞育种群中该基因的表达受温度影响不明显。【结论】结果说明TIME-EA4基因与二化螟的抗低温胁迫以及低温滞育相关。本研究为TIME-EA4在农业害虫滞育的分子作用机制研究奠定了基础。

【Aim 】 Time interval measuring enzyme（ TIME-EA4） is a diapause bioclock protein in insects. This study aims to clone TIME-EA4 c DNA and analyze its spatiotemporal expression patterns in various developmental stages and different tissues of Chilo suppressalis,including the populations collected in the fields,and diapause and non-diapause populations induced at various temperatures. 【Methods】Full-length c DNA sequence of TIME-EA4 gene of C. suppressalis was cloned by using RACE technique.The relative expression levels of TIME-EA4 in different developmental stages,different tissues of the 6th instar larvae,larvae of field populations collected in different seasons and the 6th instar larvae from different populations of the moth treated for one hour at different temperatures were assayed by real-time PCR. 【Results】The full-length c DNA of C. suppressalis TIME-EA4 is 821 bp,with an open reading frame of 516 bp encoding 172 aa. TIME-EA4 was ubiquitously expressed,but with higher expression levels in early pupae and adults,and in the head,fat body and midgut of the 6th instar larvae. And the expression level of TIME-EA4 in the tissues of diapause larvae was much higher than that in the nondiapause larvae. Interestingly,the expression level of TIME-EA4 was significantly increased 3-fold in diapause groups compared with that in the non-diapause individuals. The abundance of TIME-EA4 gene was elevated at ≤10℃ in the non-diapause population,and reached the peak at 10℃. However,TIMEEA4 expression was not significantly altered by temperature change in the diapause population.【Conclusion】These results suggest that the enhanced expression of TIME-EA4 correlates with the low temperature stress or low temperature-induced diapause in C. suppressalis. This study forms a basis for further investigation of TIME-EA4 in the diapause of agricultural pests.