巨尾阿丽蝇卵不同发育时间形态变化及基因表达

Morphological characteristics and gene expression of Aldrichina grahami egg in different developmental stages

目的探讨巨尾阿丽蝇（Aldrichina grahami）卵不同发育时间的形态变化与基因表达差异规律。方法从巨尾阿丽蝇成虫产完卵开始记为0 h,每隔4 h取1次卵,直至幼虫孵出,用扫描电镜观察不同发育时期形态学变化特点;提取0、4、8、12、16 h的蝇卵RNA,应用Real-time PCR比较循环阈值（CT值）法测定bicoid、slalom、几丁质合成酶（chitin synthase）基因相对表达量,用SPSS 19.0软件对CT值及时间进行曲线拟合。结果扫描电镜结果显示,发育0～4 h时,巨尾阿丽蝇卵变化不明显,卵孔周围有突起,8 h卵孔周围变光滑,卵孔可见,12 h卵孔消失,周围皱缩,16 h卵孔周围皱缩明显,18 h发育为幼虫;RT-PCR结果显示,bicoid基因在发育0～8 h表达变化不明显、12h表达水平最高,CT值为（4.19±0.04）;slalom基因4 h表达水平最低、12 h表达最高,CT值分别为（4.05±0.01）、（6.20±0.03）;chitin synthase基因8 h表达量最高、12 h最低,CT值分别为（5.80±0.01）、（4.38±0.02）;不同发育阶段各基因CT值不同,差异均有统计学意义（均P〈0.05）。结论巨尾阿丽蝇卵形态结构随发育时间而发生变化,不同发育时期bicoid、slalom、chitin synthase基因表达量存在差异。

Objective To examine the morphological characteristics and differential gene expression of Aldrichina grahami egg in the different developmental stages. Methods After Aldrichina grahami layed eggs（ 0 h）,the eggs were collected every 4 hours until the eggs hatched into larvae. The morphological characteristics of Aldrichina grahami egg in different developmental stages were observed with scanning electron microscopy. The total RNA the eggs at every develop-mental stage was extracted. The expression levels of bicoid,slalom,and chitin synthase gene were determined with real-time flourescence quantitative PCR. Statistic analyses were performed with SPSS 19. 0. Results The scanning electron microscope images show ed that the morphological change of the eggs were not obvious in the first 4 hours,with protuberance around the micropyle. At the 8th hour the surface around the micropyle was smooth and the shape of micropyle was visible. At the 12 th hour,the micropyle disappeared,with shrinking around the place of the micropyle and the shrinking was more obvious at the 16 th hour. After 18 hours the larvae hatched from the eggs. Real-time fluorescence quantitative PCR results indicated that the expression levels of bicoid,slalom and chitin synthase genes in the Aldrichina grahami eggs changed regularly with the developmental stages; the change of bicoid gene expression was not obvious from 0 to 8th hour and at 12 th hour the expression level was the highest,with a CT value of 4. 19 ± 0. 04; the low est slalom gene expression appeared at 4th hour and the highest at 12 th hour,with the CT values of 4. 05 ± 0. 01 and 6. 20 ±0. 03; the highest chitin synthase gene expression appeared at 8th hour and the low est at 12 th hour,with the CT values of5. 80 ± 0. 01 and 4. 38 ± 0. 02,respectively; the expression levels were different in different development stages and there were significant difference in CT values between different developmental stages of Aldrichina grahami eggs（ all P〈0. 05）.Conclusion The morphological characteristics of Aldrichina grahami eggs changes with the development stage. The expression levels of bicoid,slalom,and chitin synthase genes of Aldrichina grahami eggs are different in various development period.