大头金蝇卵不同发育时间形态变化及基因表达差异研究

Morphological Characteristics and Gene Expression of Chrysomya megacephala Eggs in Different Developmental Stages

目的 研究大头金蝇（Chrysomya megacephala）卵不同发育时间的形态变化和基因表达差异规律。方法 从大头金蝇成虫产完卵后,取出卵块,记时为0 h,每隔2 h取卵10枚,直至有幼虫孵出,分别用体视显微镜和扫描电镜观察其不同发育时间的形态学变化。提取0、2、4、6和8 h等5个发育时间点的蝇卵RNA,应用RT-PCR比较循环阈值（threshold cycle,CT值）法测定bicoid、slalom和几丁质合成酶（chitin synthase）基因相对表达水平,SPSS19.0统计学软件对CT值及时间进行拟合直线方程、等比级数曲线方程和指数方程分析。结果 体视显微镜下观察,大头金蝇卵产后0～4h形态学变化不明显,6h有体节形成,8 h开始出现皱缩,9 h有幼虫孵出;扫描电镜下观察,卵产后0～4 h时变化不明显,卵孔一端略向外突出,卵孔周围光滑,6 h后卵孔一端向内凹陷,卵孔周围有不规则突起形成,8 h时凹陷明显,9 h已发育为幼虫。大头金蝇卵在不同发育时间bicoid、slalom和chitin synthase等3个基因的相对表达水平随着时间变化呈一定的规律变化,相关回归方程式3个基因CT值的差异均有统计学意义（P〈0.05）。结论 体视显微镜和扫描电镜下大头金蝇卵形态结构随时间改变而发生变化。大头金蝇卵不同发育时间的bicoid、slalom和chitin synthase基因表达水平有差异。

Objective To research the morphological characteristics and differential gene expression of Chrysomya megacephala eggs in different developmental stages. Methods After C. megacephala laid eggs（0 h）, the eggs were collected every 2 h until eggs hatched into larvae. The morphological characteristics of C. megacephala eggs in different developmental stages were observed by stereo microscopy and scanning electron microscopy. The total RNA of the fly eggs was extracted. The expression levels of bicoid, slalom and chitin synthase genes was determined by Real-time flourescence quantitative PCR. Statistic analyses were performed with SPSS 19.0. Results Under the stereomicroscope, at 0-4 h after egg laying, the morphological change of C. megacephala eggs was not obvious. At the 6th hour after egg laying, somites were formed. After 8 hours the eggs shriveled. At the 9th hour after egg laying, the eggs hatched into larvae. The scanning electron microscope images showed that the morphological change of eggs was not obvious in the first 4 hours,the end of micropyle slightly outward, the surface around the micropyle was smooth. At the 6th hour after egg laying, the end of micropyle began to sag and irregular protrusions formed around the micropyle. At the 8th hour the end of micropyle was obviously dented. After 9 hours larvae hatched from eggs. Real-time fluorescence quantitative PCR indicated that the expression levels of bicoid, slalom and chitin synthase genes from C. megacephala eggs regularly changed with the developmental stages. There was a significant difference in threshold cycle values among the three genes（ P 0.05）.Conclusion The morphological characteristics of C. megacephala eggs change with the development stage. The levels of gene expression in different development period of C. megacephala eggs are different.