【摘要】 通过对青虾性别调控相关基因transformer-2(tra-2)的RNA干扰规律进行研究,探讨了tra-2基因RNA干扰的最适注射部位、注射剂量、干扰效果以及干扰规律等。通过对青虾肌肉、心脏、围心腔和眼柄基部注射比较发现,围心腔注射死亡率最低,适宜RNA干扰研究。采用4μg/g的dsRNA剂量进行时间依赖性干扰试验,RT-PCR检验注射后第1,7,14天的卵巢tra-2表达量,结果显示第7天基因表达量出现显著降低(P<0.05)。不同剂量水平(0.4μg/g、4μg/g以及12μg/g)干扰研究发现,0.4μg/g dsRNA没有干扰结果,4μg/g和12μg/g剂量的干扰效果均显著。与4μg/g的剂量相比,12μg/g的dsRNA能在较短时间内起到明显的干扰效果,且能持续较长的时间,表明干扰规律会随着剂量的改变而变化。但两组tra-2的最低表达量分别为正常水平的23%和21%,无显著差异(P>0.05)。更多还原

【Abstract】 The RNA interference regularity of transformer- 2(tra- 2) gene of oriental river prawn Macrobrachium nipponense was studied in this paper to find the injection sites, dose, interference effects and interference regularity. The optimal injecting sites were chosen from muscle, heart, pericardial cavity and the base of eyestalk. The pericardial cavity was the suitable site for injection due to the lowest mortality rate. For the time-dependent silencing experiment, the dsRNA quantity was calculated as 4 μg dsRNA/g body wt. The results of 1,7,or 14 days by Real- time RT- PCR showed that tra-2 gene relative mRNA expression was significantly reduced(P<0.05) on the 7th day. Experiments of different dose levels showed that the treatment group which was injected at a dose of 0.4μg/g had no interference effect; the groups with dose of 4 μg/g and 12 μg/g had significant interference effect. Compared to dose of 4 μg/g, the dose of 12 μg/g could have interference effect in a relatively short time and last for a longer time. It indicated that the interference pattern would change as the dose changed. But the lowest expression level of the two groups were 23% and 21% of the normal level, with no significant difference(P>0.05).更多还原