家蚕滞育解除过程中相关氯离子通道蛋白基因及辅酶研究

发布时间：2018-04-16 06:09

  本文选题：家蚕 + 滞育解除　； 参考：《华南农业大学》2016年硕士论文

【摘要】：家蚕是研究卵滞育的重要模式昆虫之一,具有良好的可调控性和操作性并取得了相当多的研究成果。本研究以932品种的蚕卵为实验材料,通过多种不同方式处理蚕卵,获得最佳的解除滞育的方式;利用生物信息学、分子生物学和基因工程等技术方法,筛选出盐酸解除滞育过程中的氯离子通道蛋白基因,进行克隆和表达分析;同时研究了胚胎发育过程四种辅酶的变化,获得主要结果如下:(1)蚕卵在盐酸和DMSO中浸渍不同时间得到的孵化率不同。蚕卵产后20h进行浸酸,浸酸时间0.5~1.5min时,随着浸酸时间的增加孵化率逐渐上升;浸酸时间1.5~6min时,孵化率保持在95%~97%之间。蚕卵产后20h利用DMSO浸渍,浸渍时间15~40min时,孵化率逐渐上升;浸渍时间40~50min时,孵化率保持在88%~90%之间;浸渍时间60min时,孵化率下降为86%。蚕卵产后10d完全进入滞育,置于5℃冷库保存90d,再用盐酸进行刺激,浸酸时间4~6min时,孵化率维持在94%~95%之间;浸酸7min时,孵化率下降为93%,发现蚕卵死亡现象。(2)克隆家蚕细胞内氯离子通道(BmCLIC)、家蚕谷氨酸氯离子通道(BmGluCl)和家蚕γ-氨基丁酸氯离子通道受体(Bmγ-Cl)三个氯离子通道基因。根据候选基因的CDS序列设计引物,通过PCR、琼脂糖电泳、胶回收、连接载体、酶切和测序等技术,成功克隆出BmCLIC、BmGluCl和Bmγ-Cl 3个目的基因。(3)通过实时荧光定量PCR技术,检测了BmCLIC、BmGluCl和Bmγ-Cl基因在不同处理卵和胚胎发育过程中的转录表达情况。发现产后20h卵和冷藏卵浸酸处理后BmCLIC、BmGluCl和Bmγ-Cl基因相对表达量都相应提高,表明浸酸可以诱导目的基因上调。在滞育卵中,BmCLIC基因第1d相对表达量最大,随着发育经过逐渐减小;BmGluCl基因第1d相对表达量最大,在发育过程中先减小后增加;Bmγ-Cl基因第2d相对表达量最大值为2.04,随着发育经过逐渐减小。在即时浸酸卵和DMSO处理卵发育过程中的三个候选基因都有相同的变化趋势。(4)检测了滞育卵和即时浸酸卵的胚胎发育1~9d中的乙酰辅酶A、黄素腺嘌呤二核苷酸、辅酶Ⅰ和辅酶Ⅱ四种辅酶的变化。乙酰辅酶A在滞育卵发育过程中,乙酰辅酶A呈现持续下降;在浸酸卵发育前期持续下降,第5d后缓慢回升。FAD在滞育卵发育前期呈现上升,第5d后呈现下降趋势;在即时浸酸卵发育过程中,每克蛋白中的FAD量从第1d的0.00024pg一直上升到第9d的0.00102pg。NAD在滞育卵发育过程中,从第1d的41.54952下降到第4d的13.45342下降,第4d后保持在相对较低的稳定状态;在浸酸卵发育过程中,呈现上升趋势,第9d时峰值为122.04747mol/g。NADP在滞育卵发育过程中,保持稳定的水平;在浸酸卵发育过程中,呈现上升趋势,第9d时峰值为59.45774 mol/g。
[Abstract]:Silkworm (Bombyx mori) is one of the most important model insects in the study of diapause.In this study, 932 varieties of silkworm eggs were used as experimental materials to obtain the best way to remove diapause by treating the eggs in many different ways, and the methods of bioinformatics, molecular biology and genetic engineering were used.The chloride channel protein gene during diapause was screened, cloned and expressed, and the changes of four coenzymes during embryonic development were studied.The main results are as follows: (1) the hatching rate of silkworm eggs impregnated in hydrochloric acid and DMSO is different.The hatching rate of eggs increased with the increase of acid-soaking time (0.5~1.5min), and the hatching rate of 1.5~6min kept between 95% and 97% at the time of soaking at 20 h postpartum.When eggs were impregnated with DMSO at 20 h after delivery, the hatching rate of 15~40min increased gradually, while that of 40~50min at impregnation time kept between 88% and 90%, and that of incubation rate decreased to 86% during impregnation time 60min.Silkworm eggs entered diapause completely after 10 days postpartum, stored in 5 鈩，

本文编号：1757604