發(fā)布時(shí)間：2018-04-16 06:09

  本文選題：家蠶 + 滯育解除��； 參考：《華南農(nóng)業(yè)大學(xué)》2016年碩士論文

【摘要】：家蠶是研究卵滯育的重要模式昆蟲(chóng)之一,具有良好的可調(diào)控性和操作性并取得了相當(dāng)多的研究成果。本研究以932品種的蠶卵為實(shí)驗(yàn)材料,通過(guò)多種不同方式處理蠶卵,獲得最佳的解除滯育的方式;利用生物信息學(xué)、分子生物學(xué)和基因工程等技術(shù)方法,篩選出鹽酸解除滯育過(guò)程中的氯離子通道蛋白基因,進(jìn)行克隆和表達(dá)分析;同時(shí)研究了胚胎發(fā)育過(guò)程四種輔酶的變化,獲得主要結(jié)果如下:(1)蠶卵在鹽酸和DMSO中浸漬不同時(shí)間得到的孵化率不同。蠶卵產(chǎn)后20h進(jìn)行浸酸,浸酸時(shí)間0.5~1.5min時(shí),隨著浸酸時(shí)間的增加孵化率逐漸上升;浸酸時(shí)間1.5~6min時(shí),孵化率保持在95%~97%之間。蠶卵產(chǎn)后20h利用DMSO浸漬,浸漬時(shí)間15~40min時(shí),孵化率逐漸上升;浸漬時(shí)間40~50min時(shí),孵化率保持在88%~90%之間;浸漬時(shí)間60min時(shí),孵化率下降為86%。蠶卵產(chǎn)后10d完全進(jìn)入滯育,置于5℃冷庫(kù)保存90d,再用鹽酸進(jìn)行刺激,浸酸時(shí)間4~6min時(shí),孵化率維持在94%~95%之間;浸酸7min時(shí),孵化率下降為93%,發(fā)現(xiàn)蠶卵死亡現(xiàn)象。(2)克隆家蠶細(xì)胞內(nèi)氯離子通道(BmCLIC)、家蠶谷氨酸氯離子通道(BmGluCl)和家蠶γ-氨基丁酸氯離子通道受體(Bmγ-Cl)三個(gè)氯離子通道基因。根據(jù)候選基因的CDS序列設(shè)計(jì)引物,通過(guò)PCR、瓊脂糖電泳、膠回收、連接載體、酶切和測(cè)序等技術(shù),成功克隆出BmCLIC、BmGluCl和Bmγ-Cl 3個(gè)目的基因。(3)通過(guò)實(shí)時(shí)熒光定量PCR技術(shù),檢測(cè)了BmCLIC、BmGluCl和Bmγ-Cl基因在不同處理卵和胚胎發(fā)育過(guò)程中的轉(zhuǎn)錄表達(dá)情況。發(fā)現(xiàn)產(chǎn)后20h卵和冷藏卵浸酸處理后BmCLIC、BmGluCl和Bmγ-Cl基因相對(duì)表達(dá)量都相應(yīng)提高,表明浸酸可以誘導(dǎo)目的基因上調(diào)。在滯育卵中,BmCLIC基因第1d相對(duì)表達(dá)量最大,隨著發(fā)育經(jīng)過(guò)逐漸減小;BmGluCl基因第1d相對(duì)表達(dá)量最大,在發(fā)育過(guò)程中先減小后增加;Bmγ-Cl基因第2d相對(duì)表達(dá)量最大值為2.04,隨著發(fā)育經(jīng)過(guò)逐漸減小。在即時(shí)浸酸卵和DMSO處理卵發(fā)育過(guò)程中的三個(gè)候選基因都有相同的變化趨勢(shì)。(4)檢測(cè)了滯育卵和即時(shí)浸酸卵的胚胎發(fā)育1~9d中的乙酰輔酶A、黃素腺嘌呤二核苷酸、輔酶Ⅰ和輔酶Ⅱ四種輔酶的變化。乙酰輔酶A在滯育卵發(fā)育過(guò)程中,乙酰輔酶A呈現(xiàn)持續(xù)下降;在浸酸卵發(fā)育前期持續(xù)下降,第5d后緩慢回升。FAD在滯育卵發(fā)育前期呈現(xiàn)上升,第5d后呈現(xiàn)下降趨勢(shì);在即時(shí)浸酸卵發(fā)育過(guò)程中,每克蛋白中的FAD量從第1d的0.00024pg一直上升到第9d的0.00102pg。NAD在滯育卵發(fā)育過(guò)程中,從第1d的41.54952下降到第4d的13.45342下降,第4d后保持在相對(duì)較低的穩(wěn)定狀態(tài);在浸酸卵發(fā)育過(guò)程中,呈現(xiàn)上升趨勢(shì),第9d時(shí)峰值為122.04747mol/g。NADP在滯育卵發(fā)育過(guò)程中,保持穩(wěn)定的水平;在浸酸卵發(fā)育過(guò)程中,呈現(xiàn)上升趨勢(shì),第9d時(shí)峰值為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，

本文編號(hào)：1757604