二化螟中肠干细胞的增殖和分化及其与Cry1Ab耐受性的关系

发布时间：2018-05-17 03:07

本文选题：二化螟 + 中肠干细胞　；参考：《山西农业大学》2015年硕士论文

【摘要】：二化螟(Chilo suppressalis Walker)是水稻上的重要害虫,转Bt基因水稻的研制为二化螟的防治提供了新策略,但二化螟对Bt水稻的抗性风险是制约其可持续应用的关键因素。干细胞是一类能自我更新并具有多向性分化潜能的细胞,其增殖和分化有利于昆虫中肠的再生和修复,而中肠的再生和修复与昆虫Bt抗性的产生密切相关,基于此,本文开展了不同因素包括龄期、培养基添加物对二化螟中肠干细胞增殖与分化影响的研究,及干细胞的增殖与分化与二化螟Cry1Ab耐受性的关系,主要研究结果如下1.分别采用干细胞分离法和胶原酶消化法提取中肠细胞,发现0.1%胶原酶消化法提取的细胞较前者纯度高；进而根据干细胞和成肠细胞形态结构和密度的差异,以及由此产生的免疫荧光反应的差异,进一步采用流式细胞术的方法检测了干细胞的增殖与分化。2.本研究测定了3、4和5龄幼虫的干细胞增殖与分化能力,发现3龄幼虫中肠中的干细胞较多,进而以3龄幼虫为实验材料进行后续研究；通过在培养基中加入二化螟幼虫的脂肪体提取物和蜕皮激素,明确了其具有促进干细胞增殖的作用。3.通过比较Cry1Ab对二化螟敏感和Cry1Ab筛选种群中肠干细胞增殖与分化的影响,得出敏感种群中肠的增殖与分化受不同浓度的Cry1Ab影响较大,且在Cry1Ab溶液浓度为0.15μg/mL时,干细胞增殖最快,而Cry1Ab筛选种群试虫在0.3μg/mL时增殖和分化速度最快,从细胞水平证实敏感比Cry1Ab筛选种群对Cry1Ab的敏感性高。4.先饥饿试虫5h再喂食亚致死剂量(LC10)的Cry1Ab溶液,之后解剖中肠做切片,进行透射电镜观察,与对照组相比,发现0.3 μg/mL Cry1Ab处理组细胞的变化比较明显,包括中肠细胞微绒毛脱落、线粒体数目减少,并且出现自我吞噬、异染色质以及涡旋膜结构,明确了在Cry1Ab毒素诱导下二化螟体内中肠细胞的变化。5.在培养基中加入不同浓度的Cry1Ab溶液,在各浓度的细胞悬浮液中都检测到中肠细胞的凋亡,且Cry1Ab浓度为0.06 μg/mL时凋亡最严重。在不更换培养基的条件下,凋亡细胞的数目随着时间的推移而增加,明确了Cry1Ab毒素对离体状态下二化螟中肠细胞的毒害作用。
[Abstract]:Chilo suppressalis Walker is an important pest in rice. The development of transgenic BT rice provides a new strategy for the control of Chilo suppressalis, but the risk of resistance to BT rice is the key factor restricting its sustainable application. Stem cells are a kind of cells which can regenerate themselves and have the potential of multidirectional differentiation. The proliferation and differentiation of stem cells are beneficial to the regeneration and repair of insect midgut, and the regeneration and repair of midgut is closely related to the production of insect BT resistance. In this paper, the effects of different factors, including age, medium addition on the proliferation and differentiation of stem cells in midgut of Chilo suppressalis, and the relationship between proliferation and differentiation of stem cells and Cry1Ab tolerance of Chilo suppressalis were studied. The main results are as follows: 1. Stem cell isolation and collagenase digestion were used to extract mesenteric cells. The results showed that the cells extracted by 0.1% collagenase digestion were of higher purity than that of the former, and then according to the difference of morphology, structure and density of stem cells and intestinal cells. Furthermore, flow cytometry was used to detect the proliferation and differentiation of stem cells. In this study, the ability of stem cell proliferation and differentiation of 3rd and 5th instar larvae was measured. It was found that there were more stem cells in the midgut of 3rd instar larva, and then the third instar larva was used as experimental material for further study. By adding fat body extract and ecdysone from Chilo suppressalis larva in culture medium, it was determined that it could promote stem cell proliferation. By comparing the effects of Cry1Ab on the proliferation and differentiation of stem cells from midgut of Chilo suppressalis and Cry1Ab screening population, it was concluded that the proliferation and differentiation of mesenteric stem cells in sensitive populations were greatly affected by different concentrations of Cry1Ab, and the proliferation of stem cells was the fastest when the concentration of Cry1Ab solution was 0.15 渭 g/mL. The rate of proliferation and differentiation was the fastest at 0.3 渭 g/mL for Cry1Ab screening population, and the sensitivity of Cry1Ab screening population to Cry1Ab was higher than that of Cry1Ab screening population. The Cry1Ab solution of sublethal dose (LC10) was fed to the starved insect for 5 h, then the midgut was sectioned and observed by transmission electron microscope. Compared with the control group, the changes of the cells in the 0.3 渭 g/mL Cry1Ab treatment group were obvious, including the microvilli of the midgut cells shedding. The decrease in the number of mitochondria and the appearance of self-phagocytosis, heterochromatin and vortex membrane structure indicate the changes of midgut cells induced by Cry1Ab toxin. The apoptosis of midgut cells was detected in the cell suspensions with different concentrations of Cry1Ab solution, and the apoptosis was the most serious when the concentration of Cry1Ab was 0.06 渭 g/mL. The number of apoptotic cells increased with the passage of time without changing the culture medium. The toxic effect of Cry1Ab toxin on the midgut cells of Chilo suppressalis in vitro was confirmed.
【学位授予单位】：山西农业大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S433.4

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