本文選題：二化螟 + 中腸干細(xì)胞��； 參考：《山西農(nóng)業(yè)大學(xué)》2015年碩士論文

【摘要】：二化螟(Chilo suppressalis Walker)是水稻上的重要害蟲,轉(zhuǎn)Bt基因水稻的研制為二化螟的防治提供了新策略,但二化螟對Bt水稻的抗性風(fēng)險是制約其可持續(xù)應(yīng)用的關(guān)鍵因素。干細(xì)胞是一類能自我更新并具有多向性分化潛能的細(xì)胞,其增殖和分化有利于昆蟲中腸的再生和修復(fù),而中腸的再生和修復(fù)與昆蟲Bt抗性的產(chǎn)生密切相關(guān),基于此,本文開展了不同因素包括齡期、培養(yǎng)基添加物對二化螟中腸干細(xì)胞增殖與分化影響的研究,及干細(xì)胞的增殖與分化與二化螟Cry1Ab耐受性的關(guān)系,主要研究結(jié)果如下1.分別采用干細(xì)胞分離法和膠原酶消化法提取中腸細(xì)胞,發(fā)現(xiàn)0.1%膠原酶消化法提取的細(xì)胞較前者純度高；進(jìn)而根據(jù)干細(xì)胞和成腸細(xì)胞形態(tài)結(jié)構(gòu)和密度的差異,以及由此產(chǎn)生的免疫熒光反應(yīng)的差異,進(jìn)一步采用流式細(xì)胞術(shù)的方法檢測了干細(xì)胞的增殖與分化。2.本研究測定了3、4和5齡幼蟲的干細(xì)胞增殖與分化能力,發(fā)現(xiàn)3齡幼蟲中腸中的干細(xì)胞較多,進(jìn)而以3齡幼蟲為實驗材料進(jìn)行后續(xù)研究；通過在培養(yǎng)基中加入二化螟幼蟲的脂肪體提取物和蛻皮激素,明確了其具有促進(jìn)干細(xì)胞增殖的作用。3.通過比較Cry1Ab對二化螟敏感和Cry1Ab篩選種群中腸干細(xì)胞增殖與分化的影響,得出敏感種群中腸的增殖與分化受不同濃度的Cry1Ab影響較大,且在Cry1Ab溶液濃度為0.15μg/mL時,干細(xì)胞增殖最快,而Cry1Ab篩選種群試蟲在0.3μg/mL時增殖和分化速度最快,從細(xì)胞水平證實敏感比Cry1Ab篩選種群對Cry1Ab的敏感性高。4.先饑餓試蟲5h再喂食亞致死劑量(LC10)的Cry1Ab溶液,之后解剖中腸做切片,進(jìn)行透射電鏡觀察,與對照組相比,發(fā)現(xiàn)0.3 μg/mL Cry1Ab處理組細(xì)胞的變化比較明顯,包括中腸細(xì)胞微絨毛脫落、線粒體數(shù)目減少,并且出現(xiàn)自我吞噬、異染色質(zhì)以及渦旋膜結(jié)構(gòu),明確了在Cry1Ab毒素誘導(dǎo)下二化螟體內(nèi)中腸細(xì)胞的變化。5.在培養(yǎng)基中加入不同濃度的Cry1Ab溶液,在各濃度的細(xì)胞懸浮液中都檢測到中腸細(xì)胞的凋亡,且Cry1Ab濃度為0.06 μg/mL時凋亡最嚴(yán)重。在不更換培養(yǎng)基的條件下,凋亡細(xì)胞的數(shù)目隨著時間的推移而增加,明確了Cry1Ab毒素對離體狀態(tài)下二化螟中腸細(xì)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.
【學(xué)位授予單位】：山西農(nóng)業(yè)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2015
【分類號】：S433.4

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本文編號：1899614