本文選題：果實(shí)特異啟動(dòng)子 + 農(nóng)桿菌介導(dǎo)法��； 參考：《云南農(nóng)業(yè)大學(xué)》2016年碩士論文

【摘要】：番茄(Lycopersicon esculentum Mill.)是茄科(Solanaceae),番茄屬的植物,是原產(chǎn)自南美亞熱帶地區(qū),一年生植物,是世界各地廣泛種植的一種重要蔬菜作物之一。在番茄上開(kāi)展表達(dá)口服疫苗的研究,可大大提升番茄的應(yīng)用價(jià)值。不同類(lèi)型的果實(shí)特異性啟動(dòng)子在果實(shí)中的發(fā)育時(shí)期及調(diào)控機(jī)理不同,篩選理想的果實(shí)特異性啟動(dòng)子可為利用番茄作為載體來(lái)表達(dá)外源蛋白奠定前期基礎(chǔ)。IGF-1是調(diào)節(jié)機(jī)體生長(zhǎng)發(fā)育、生殖、免疫和機(jī)體代謝的關(guān)鍵因子,在治療糖尿病、骨質(zhì)疏松、肌營(yíng)養(yǎng)不良性側(cè)索硬化病等疾病具有廣闊的應(yīng)用前景。本研究以番茄為材料,構(gòu)建了包含不同果實(shí)特異性的5個(gè)啟動(dòng)子表達(dá)載體,采用農(nóng)桿菌介導(dǎo)法,將IGF-1基因?qū)敕阎?并進(jìn)行了分子檢測(cè),獲得了轉(zhuǎn)基因番茄植株,為利用番茄特異表達(dá)外源蛋白提供技術(shù)和理論基礎(chǔ),也為利用番茄口服治療糖尿病奠定材料基礎(chǔ)。具體研究結(jié)果如下:(1)表達(dá)載體的構(gòu)建:本研究構(gòu)建了5個(gè)果實(shí)特異性啟動(dòng)子的植物表達(dá)載體:pCAP-IGF-1、pCAPG-IGF-1、pCAGP-IGF-1、pCATP-IGF-1和pCAOC-IGF-1。并將5個(gè)植物表達(dá)載體導(dǎo)入農(nóng)桿菌LBA4404菌株中。(2)番茄遺傳轉(zhuǎn)化體系的優(yōu)化:試驗(yàn)采用AC++和1448兩種番茄材料,分別進(jìn)行再生體系的培養(yǎng),結(jié)果表明:番茄材料AC++誘導(dǎo)分化的效果明顯高于1448,進(jìn)而確立了最佳再生體系培養(yǎng)的材料為番茄材料AC++。采用正交設(shè)計(jì)法,研究了不同的激素水平(IAA、ZT、AgNO3)對(duì)番茄再生體系的影響。確定了誘導(dǎo)愈傷組織及不定芽分化的最適宜培養(yǎng)基為:MS+ZT 2.0mg/L+IAA0.5mg/L+AgNO3 3.0mg/l;誘導(dǎo)生根的最適宜培養(yǎng)基為:1/2 MS+IAA 0.3mg/L。采用正交設(shè)計(jì)法,對(duì)影響轉(zhuǎn)化效率的各參數(shù)(浸染液、乙酰丁香酮、浸染時(shí)間、共培養(yǎng)天數(shù))進(jìn)行優(yōu)化,確定了最適宜的轉(zhuǎn)化參數(shù):以浸染液為MS的液體培養(yǎng)基,不添加乙酰丁香酮、浸染時(shí)間10 min、共培養(yǎng)天數(shù)2d為最佳轉(zhuǎn)化條件。(3)抗性植株的PCR鑒定:經(jīng)轉(zhuǎn)化體系的培養(yǎng),最終獲得抗性苗共41株,其中AGPL1啟動(dòng)子獲得抗性植株15株,黃瓜素啟動(dòng)子獲得抗性植株12株,甜瓜ACO啟動(dòng)子獲得抗性植株6株,PG啟動(dòng)子獲得抗性植株6株,AP啟動(dòng)子獲得2株抗性植株。對(duì)獲得的抗性植株進(jìn)行PCR分子檢測(cè),共21株陽(yáng)性植株,陽(yáng)性率達(dá)51.21%。證明外源基因IGF-1已成功整合進(jìn)入番茄基因組中。其中AGPL1啟動(dòng)子最終獲得陽(yáng)性植株8株,黃瓜素啟動(dòng)子獲得陽(yáng)性植株6株,甜瓜ACO啟動(dòng)子獲得陽(yáng)性植株3株,PG啟動(dòng)子獲得陽(yáng)性植株3株,AP啟動(dòng)子獲得1株陽(yáng)性植株。
[Abstract]:Lycopersicon esculentum Mill.) Solanaceaeus, a plant of Solanaceaeus, originated from the subtropical region of South America, is an annual plant. It is one of the important vegetable crops widely planted all over the world. The study on the expression of oral vaccine on tomato can greatly enhance the application value of tomato. Different types of fruit specific promoters have different developmental stages and regulation mechanisms in the fruit. Screening ideal fruit specific promoters can lay the foundation for the expression of exogenous proteins by using tomato as a carrier. IGF-1 regulates the growth and development of the body. The key factors of reproduction, immunity and body metabolism have a broad application prospect in the treatment of diabetes mellitus, osteoporosis, muscular dystrophy, lateral sclerosis and other diseases. In this study, five promoter expression vectors containing different fruit specificity were constructed. IGF-1 gene was introduced into tomato by Agrobacterium tumefaciens, and the transgenic tomato plants were obtained by molecular detection. It provides the technical and theoretical basis for the specific expression of exogenous protein in tomato, and also lays the material foundation for oral treatment of diabetes mellitus with tomato. The specific results were as follows: in this study, five plant expression vectors of fruit specific promoters, pCAP-IGF-1, pCAPG-IGF-1, pCAGP-IGF-1pCATP-IGF-1 and pCAOC-IGF-1, were constructed. Five plant expression vectors were introduced into Agrobacterium tumefaciens LBA4404 strain. The results showed that the differentiation effect of tomato material AC was obviously higher than that of 1448, and the best regeneration system was established as tomato material AC. The effects of different hormone levels on tomato regeneration system were studied by orthogonal design. The optimum medium for callus induction and adventitious bud differentiation was: 1 MS ZT 2.0 mg / L IAA 0.5 mg / L Agno 3 3.0 mg / L, and 1 / 2 MS IAA 0.3 mg / L for rooting. Orthogonal design method was used to optimize the parameters (dipping solution, acetyl eugenone, soaking time, co-culture days) that affect the conversion efficiency. The most suitable transformation parameters were determined as follows: the liquid medium with soaking solution as MS, The resistant plants were identified by PCR without acetyl eugenone, soaking time for 10 minutes and co-culture days for 2 days. The resistant plants were identified by PCR. 41 resistant plants were obtained by culture of transformation system, 15 of which were obtained by AGPL1 promoter. 12 resistant plants were obtained by cucumber hormone promoter, 6 resistant plants were obtained by ACO promoter and 6 by PG promoter and 2 resistant plants were obtained by AP promoter. A total of 21 positive plants were detected by PCR, and the positive rate was 51.21%. It was proved that the exogenous gene IGF-1 had been successfully integrated into tomato genome. Among them, 8 positive plants were obtained from AGPL1 promoter, 6 positive plants from cucumber hormone promoter, 3 positive plants from ACO promoter of muskmelon, 3 positive plants from PG promoter and 1 positive plant from AP promoter.
【學(xué)位授予單位】：云南農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2016
【分類(lèi)號(hào)】：S641.2

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