【摘要】：百合是著名的球根花卉,具有較高的觀賞、食用和藥用價(jià)值。百合鱗莖在發(fā)育成熟時(shí)會(huì)逐漸進(jìn)入休眠狀態(tài),未解除休眠的百合鱗莖在種植后會(huì)出現(xiàn)發(fā)芽率不高和盲花現(xiàn)象。ARF基因的表達(dá)會(huì)影響蔗糖磷酸合成酶的活性,參與生長(zhǎng)素的合成,促進(jìn)植物的發(fā)育,ArfGTP酶激活蛋白會(huì)影響生長(zhǎng)素的極性運(yùn)輸。本研究以細(xì)葉百合鱗莖為試驗(yàn)材料,分析休眠解除過(guò)程中的四個(gè)重要時(shí)期淀粉和蔗糖酶活性變化,以及ARF基因的表達(dá),克隆ArfGTPase激活蛋白LpAGD14基因,并進(jìn)行生物信息學(xué)分析及植物表達(dá)載體構(gòu)建,為后續(xù)的功能驗(yàn)證奠定基礎(chǔ)。主要試驗(yàn)結(jié)果如下:1、休眠期、萌動(dòng)期、芽迅速增長(zhǎng)期、出芽期4個(gè)時(shí)期的淀粉酶、淀粉磷酸化酶、蔗糖合成酶、蔗糖磷酸化酶的活性分析結(jié)果表明,在鱗莖低溫儲(chǔ)藏初期,淀粉代謝相關(guān)酶活性不高,是該時(shí)期百合鱗莖休眠程度較高,新陳代謝活動(dòng)緩慢,可溶性碳水化合物消耗水平低,淀粉轉(zhuǎn)化成可溶性糖的速率低,導(dǎo)致初期蔗糖磷酸合成酶活性升高,促進(jìn)蔗糖磷酸合成蔗糖。在休眠開(kāi)始解除至完全解除的時(shí)期,新陳代謝活動(dòng)迅速,相關(guān)酶活性升高,可溶性糖消耗快,蔗糖酶合成酶活性達(dá)到最大。外層鱗片淀粉酶普遍高于內(nèi)層鱗片,說(shuō)明內(nèi)層鱗片的新陳代謝活動(dòng)低于外層鱗片。2、從細(xì)葉百合轉(zhuǎn)錄組等得到69個(gè)ADP核糖基化因子(ARF)相關(guān)基因,通過(guò)生物信息學(xué)分析,發(fā)現(xiàn)這些基因主要參與氨基酸代謝,輔酶合成,糖代謝,信號(hào)轉(zhuǎn)導(dǎo)等生物過(guò)程。選取上調(diào)表達(dá)的15個(gè)基因,設(shè)計(jì)特異性引物,以低溫儲(chǔ)藏四個(gè)時(shí)期的細(xì)葉百合鱗莖cDNA為模板,進(jìn)行實(shí)時(shí)熒光定量試驗(yàn),驗(yàn)證這些基因在4個(gè)不同休眠時(shí)期的表達(dá)。試驗(yàn)結(jié)果發(fā)現(xiàn),所有基因在四個(gè)時(shí)期的表達(dá)量均呈上升趨勢(shì),儲(chǔ)藏初期休眠程度最高,基因表達(dá)量最低,休眠完全解除時(shí),相關(guān)基因表達(dá)量最大,說(shuō)明這些基因?yàn)樯险{(diào)基因,有進(jìn)一步研究的意義。3、從細(xì)葉百合cDNA中克隆的LpAGD14基因?qū)儆贏RF基因家族,具有典型的ArfGAP保守結(jié)構(gòu)域,通過(guò)蛋白功能預(yù)測(cè),發(fā)現(xiàn)其與油棕、芭蕉和蘆筍等多種植物的GTP激活蛋白AGD14基因具有較高的同源性,推測(cè)出該蛋白屬于Arf類GTP酶激活蛋白,能夠調(diào)節(jié)生長(zhǎng)素極性運(yùn)輸、囊泡運(yùn)輸和根發(fā)育,為進(jìn)一步研究ARF基因的功能奠定基礎(chǔ)。4、構(gòu)建植物表達(dá)載體pBI121-LpAGD14-GFP,利用高壓基因槍法將此GFP融合蛋白打入洋蔥表皮細(xì)胞內(nèi)做瞬時(shí)表達(dá),發(fā)現(xiàn)在洋蔥表皮細(xì)胞的的細(xì)胞膜和細(xì)胞核中均有綠色熒光,表明LpAGD14基因在細(xì)胞膜表達(dá)。構(gòu)建植物表達(dá)載體pCXSN-LpAGD14,利用農(nóng)桿菌介導(dǎo)法將此載體轉(zhuǎn)入野生型擬南芥中獲得抗性植株,通過(guò)初步PCR鑒定,證實(shí)了該外源基因已成功轉(zhuǎn)入到擬南芥的基因組中。
[Abstract]:Lily is a famous bulb flower with high ornamental, edible and medicinal value. Lily bulbs will gradually enter dormancy state during development and maturation. The low germination rate and blind flower phenomenon. ARF gene expression will affect the activity of sucrose phosphate synthase and participate in the synthesis of auxin. The arfGTP activator protein, which promotes plant development, affects the polar transport of auxin. In this study, the changes of starch and sucrase activities and the expression of ARF gene in bulb of Lilium tenuifolia were analyzed in four important stages of dormancy release, and the ArfGTPase activator protein LpAGD14 gene was cloned. Bioinformatics analysis and construction of plant expression vector were carried out, which laid a foundation for subsequent functional verification. The main results were as follows: the activities of amylase, starch phosphorylase, sucrose synthase and sucrose phosphorylase in the four stages of 1: 1, dormancy, germinating, sprout, budding, and early stage of bulb storage at low temperature, the results showed that the activity of amylase, starch phosphorylase, sucrose synthase and sucrose phosphorylase in the early stage of bulb cryopreservation. The activity of starch metabolism related enzymes was not high, the dormancy degree of lily bulb was high, the metabolism activity was slow, the consumption level of soluble carbohydrate was low, and the rate of conversion of starch to soluble sugar was low. The activity of sucrose phosphate synthase increased in the initial stage, and promoted the synthesis of sucrose from sucrose phosphate. During the period from the beginning of dormancy to the complete release of dormancy, the metabolic activity was rapid, the activity of related enzymes was increased, the consumption of soluble sugar was fast, and the activity of sucrase synthase reached the maximum. Amylase in outer layers was generally higher than that in inner scales, indicating that the metabolic activity of inner scales was lower than that of outer scales. 69 genes related to ADP ribosylation factor (ARF) were obtained from transcriptome of Lilium tenuifolia, and analyzed by bioinformatics. These genes are mainly involved in amino acid metabolism, coenzyme synthesis, glucose metabolism, signal transduction and other biological processes. Fifteen up-regulated genes were selected and specific primers were designed. The cDNA of bulb of Lilium tenuifolia was used as template in four periods of low temperature storage, and real-time fluorescence quantitative test was carried out to verify the expression of these genes in four different dormancy stages. The results showed that the expression of all genes showed an upward trend in the four periods, the dormancy was the highest and the gene expression was the lowest in the early storage period. When dormancy was completely released, the expression of the related genes was the largest, which indicated that these genes were up-regulated because of the up-regulation of the genes. The LpAGD14 gene cloned from the cDNA of Lilium tenuifolia belongs to the ARF gene family and has a typical conserved domain of ArfGAP. The AGD14 gene of banana and asparagus has high homology. It is inferred that AGD14 gene belongs to Arf class GTPase activator protein, which can regulate polar transport of auxin, vesicle transport and root development. In order to further study the function of ARF gene, a plant expression vector pBI121-LpAGD14-GFPP was constructed, and the GFP fusion protein was inserted into onion epidermal cells for transient expression by high-pressure gene gun method. Green fluorescence was found in the cell membrane and nucleus of onion epidermal cells, indicating that LpAGD14 gene was expressed on the cell membrane. A plant expression vector pCXSN-LpAGD14 was constructed and transferred into wild type Arabidopsis thaliana by Agrobacterium tumefaciens to obtain resistant plants. It was confirmed by PCR that the foreign gene had been successfully transferred into Arabidopsis genomes.
【學(xué)位授予單位】：東北林業(yè)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：S682.29

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