百合鳞茎休眠过程中淀粉和蔗糖酶活性变化及ARF基因克隆

发布时间：2018-07-15 21:21

【摘要】：百合是著名的球根花卉,具有较高的观赏、食用和药用价值。百合鳞茎在发育成熟时会逐渐进入休眠状态,未解除休眠的百合鳞茎在种植后会出现发芽率不高和盲花现象。ARF基因的表达会影响蔗糖磷酸合成酶的活性,参与生长素的合成,促进植物的发育,ArfGTP酶激活蛋白会影响生长素的极性运输。本研究以细叶百合鳞茎为试验材料,分析休眠解除过程中的四个重要时期淀粉和蔗糖酶活性变化,以及ARF基因的表达,克隆ArfGTPase激活蛋白LpAGD14基因,并进行生物信息学分析及植物表达载体构建,为后续的功能验证奠定基础。主要试验结果如下:1、休眠期、萌动期、芽迅速增长期、出芽期4个时期的淀粉酶、淀粉磷酸化酶、蔗糖合成酶、蔗糖磷酸化酶的活性分析结果表明,在鳞茎低温储藏初期,淀粉代谢相关酶活性不高,是该时期百合鳞茎休眠程度较高,新陈代谢活动缓慢,可溶性碳水化合物消耗水平低,淀粉转化成可溶性糖的速率低,导致初期蔗糖磷酸合成酶活性升高,促进蔗糖磷酸合成蔗糖。在休眠开始解除至完全解除的时期,新陈代谢活动迅速,相关酶活性升高,可溶性糖消耗快,蔗糖酶合成酶活性达到最大。外层鳞片淀粉酶普遍高于内层鳞片,说明内层鳞片的新陈代谢活动低于外层鳞片。2、从细叶百合转录组等得到69个ADP核糖基化因子(ARF)相关基因,通过生物信息学分析,发现这些基因主要参与氨基酸代谢,辅酶合成,糖代谢,信号转导等生物过程。选取上调表达的15个基因,设计特异性引物,以低温储藏四个时期的细叶百合鳞茎cDNA为模板,进行实时荧光定量试验,验证这些基因在4个不同休眠时期的表达。试验结果发现,所有基因在四个时期的表达量均呈上升趋势,储藏初期休眠程度最高,基因表达量最低,休眠完全解除时,相关基因表达量最大,说明这些基因为上调基因,有进一步研究的意义。3、从细叶百合cDNA中克隆的LpAGD14基因属于ARF基因家族,具有典型的ArfGAP保守结构域,通过蛋白功能预测,发现其与油棕、芭蕉和芦笋等多种植物的GTP激活蛋白AGD14基因具有较高的同源性,推测出该蛋白属于Arf类GTP酶激活蛋白,能够调节生长素极性运输、囊泡运输和根发育,为进一步研究ARF基因的功能奠定基础。4、构建植物表达载体pBI121-LpAGD14-GFP,利用高压基因枪法将此GFP融合蛋白打入洋葱表皮细胞内做瞬时表达,发现在洋葱表皮细胞的的细胞膜和细胞核中均有绿色荧光,表明LpAGD14基因在细胞膜表达。构建植物表达载体pCXSN-LpAGD14,利用农杆菌介导法将此载体转入野生型拟南芥中获得抗性植株,通过初步PCR鉴定,证实了该外源基因已成功转入到拟南芥的基因组中。
[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.
【学位授予单位】：东北林业大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S682.29

【相似文献】