【作者】 师守国；

【导师】 马锋旺；

【作者基本信息】 西北农林科技大学 ， 园林植物与观赏园艺， 2011， 博士

【摘要】 兰州百合（Lilium davidii var. unicolor）是百合科百合属川百合一个变种,是我国特有的一种百合,集食用、药用和观赏于一身,同时也是我国栽培面积最大的百合。目前关于兰州百合遗传转化方面的研究尚未见报道。本研究以兰州百合为材料,通过农杆菌介导法和基因枪介导法分别将苹果抗坏血酸合成和代谢关键酶基因抗坏血酸过氧化物酶（APX）和L-半乳糖-1,4-内酯脱氢酶（GLDH）转入兰州百合,通过分子筛选、PCR和Southern杂交检测,获得转苹果APX和GLDH基因兰州百合植株,并研究了转基因植株对盐胁迫抗性的影响,从而为提高兰州百合抗逆性,为兰州百合品种的抗性改良和营养价值的改良奠定基础。主要研究结果如下:1.分别构建了苹果APX基因植物表达载体pCAMBIA-APX和GLDH基因植物表达载体pCAMBIA-GLDH。该载体的目的基因启动子为CaMV35s启动子,终止子为胭脂碱合成酶,抗性选择标记为Hyg基因,同时含有GUS标记基因。2.研究了APX基因转化中影响鳞片叶转化效率的多种因素,包括Hyg浓度、Cef浓度等,确立了10mg L-1 Hyg,500mg/LCef,2d的预培养,3d的共培养,OD600为0.4,侵染20min的高效兰州百合遗传转化体系,并获得了兰州百合抗性植株。3.对GUS染色呈阳性的10个转苹果APX基因百合抗性株系进行PCR扩增,10个株系均扩增出900bp大小的特异条带,而对照无特异性条带出现,初步证明外源基因已转入兰州百合植株中。分别对PCR检测呈阳性的10个株系进行Southern杂交检测,有2个株系检测到一条杂交带,一个株系两条杂交带,证明不同拷贝的APX基因已经整合到兰州百合基因组中。4.测定了转APX基因和对照兰州百合在200 mM盐胁迫下的生理反应,结果显示,转基因兰州百合的APX活性始终高于对照植株,细胞膜透性和MDA的含量上升的速度低于对照植株,表明转基因植株的耐盐性高于对照植株,而转基因植株的SOD活性和对照植株无明显差异。5.通过研究GLDH基因转化中影响鳞片叶转化效率的多种因素,包括轰击压力和轰击距离等,建立了10 mg L^-1 Hyg,500mg L^-1Cef,轰击距离为6 cm,轰击压力为1100 psi,每枪金粉用量为300μg的兰州百合基因枪转化体系,并获得了兰州百合抗性植株。6.对GUS染色为阳性的35个转苹果GLDH基因百合抗性株系进行PCR扩增,结果35个株系均扩增出850bp大小的特异条带,而对照百合植株无特异性条带出现,初步证明外源基因已转入兰州百合植株中。分别对PCR检测呈阳性的35个兰州百合株系进行Southern杂交检测,有7个株系检测到一条杂交带,一个株系两条杂交带,证明GLDH基因已经整合到兰州百合基因组中。7.测定了转基因和对照兰州百合GLDH酶活性和ASA含量,结果表明GLDH活性提高了5-6倍,ASA含量提高了2-7倍。8.研究了兰州百合叶片抗氧化酶活性和抗氧化剂对干旱胁迫的反应,在干旱胁迫下,兰州百合叶片SOD、CAT表现为先升后降,干旱第4 d活性达到最大,POD也表达为相同的趋势,在干旱第5 d天达到最大;ASA相关的APX、DHAR、MDHAR和GR表现为先升后降,其中DHAR和MDHAR在第5 d达到最大,APX和GR在第4 d达到最大。抗氧化剂ASA和GSH的含量在第4 d达到最高。更多还原

【Abstract】 Lanzhou lily （Lilium davidii var. unicolor）, which is a variety of Lilium davidii, belongs to genus Lilium of family Liliaceae. It is a peculiar variety in China and can be used as a kind of food, medicine and ornamental plant. What is more, it is the crown of the edible lily. Lanzhou lily is the most widely cultivated lily used for vegetable. However, there is no report about the genetic transformation of Lanzhou lily worldwide.This study took the edible Lanzhou lily as material. ASA biosynthesis and metabolism related enzyme genes （Ascorbate peroxidase APX and L-gactono-1, 4-lactone dehydrogenase GLDH） were transferred into Lanzhou lily. Transgenic plants of APX and GLDH were gained by screening and detection. Meanwhile, plant physiological mechanism of the transgenic and non-transgenic lily was studied under salt stress. Through genetic modification, the study aiming of increasing the salt tolerance of Lanzhou lily, and improve the resistance and the nutritional value.The main content and result of experiment are as follows:1. Two plant expression vector apple APX plant expression vector pCAMBIA-APX and GLDH expression vector pCAMBIA-GLDH were constructed. The target gene promoter was the CaMV35s gene, the terminator for the nopaline synthase. Hyg was used as a resistant selection marker together with a GUS marker gene2. By studying the effects of transgenic APX conversion efficiency of scale leaves, a number of factors, including Hyg concentration, the concentration of inhibitory factors, were established, and it is concluded that 10 mg L^-1 Hyg, 500mg L^-1Cef, 2d pre-culture, 3d of the co-culture, OD600 value 0.4 and 20 min of Lily infection were the best genetic transformation system, and gained the highest number of resistant plants after selection.3. Ten resistant lines, which were positive on the GUS staining, were identified by PCR amplification, the results showed that 10 strains amplified the specific bands of about 900 bp, while the control had no specific bands. The results proved preliminarily that the APX gene had been successfully transformed to Lanzhou lily. Respectively, 10 PCR-positive lines were further identified with Southern blot ting analysis; there are two strains with one hybrid zone, and one strain with two lines. This confirmed that APX gene was transferred into the genome of Lanzhou lily with different copies.4. Physiological responses under 200 mM salt stresses were determined in transgenic and control plant. The results showed that the APX activity of transgenic plant was significantly higher than the control plants; the speed of cell membrane permeability and MDA contents increasement was much lower than the control plants. This indicates that the salt tolerance of transgenic plants was higher than control plants. The SOD activity had no significant difference between the transgenic and control plants.5. The impact of GLDH gene conversion in the conversion efficiency of scale leaves, including the bombardment pressure and bombardment distance, was studied, it was established that 10 mg L^-1 Hyg, 500 mg L^-1 Cef, bombardment distance of 6 cm, bombardment pressure of 1100 psi and an amount of 300μg per gun of Lanzhou lily for the gene gun transformation system.6. Thirty-five positive strains with GUS staining were examined with PCR amplification, and all of them amplified the specific bands of around 850bp, while the control plants had no specific bands. This initially proved that the foreign gene was transferred into lily successfully. Respectively, 35 PCR-positive lines of Lily were detected by Southern blotting; one hybrid zone was detected in 7 strains, and two hybrid zones in 1 strain. This confirmed that GLDH gene has been transferred to the genome of Lanzhou lily.7. GLDH activity and ASA contents of the control and transgenic lily were determined, the results showed that GLDH activity increased 5-6 times, ASA contents increased by 2-7 times.8. The trends of antioxidant activity of antioxidant enzymes under drought stress in Lanzhou lily. The activity of SOD and CAT rose and then dropped and were highest at the fourth day. The activity of POD was the trends and highest at the fifth day. APX, DHAR, MDHAR and GR showed rose and then decreased, which DHAR and MDHAR maximum in the fifth day, APX and GR in the fourth day maximum. ASA and GSH antioxidant contents were the highest in the fourth day.更多还原