【作者】 刘峰；

【导师】 郑金贵；

【作者基本信息】 福建农林大学 ， 生物化学与分子生物学， 2003， 硕士

【摘要】 在谷物、豆类和油料等作物粒实中，约有60-80％磷素是以植酸的形式存在，不能被人与单胃动物吸收、利用。从而造成了许多问题：1．导致磷源浪费，一方面饲料中的磷素不能得到有效利用，另一方面为了满足动物对磷的需求，又必须在饲料中添加无机磷，提高了饲料成本；2．形成高磷粪便污染环境；3．植酸在食物或饲料中还会与Ca²⁺、Mg²⁺、Fe²⁺、Zn²⁺、Cu²⁺等金属阳离子或带有正电荷基团的蛋白形成不溶的络合物，降低了这些矿质营养物质的生物利用率。 将耐酸、抗热的微生物植酸酶基因（phytase gene）转入禾谷类作物中，可望使禾谷类作物中的磷素及含有该作物的配合饲料中的其它原料的磷素从植酸中释放出来，从而可大大提高其磷素的利用率，同时还能降低动物植酸排泄所造成的环境污染。 在植物遗传转化过程中，标记基因用于识别和鉴定转基因植物。但其对植物的生长并非必要。而且它的存在还会引起安全性、妨碍多次重复转化及不利于多种优良性状的组合等问题。 本研究采用剔除标记基因的共转化系统，构建了无选择标记基因的植酸酶基因植物表达载体p1300UbiPHYTASE，该表达载体选用植物高效遗传转化载体：pCAMBIA载体系列中的pCAMBIA1300，剔除了pCAMBIA1300载体上T-DNA区内的选择标记基因hpt和花椰菜花叶病毒35S启动子，使T-DNA区仅含有目的基因phytase（目的基因为原长基因，含有能增强基因表达的信号肽和内含子序列）。选用单子叶植物高效表达启动子玉米Ubiquitin启动子，经酶切、PCR鉴定表明：该载体构建正确，适合于禾谷类作物的遗传转化。 利用冻融法将表达载体p1300UbiPHYTASE导入农杆菌EHA105中，拟采用双农杆菌（two-plasmid／two-strain）介导法对植物细胞进行共转化，以期获得无选择标记的转植酸酶基因植株。更多还原

【Abstract】 Phosphorus is an important constituent of cereals, legumes, and oilseed crops, which serve as a major source of nutrients for humans and animals. Accounting for about 60-80% of the total phosphorus presenting in these crops is in the form of phytic acid. Monogastric animals are unable to metabolize phytic acid and largely excrete it in their mature, causing environmental pollution in areas of intensive livestock production. Furthermore, in order to compensate for the low available phosphorus, inorganic phosphate has to be supplemented to the feed, which increases feed cost. In addition, phytic acid can chelate various metal ions, such as calcium, copper, zinc, etc., and proteins with positive ligands forming insoluble chelates, which decrease mineral nutrients availability.Phytases can catalyze the hydrolysis of phytase into inorganic phosphates. Genetically engineered crops including rice that contain phytase gene derived from microorganisms will resolve the above problems. The phytase originated from Aspergillus Nigar has a high intrinsic resistance to heat and acid inactivation. Adding phytase to the feed for monogastric animals will reduce the amount of phosphate excreted in the mature and to circumvent supplementation of the feed with inorganic phosphate.During plant genetic transformation, selectable marker is used to identify transgenic plants. However, it is not a necessity for plant growth and it causes public concern about bio-safety, interferes with multiple transformations and hampers the combination of superior characters.The research established a marker-free phytase gene expression vector p1300UbiPHYTASE via co-transformation system with selectable marker gene that can be easily excised. pCAMBIA1300, a member of high-efficient plant genetic transformation expression vector family pCAMBIA, was appliedto construct the target vector, hpt gene, which is a selectable marker gene with T-DNA of pCAMBIA1300 vector, and CaMV 35S promoter were excised. Thus the T-DNA zone contained only phytase gene of interest. High-efficient monocot promoter of Ubiquitin was used and results of enzyme digests and PCR analysis showed that the vector was well established and suitable for the genetic transformation of cereal crops.In this study, expression vector pCAMBIA1300 was transformed to Agrobacterium EHA105 through liquid nitrogen freezing and co-transformation of plant cell conducted via two-plasmid-mediated method, which aimed to obtain marker-free transgenic plants.更多还原