拟南芥NaKR2超表达转基因株系的构建
时间:2020-03-31 21:06 来源:毕业论文 作者:毕业论文 点击:次
| 摘要SODIUM POTASSIUM ROOT DEFECTIVE1（NaKR1；以前被称为NPCC6）编码一种在韧皮部伴细胞特异表达的一种可溶性的金属结合蛋白。nakr1-1突变体的表型包括高Na+、K+、Rb+，和叶片中淀粉的积累，根短，开花延迟，蔗糖的长距离输送缩短。基于基因芯片的缺失定位，一个7 bp的缺失在NaKR1的一个外显子上被发现，相当于插入了一个提前终止密码子。突变体的表型是由转化的天然基因或NaKR1-GFP（绿色荧光蛋白）和NaKR1 -β-葡萄糖醛酸酶融合启动子驱动。NAKR1-GFP在韧皮部移动；它从伴细胞进入筛元件并进入根分生组织的一个以前未发现的共质域。嫁接实验表明，高Na+积累主要是由于叶片中nakr1的功能丧失造成的。这支持了在循环中的韧皮部的作用Na+到根限制叶片中钠的积累。根表型的发病恰逢发芽后NaKR1的表达。nakr1-1短根的表型主要是由于在根分生组织的细胞的分裂率下降，表明了在韧皮部NaKR1的表达在根分生组织维持中的作用。为更深入地研究其作用机制，通过PCR，酶切，连接等技术手段构建了拟南芥NaKR1的超表达载体，测序正确后，利用农杆菌介导花序转化法，转入拟南芥野生型Col中，通过筛选得到该基因的单拷贝纯和株系，为进一步对 NaKR基因功能进行研究提供良好材料。47207 Abstract: SODIUM POTASSIUM ROOT DEFECTIVE1 (NaKR1; previously called NPCC6) encodes a soluble metal binding protein that is specifically expressed in companion cells of the phloem. The nakr1-1 mutant phenotype includes high Na+, K+, Rb+, and starch accumulation in leaves, short roots, late flowering, and decreased long-distance transport of sucrose. Using traditional and DNA microarray-based deletion mapping, a 7-bp deletion was found in an exon of NaKR1 that introduced a premature stop codon. The mutant phenotypes were complemented by transformation with the native gene or NaKR1-GFP (green fluorescent protein) and NaKR1-β-glucuronidase fusions driven by the native promoter. NAKR1-GFP was mobile in the phloem; it moved from companion cells into sieve elements and into a previously undiscovered symplasmic domain in the root meristem. Grafting experiments revealed that the high Na+ accumulation was due mainly to loss of NaKR1 function in the leaves. This supports a role for the phloem in recirculating Na+ to the roots to limit Na+ accumulation in leaves. The onset of root phenotypes coincided with NaKR1 expression after germination. The nakr1-1 short root phenotype was due primarily to a decreased cell pision rate in the root meristem, indicating a role in root meristem maintenance for NaKR1 expression in the phloem.[1] To research mechanisms of NaKR2 more deeply ,in this paper ,overexpression vector of NaKR1 gene was constructed .By the way of A grobacterium-mediated floral dipping methed , NaKR2-overexpression vector was transformed into Arabidopsis wild-type(Col) plants .the single cope homozygous were obtained by screening. These screened transgenic lines will be good materials for functional research of NaKR2. 毕业论文关键词：NAKR2；拟南芥； 超表达载体； PCR Keyword：NAKR1； Arabidopsis；over-expression vector ；PCR 目 录 引言 4 1. 材料与方法 6 1.1. 实验材料 6 1.1.1. 植物材料 6 1.1.2. 菌株与载体 6 1.1.3. 试剂与仪器 6 1.2. 基本实验方法 7 1.2.1. 拟南芥种子处理 7 1.2.2. 大肠杆菌感受态细胞的制备 7 1.2.3. 质粒提取 7 1.2.4. 植物总 RNA 提取 8 (责任编辑：qin) |