中文摘要
大豆原生质体瞬时表达体系的建立及大豆隐花色素的亚细胞定位
隐花色素是植物体内的一类重要的蓝光受体，能够介导光信号调控植物的去黄化、开花、避荫反应等多种生长发育过程。隐花色素在模式植物拟南芥中得到了较好的研究，而在大豆等主要作物中的研究较少。原生质体瞬时表达体系能够快速、高效地实现目标蛋白的亚细胞定位分析。本文希望利用大豆原生质瞬时表达体系对大豆隐花色素在黑暗和蓝光下的亚细胞定位进行分析，为大豆隐花色素的功能预测提供信息。由于尚无大豆原生质体转化方法的报道，本文尝试利用酶解法裂解大豆叶片细胞壁获得了原生质体，并用PEG介导的方法成功将外源载体转入大豆叶片原生质体并表达。进一步对酶解液组成、PEG分子量、叶片时期、质粒浓度和转化时间等影响因素进行分析，确定了大豆叶片原生质体提取和转化的最佳条件，建立了高效的大豆叶片原生质体瞬时转化体系，转化效率达50 %以上。通过该体系将大豆7个隐花色素(GmCRYs)转入大豆叶片原生质体并观察了其在黑暗和蓝光下的亚细胞定位情况。具体结果如下：
1）最佳叶片时期：出土10天的真叶或刚展开的第一片三出复叶。
2）最佳裂解酶成分：1.0 %纤维素酶，0.2~0.4 %果胶酶。
3）最佳PEG分子量：4000。
4）最佳质粒浓度：大于或等于1 μg/μL。
5）最佳转化时间：15分钟。
6）大豆隐花色素的亚细胞定位：GmCRY1a、GmCRY1b、GmCRY1c和GmCRY1d在黑暗下分布于细胞核和细胞质中，见蓝光后迅速在细胞质
中形成类似光小体的结构。GmCRY2a、GmCRY2b和GmCRY2c持续定
位在细胞核中，见蓝光后没有观察到明显的光小体结构形成。
关键词：
大豆，隐花色素，原生质体，亚细胞定位
Abstract
Establishment of the Protoplasts Transient Gene Expression System and the Subcellular Localization of Cryptochromes in Soybean
Cryptochromes are a kind of blue light receptors which mediate the regulation of growth and develop
ment in plants by mediating various light respons including de-etiolation of edlings, flowering time, shade avoidance respon, and so on. Many cret of cryptochromes have been revealed in Arabidopsis, however, it is little known about the cryptochromes in soybean. The transient gene expression system using protoplasts has provided a rapid and highly efficient way for the subcellular localization.We want to investigate the subcellular localization of soybean cryptochromes in darkness and under blue light using soybean protoplasts and provide information on the function of cryptochromes in soybean. There is little report about soybean protoplasts transformation, however, we have successfully isolated the protoplasts by enzymatic digestion of mesophyll cells from soybean and transformed the expression vector to the protoplasts for subquent transient expression, which is mediated by PEG. Moreover, we optimized the main influencing factors including the suitable enzyme digestion system, the molecular weight of PEG, the different growth stages of leaves, the plasmids concentrations and the incubation time, and developed an efficient transient gene expression system using soybean mesophyll protoplasts who transformation efficiency is over 50 %. We studied the subcellular localization of cryptochromes in soybean in darkness and under blue light using this system. The main results are showed as follows:
1)The best growth stages of leaves: the 10-day-old unifoliolates after
emergence and the first trifoliolates which is just unfolding.
2)The best enzyme digestion system: Cellua 1.0 %, Pectola Y-23
0.2~0.4 %.
3)The best molecular weight of PEG: 4000.
4)The best plasmids concentrations: greater than or equal to 1 μg/μL.
5)The best incubation time: 15 min.
6)The subcellular localization of cryptochromes in soybean: The GmCRY1a、
GmCRY1b、GmCRY1c and GmCRY1d are located in the nucleus and the
cytoplasm of cells in darkness，and formed photobody-like structure in
肩膀长痣cytoplasm while transferred to blue light. The GmCRY2a、GmCRY2b and
GmCRY2c are always prent in the nucleus, and it is difficult to obrve the
photobody formation of the GmCRY2a、GmCRY2b and GmCRY2c.
Keywords:
Soybean, Cryptochromes, Protoplast, Subcellular localization
中英文对照表
英文缩写英文全称中文
CRY Cryptochrome 隐花色素
PHY Phytochrome 光敏色素
Phot Phototropin 向光素
PEG polyethylene glycol 聚乙二醇
FAD Flavin Adenine Dinucleotide 黄素腺嘌呤二核苷酸SPA Suppressor of Phytochrome A 光敏色素A抑制子COP Constitutive Photomorphogenic 持续性光形态建成
CIB cryptochrome-interacting
红色龙沙宝石basic-helix-loop-helix 隐花色素互作bHLH转录因子小提琴弓>怎么化妆
BIC Blue-light Inhibitor of Cryptochromes 隐花色素的蓝光抑制子YFP Yellow Fluorescent Protein 黄色荧光蛋白
颓废英文
DAE Days After Emergence 出土后天数
目录
第1章 绪论 (1)
1.1 隐花色素 (1)期刊分级
1.1.1 隐花色素的发现 (1)
1.1.2 隐花色素的结构 (2)
1.1.3 隐花色素的功能 (3)
1.1.4 植物隐花色素的光激活及光信号传递机制 (8)
1.2 植物原生质体的基因瞬时表达体系 (12)
1.2.1 原生质体的分离 (12)
1.2.2 原生质体的转化 (13)
1.2.3 原生质体的培养 (14)
1.3 研究目的与意义 (15)
第2章 材料与方法 (16)
2.1 试验材料 (16)
2.2 主要试剂的配制 (17)
2.3 大豆原生质体瞬时表达体系的建立 (19)
2.3.1 pA7-YFP质粒的扩繁与提取 (19)
2.3.2 大豆叶片原生质体的提取 (22)
2.3.3 大豆叶片原生质体的转化 (23)
花生发芽能吃吗
贵港电视台
2.4 大豆隐花色素的亚细胞定位观察 (26)
2.4.1 大豆隐花色素进化树及蛋白序列分析 (26)
2.4.2 pA7-GmCRYs-YFP载体的构建 (26)
2.4.3 pA7-GmCRYs-YFP载体转化大豆叶片原生质体 (29)
2.4.4 大豆隐花色素亚细胞定位的观察 (29)
第3章 结果与分析 (30)
3.1 大豆原生质体的提取与转化 (30)
3.1.1 大豆叶片裂解酶浓度的选择 (30)
3.1.2 PEG分子量对大豆叶片原生质转化的影响 (31)
