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

发布时间：2019-04-03 16:52

【摘要】：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1μg/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、Gm CRY1b、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。
[Abstract]:Cryptoanthocyanins are a kind of important blue light receptors in plants, which can mediate light signals to regulate the growth and development of plants such as yellowing, flowering, shade avoidance and so on. Cryptoanthocyanin has been well studied in Arabidopsis thaliana, a model plant, but less in soybean and other major crops. Protoplast transient expression system can quickly and efficiently realize the subcellular localization analysis of target proteins. In this paper, we hope to analyze the subcellular localization of soybean crypto pigment in dark and blue light by using the transient expression system of soybean protoplast, so as to provide information for the function prediction of soybean crypto pigment. Since there is no report on transformation methods of soybean protoplasts, protoplasts were obtained by enzymatic lysis of soybean leaf cell wall. The exogenous vectors were successfully transferred into soybean leaf protoplasts and expressed by PEG-mediated method. The optimal conditions for extraction and transformation of protoplasts from soybean leaves were determined by analyzing the composition of enzyme hydrolysate, molecular weight of PEG, leaf stage, plasmid concentration and transformation time. An efficient transient transformation system of soybean leaf protoplasts was established, and the transformation efficiency was more than 50%. Seven cryptochrome (GmCRYs) were transferred into protoplasts of soybean leaves in this system and their subcellular localization in dark and blue light was observed. The results are as follows: 1) optimal leaf stage: true leaves unearthed for 10 days or newly developed first triplicate leaves. 2) optimal lyase composition: 1.0% cellulase, 0.2% 0.4% Pectinase. 3) optimal PEG molecular weight: 4000.4) optimal plasmid concentration: greater than or equal to 1 渭 g / 渭 L.5) optimal transformation time: 15 minutes. 6) Subcellular localization of soybean cryptoanthocyanin: GmCRY1a,Gm CRY1b, GmCRY1c and GmCRY1d were distributed in the nucleus and cytoplasm in the dark. The photosome-like structure was rapidly formed in the cytoplasm after blue light. GmCRY2a, GmCRY2b and GmCRY2c continued to locate in the nucleus, but no obvious photosome formation was observed after blue light.
【学位授予单位】：吉林大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S565.1

【相似文献】