番茄E3泛素连接酶基因SINA1的功能解析

发布时间：2018-03-10 00:08

本文选题：番茄　切入点：SlSINA1　出处：《华中农业大学》2017年硕士论文　论文类型：学位论文

【摘要】：果实大小作为番茄重要的商品性状,也是生物学研究和番茄育种的重要表型性状。通过分子生物学发掘调控果实大小的基因,阐释调控番茄果实大小的分子机理,不仅对番茄分子育种具备关键的指导性作用,也为新品种的鉴定提供了重要的科学依据。番茄中关于SINA(Seven in absentia)的研究很少,SINA家族含有高度保守的Sina和RING功能域,其RING功能域能够行使E3泛素连接酶活性。通过功能研究发现,在番茄中超量表达SlSINA1导致番茄的果实变小。主要研究结果如下:1.通过组织表达谱分析表明,SlSINA1在所有组织器官中均有表达,在花中表达量最高,在花蕾中表达量次之,在根和红熟期(RR)果实中表达量最低。随着果实的发育,SlSINA1在17DPA(Days post-anthesis)时的果实中表达量达到峰值,然后表达量不断降低,在红熟期降至最低。2.与对照(AC,Ailsa Craig)相比,SlSINA1超量转基因材料T1代和T3代果实纵径、横径和果实重量显著减小。3.细胞生物学观察发现,0DPA、7DPA、24DPA和35DPA的超量转基因株系番茄果实的果皮厚度、果皮细胞层数与对照相比显著减小,7DPA、24DPA和35DPA的果皮细胞平均面积较对照显著的减小。随着时间的推移,果皮的厚度、细胞层数和细胞平均面积均不断增加,并且在7DPA至24DPA期间这三者增加了至少10-15倍,其他时间内这三者虽然有所增加,但是变化较小。因此推测SlSINA1可能通过影响细胞的大小和数量影响果皮厚度而调控番茄果实大小的发育。4.酵母双杂交实验表明,SlSINA1能够与RAP30、SPG和FW2.2蛋白互作,SlSINA1可能与这些蛋白形成复合体发挥功能。5.在SlSINA1超量转基因系中进行表达量分析结果显示,较对照而言,SlSINA1表达量显著增加,而RAP30、SPG和FW2.2的表达量均显著下降。6.通过Western-blot实验,未能检测到SPG和FW2.2蛋白的表达,Co-IP验证未发现SlSINA1与RAP30在植物体内存在互作关系。
[Abstract]:Fruit size, as an important commodity trait of tomato, is also an important phenotypic trait in biological research and tomato breeding. It not only has a key guiding role in tomato molecular breeding, but also provides an important scientific basis for the identification of new varieties. Few studies on SINA(Seven in absentia in tomato show that Sina family contains highly conserved Sina and RING functional domains. Its RING domain can exercise the activity of E3 ubiquitin ligase. The main results were as follows: 1.The results of tissue expression analysis showed that SlSINA1 was expressed in all tissues and organs, the highest in flower and the second in flower bud, the main results were as follows: 1.The main results were as follows: 1. Through tissue expression analysis, SINA1 was expressed in all tissues and organs, the highest in flower, and the second in flower bud. The expression level of SlSINA1 reached its peak value in the fruit of 17DPADDays post-anthesism, and then decreased, with the development of the fruit, the expression of SlSINA1 was the lowest in the root and red mature fruit, and the expression of SlSINA1 reached its peak value in the fruit of 17DPADDays post-anthesism. The fruit diameter, transverse diameter and fruit weight of T _ 1 and T _ 3 generations decreased significantly compared with those of the control, Ailsa Craigi. The fruit skin thickness of transgenic tomato lines were observed by cell biology observation, and the results showed that the fruit skin thickness of the transgenic lines was 7DPA-7DPA-24DPA and 35DPA, and the fruit diameter was significantly lower than that of the control (Ailsa Craigg), and the fruit diameter, transverse diameter and fruit weight of T _ 1 and T _ 3 generations were significantly decreased. The average area of pericarp cells of 7DPA-24DPA and 35DPA decreased significantly compared with the control, and the thickness of pericarp, the number of cell layers and the average area of cells increased with the passage of time. And between 7DPA and 24DPA, these three increased by at least 10-15 times, but at other times these three increased, But the change is small. Therefore, it is speculated that SlSINA1 may regulate the development of tomato fruit size by affecting the cell size and the number of the fruit peel thickness. 4. Yeast two-hybrid experiment shows that SlSINA1 can interact with RAP30SINA1 and FW2.2 protein and SlSINA1 may interact with this. The expression of these protein forming complexes in SlSINA1 superabundant transgenic lines was analyzed, and the results showed that, Compared with the control, the expression of SlSINA1 was significantly increased, while the expression of SPG and FW2.2 was decreased significantly. 6. The expression of SPG and FW2.2 protein was not detected by Western-blot test. Co-IP verification showed that there was no interaction between SlSINA1 and RAP30 in plants.
【学位授予单位】：华中农业大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S641.2

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