Abstract:

E3 ubiquitin ligases are very important regulatory components for the ubiquitin proteasome pathway. Most of E3 ligases comprise multiple groups and contain scaffolding proteins known as cullins and a common catalytic subunit, RBX1. RBX1 has a function to bind the ubiquitin-conjugating enzyme E2 and makes it into close proximity with the Cullin-based E3 substrate. In this study, yeast two-hybrid showed the tomato RBX1 can be interacted with CUL4 directly. We examined the expression level of RBX1 genes in different tomato tissues of wild type, and the result showed that RBX1 gene was expressed constitutively, while higher expression level was detected in the flower and fruit, comparing with that of leaf and root. In addition, an over-expression vector composing of aimed gene fragment and PBI121 with CaMV35S promoter, named PBI121-35S-RBX1 was constructed. We introduced the constructed vector into tomato cv. Ailsa Craig via Agrobacterium-mediated transformation, and acquired 3 transgenic plants of co-suppression and 3 transgenic plants of over-expressing, respectively. An increase in leaf expansion, a reduction in apical dominance comparing with wild type and sterility were observed in transgenic plants of co-suppression, which indicated that RBX1 played an important role in plant growth and development. We speculated that plant RBX1 gene may involve with auxin and brassinosteroid response and proliferation of cells, which provided clues for further studies on RBX1 in plants.

Key words: tomato, E3 ubiquitin ligases, RBX1, yeast two-hybrid analysis, Agrobacterium-mediated transformation

摘要：

E3泛素连接酶是泛素蛋白酶体途径中非常重要的蛋白复合体,而大部分的E3连接酶都以Cullin蛋白作为支架,所有基于Cullin蛋白的 E3 连接酶都含有一个催化亚基RBX1。RBX1对于E3复合体结合E2和Cullin蛋白的活化具有重要作用。利用酵母双杂交技术鉴定出番茄RBX1蛋白与番茄CUL4蛋白有直接的相互作用。通过荧光定量PCR（Real-time PCR）技术分析野生型番茄中RBX1基因的组织特异性,结果显示RBX1基因在番茄各组织中均有表达,但在花与果实中表达量较高,茎和叶中相对较少。同时构建植物过量表达载体 PBI121-35S-RBX1并转化番茄,获得 3 株过量表达和 3 株RBX1共抑制转基因植株。研究发现共抑制的转基因植株表现出叶片变大、顶端优势变弱和不育等表型,说明RBX1基因在番茄的生长发育中发挥重要作用,推测植物RBX1基因可能与生长素和油菜素内酯等植物激素应答以及细胞增殖有关,为进一步研究该基因在植物中的生物学功能提供了线索。

关键词: 番茄;E3泛素连接酶;RBX1;酵母双杂交;转化