pEAQ: versatile expression vectors for easy and quick transient expression of heterologous proteins in plants
pEAQ: versatile expression vectors for easy and quick transient expression of heterologous proteins in plants
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DOI：
10.1111/j.1467-7652.2009.00434.x
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发表时间：
2009-09-01
影响因子：
13.8
通讯作者：
Lomonossoff, George P.
中科院分区：
文献类型：
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作者：
Sainsbury, Frank;Thuenemann, Eva C.;Lomonossoff, George P.
用含有目的序列的双元载体农杆菌渗入叶组织是在植物中表达蛋白质的快速方法。最近已经表明，在双元载体pBINPLUS内，用来自豇豆花叶病毒（CPMV）RNA-2（CPMV-HT）的修饰的5 ′-非翻译区（UTR）和3 ′-UTR侧翼待表达的序列大大提高了可以实现的表达水平[Sainsbury，F.（2008）Plant Physiol.148，1212-1218]。为了利用这一发现，已经创建了一系列针对瞬时表达定制的小二元载体（称为pEAQ载体）。在这些中，从pBINPLUS骨架和T-DNA区域去除超过7 kb的非必需序列，并且引入独特的限制性位点以允许在同一质粒上容纳多个表达盒，包括沉默抑制子的表达盒。这些载体允许在几天内从单个质粒高水平同时表达多种多肽。此外，已经开发了允许通过基于限制酶的克隆和GATEWAY重组将基因直接克隆到二元质粒中的载体。在这两种情况下，N-或C-末端组氨酸标签可以根据需要与靶序列融合。这些载体提供了一个简单和快速的工具，用于在台式规模上用标准植物研究技术从植物中生产毫克量的重组蛋白。
Agro-infiltration of leaf tissue with binary vectors harbouring a sequence of interest is a rapid method of expressing proteins in plants. It has recently been shown that flanking the sequence to be expressed with a modified 5'-untranslated region (UTR) and the 3'-UTR from Cowpea mosaic virus (CPMV) RNA-2 (CPMV-HT) within the binary vector pBINPLUS greatly enhances the level of expression that can be achieved [Sainsbury, F. and Lomonossoff, G.P. (2008) Plant Physiol. 148, 1212-1218]. To exploit this finding, a series of small binary vectors tailored for transient expression (termed the pEAQ vectors) has been created. In these, more than 7 kb of non-essential sequence was removed from the pBINPLUS backbone and T-DNA region, and unique restriction sites were introduced to allow for accommodation of multiple expression cassettes, including that for a suppressor of silencing, on the same plasmid. These vectors allow the high-level simultaneous expression of multiple polypeptides from a single plasmid within a few days. Furthermore, vectors have been developed which allow the direct cloning of genes into the binary plasmid by both restriction enzyme-based cloning and GATEWAY recombination. In both cases, N- or C-terminal histidine tags may be fused to the target sequence as required. These vectors provide an easy and quick tool for the production of milligram quantities of recombinant proteins from plants with standard plant research techniques at a bench-top scale.