Date Published: March 30, 2017
Publisher: Public Library of Science
Author(s): He Sun, Ni Zhou, Hai Wang, Dafang Huang, Zhihong Lang, Vitaly Citovsky.
In the transformation of multiple genes, gene fusion is an attractive alternative to other methods, including sexual crossing, re-transformation, and co-transformation, among others. The 2A peptide from the foot-and-mouth disease virus (FMDV) causes the co-translational “cleavage” of polyprotein and operates in a wide variety of eukaryotic cells. LP4, a linker peptide that originates from a natural polyprotein occurring in the seed of Impatiens balsamina, can be split between the first and second amino acids in post-translational processing. LP4/2A is a hybrid linker peptide that contains the first nine amino acids of LP4 and 20 amino acids of 2A. The three linkers have been used as a suitable technique to link the expression of genes in some transgenic plants, but to date the cleavage efficiency of three linkers have not been comprehensively demonstrated in the same transformation system, especially in the staple crop. To verify the functions of 2A, LP4, and LP4/2A linker peptides in transgenic maize, six fusion protein vectors that each encoded a single open reading frame (ORF) incorporating two report genes, Green Fluorescent Protein (GFP) and β-glucuronidase (GUS), separated by 2A (or modified 2A), LP4 or LP4/2A were assembled to compare the cleavage efficiency of the three linkers in a maize transient expression system. The results demonstrated the more protein production and higher cleavage splicing efficiency with the polyprotein construct linked by the LP4/2A peptide than those of the polyprotein constructs linked by 2A or LP4 alone. Seven other fusion proteins that each encoded a single ORF incorporating two different genes GFP and Red Fluorecent Protein (RFP) with different signal peptides were assembled to study the subcellular localization of genes linked by LP4/2A. The subcellular localization experiments suggested that both types of signal peptide, co-translational and post-translational, could lead their proteins to the target localization in maize protoplast transformed by LP4/2A polyprotein construct and it implied the LP4/2A linker peptide could alleviate the inhibition of 2A processing by the carboxy-terminal region of upstream protein of 2A when translocated into the ER.
2A is a peptide from the foot-and-mouth disease virus (FMDV), and its co-translational ‘cleavage’ activity is to ‘cleave’ the 2A from the FMDV polyprotein at its own carboxyl terminus. FMDV 2A does not require other domains within the FMDV polyprotein to function and is an autonomous element capable of mediating cleavage [1–5]. This cleavage can occur in a range of heterologous expression systems: viruses , insect cells , human HTK-143 cells , rabbit reticulocytes , wheat-germ extracts [10, 11] and HeLa cells . Using 2A for gene fusions has been widely applied in tomato, potato, tobacco, and others [13–15]. In recent years, researchers have attempted to use 2A for multi-gene transformations in staple crops [16, 17]. Ha et al. used 2A to link the phytoene synthase gene from Capsicum and the carotene desaturase gene from Pantoea to construct a fusion vector that was transformed into rice to generate a high carotenoid content for “Golden Rice” . The application of 2A from the model plant to staple crops indicates that the 2A linker has potential in genetic engineering and biotech breeding for crop improvement. In some cases, however, the cleavage efficiency of shorter versions of 2A may be inhibited by the C-terminus of certain gene sequences upstream of 2A. The use of longer versions of 2A with N-terminal extensions derived from FMDV capsid protein 1D located upstream of 2A (~30 aa in total) was reported to produce higher levels of cleavage [1, 2, 19]. Although the longest F2A sequence (58 aa) was tested to produce the most efficient cleavage, the C-terminal F2A extension of the upstream translation product may have a negative effect on protein confirmation and activity, such as being incapable of correctly producing monoclonal antibodies or expression of enzyme activity. To minimize this effect, a number of laboratories used shorter versions of 2A [8, 20] or incorporated a furin cleavage site between the C-terminus of the upstream protein and the N-terminus of the 2A sequence so that the C-terminal extension was trimmed away. This approach can only be used for secreted proteins because furin is primarily localized within the Golgi apparatus .
Gene stacking is one of the accomplishments in plant genetic engineering in which linker peptides have overcome the drawbacks of traditional methods to some extent and provided a new way to construct gene-stacking vectors. In this study, we compared the cleavage efficiency of five different linker peptides and analyzed the subcellular localization of genes linked by LP4/2A. The results will be a reference for multi-gene transformation research.