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Mol Genet Genomics. 2019 Mar 20. doi: 10.1007/s00438-019-01534-2. [Epub ahead of print]

Optimization of a 2A self-cleaving peptide-based multigene expression system for efficient expression of upstream and downstream genes in silkworm.

Wang Y1,2, Wang F1,2,3, Xu S1,2, Wang R1,2, Chen W1,2, Hou K1,2, Tian C1,2, Wang F3, Zhao P1,2, Xia Q4,5.

Author information

1
Biological Science Research Center, Southwest University, Chongqing, 400715, People's Republic of China.
2
Chongqing Key Laboratory of Sericultural Science, Southwest University, Chongqing, 400715, People's Republic of China.
3
Chongqing Engineering and Technology Research Center for Novel Silk Materials, Southwest University, Chongqing, 400715, People's Republic of China.
4
Biological Science Research Center, Southwest University, Chongqing, 400715, People's Republic of China. xiaqy@swu.edu.cn.
5
Chongqing Key Laboratory of Sericultural Science, Southwest University, Chongqing, 400715, People's Republic of China. xiaqy@swu.edu.cn.

Abstract

The multigene expression system is highly attractive to co-express multiple genes or multi-subunit complex-based genes for their functional studies, and in gene therapy and visual tracking of expressed proteins. However, the current multiple gene co-expression strategies usually suffer from severe inefficiency and unbalanced expression of multiple genes. Here, we report on an improved 2A self-cleaving peptide (2A)-based multigene expression system (2A-MGES), by introducing an optimized Kozak region (Ck) and altering the gene arrangement, both of which contributed to the efficient expression of two fluorescent protein genes in silkworm. By co-expressing DsRed and EGFP genes in insect cells and silkworms, the potent Ck was first found to improve the translation efficiency of downstream genes, and the expression of the flanking genes of 2A were improved by altering the gene arrangement in 2A-MGES. Moreover, we showed that combining Ck and an optimized gene arrangement in 2A-MGES could synergistically improve the expression of genes in the cell. Further, these two flanking genes, regulated by modified 2A-MGES, were further co-expressed in the middle silk gland and secreted into the cocoon, and both achieved efficient expression in the transgenic silkworms and their cocoons. These results suggested that the modified Ck-2A-MGES will be a potent tool for multiple gene expression, for studies of their functions, and their applications in insect species.

KEYWORDS:

2A self-cleaving peptide; Kozak sequence; Multiple-gene co-expression; Silkworm

PMID:
30895377
DOI:
10.1007/s00438-019-01534-2

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