Application of two bicistronic systems involving 2A and IRES sequences to the biosynthesis of carotenoids in rice endosperm.
Coordination of multiple transgenes is essential for metabolic engineering of biosynthetic pathways. Here, we report the utilization of two bicistronic systems involving the 2A sequence from the foot-and-mouth disease virus and the internal ribosome entry site (IRES) sequence from the crucifer-infecting tobamovirus to the biosynthesis of carotenoids in rice endosperm. Two carotenoid biosynthetic genes, phytoene synthase (Psy) from Capsicum and carotene desaturase (CrtI) from Pantoea, were linked via either the synthetic 2A sequence that was optimized for rice codons or the IRES sequence under control of the rice globulin promoter, generating PAC (Psy-2A-CrtI) and PIC (Psy-IRES-CrtI) constructs, respectively. The transgenic endosperm of PAC rice had a more intense golden color than did PIC rice, demonstrating that 2A was more efficient than IRES in coordinating gene expression. The 2A and IRES constructs were equally effective in driving transgene transcription. However, immunoblot analysis of CRTI, a protein encoded by the downstream open reading frame of the bicistronic constructs, revealed that 2A was ninefold more effective than IRES in driving translation. The PAC endosperms accumulated an average of 1.3 μg/g of total carotenoids, which was ninefold higher than was observed for PIC endosperms. In particular, accumulation of β-carotene was much higher in PAC endosperms than in PIC endosperms. Collectively, these results demonstrate that both 2A and IRES systems can coordinate the expression of two biosynthetic genes, with the 2A system exhibiting greater efficiency. Thus, the 2A expression system described herein is an effective new tool for multigene stacking in crop biotechnology.
Ha
Sun-Hwa
SH
National Academy of Agricultural Science, Rural Development Administration, Suwon, Korea. sunhwa@korea.kr
Liang
Ying Shi
YS
Jung
Harin
H
Ahn
Mi-Jeong
MJ
Suh
Seok-Cheol
SC
Kweon
Soon-Jong
SJ
Kim
Dong-Hern
DH
Kim
Young-Mi
YM
Kim
Ju-Kon
JK
D019883:Alkyl and Aryl Transferases / Q000235:genetics; D001709:Biotechnology / Q000379:methods*; D002338:Carotenoids / Q000096:biosynthesis*; D056625:Endosperm / Q000235:genetics / Q000378:metabolism*; D005818:Genetic Engineering / Q000379:methods; D051232:Geranylgeranyl-Diphosphate Geranylgeranyltransferase; D012275:Oryza / Q000235:genetics / Q000378:metabolism*; D010088:Oxidoreductases / Q000235:genetics; D010940:Plant Proteins / Q000235:genetics; D030821:Plants, Genetically Modified / Q000235:genetics / Q000378:metabolism*
D010940:Plant Proteins; D002338:Carotenoids; D010088:Oxidoreductases; C022398:phytoene dehydrogenase; D019883:Alkyl and Aryl Transferases; D051232:Geranylgeranyl-Diphosphate Geranylgeranyltransferase