Application of two bicistronic systems involving 2A and IRES sequences to the biosynthesis of carotenoids in rice endosperm

다국어 초록 (Multilingual Abstract)

<P>Summary</P><P>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 (<I>Psy</I>) from <I>Capsicum</I> and carotene desaturase (<I>CrtI</I>) from <I>Pantoea,</I> 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 <I>PAC</I> (<I><U>P</U>sy-2<U>A</U>-<U>C</U>rtI</I>) and <I>PIC</I> (<I><U>P</U>sy-<U>I</U>RES-<U>C</U>rtI</I>) constructs, respectively. The transgenic endosperm of <I>PAC</I> rice had a more intense golden color than did <I>PIC</I> 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 <I>PAC</I> endosperms accumulated an average of 1.3 &mgr;g/g of total carotenoids, which was ninefold higher than was observed for <I>PIC</I> endosperms. In particular, accumulation of &bgr;-carotene was much higher in <I>PAC</I> endosperms than in <I>PIC</I> 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.</P>