1. Vector for genes stacking
To assemble these four genes of astaxanthin biosynthetic pathway in rice endosperm, a modified multigene vector system, TransGene Stacking II (TGSII), was used. This system consists of a transformation-competent artificial chromosome (TAC)-based binary acceptor vector (pYLTAC380GW), together with two donor vectors (pYL322-d1/ pYL322-d2). By using the Cre/loxP recombination system and two pairs of mutant loxP sites, multiple rounds of gene assembly cycles were carried out with alternative use of the donor vectors, and multiple genes were sequentially delivered into the TAC vector(Liu et al., PNAS, 1999, 96: 6535-6540; Lin et al., PNAS, 2003, 100: 5962-5967; Zhu et al., unpublished). By this way, multiple genes and a maker-free element can be easily stacked into a TAC-based binary acceptor vector (Figure 3) You can read more details by click here! part and protocol

Figure 3   Physic map of the multigene vector 380MF-BBPC for biosynthesizing astaxanthin and marker-free deletion.

2. Experimental design
Firstly, the nucleic acid sequences of four genes have been codon optimized and directly synthesized for stable expression in rice. Then, these genes were subcloned into endosperm-specific gene cassettes of two donors. Secondly, these genes and a marker-free element were assembled into a TAC-based binary vector by using a transgene stacking II system. Finally, the obtained marker-free multigene vector was transferred into Agrobacterium tumefaciens strain EHA105 for rice callus transformation. The transgenic plants were identified by analyses of PCR, RT-PCR, qRT-PCR and HPLC. The schematic diagram of our project was shown in Figure 4.
Figure 4   The schematic diagram of Astaxanthin Rice project.

3. Marker free
In this part, we used Cre/loxP site-specific recombination method to delete the selective marker (Figure 5). To delete the selective resistance gene in transgenic rice, a marker-free element was used to assemble into four-gene multigene vector. This marker-free element was placed between two loxP sites, and consists of a HPT (hygromycin) resistance gene expression cassette and a Cre gene expression cassette controlled by anther-specific promoter. When Cre gene was expressed in transgenic rice anther, the Cre enzyme deleted the marker-free element between two loxP sites.
Figure 5   The schematic diagram of the marker-free process. PV4 is an anther-specific promoter that drives Cre gene expression in anther.

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