1. Parts Table
The parts we submitted are listed at the table below.
|BBa_K1959000||PSY CDS codon optimized for O. sativa.||1193 bp|
|BBa_K1959001||CrtI CDS codon optimized for O. sativa.||1650 bp|
|BBa_K1959002||BHY CDS codon optimized for O. sativa.||1053 bp|
|BBa_K1959003||BKT CDS codon optimized for O. sativa.||1158 bp|
2. Origin and functions of the part
This part is the coding sequence (CDS) of phytoene synthase (PSY) from Zea mays, which is codon optimized for expression in rice (Oryza sativa). PSY is part of β-carotene biosynthesis pathway and it catalyzes the conversion of geranylgeranyl diphosphate to phytoene.
This part is the phytoene desaturase (CrtI) from Erwinia uredovora fused with Pea transit peptide. It is codon optimized for rice. Bacterial phytoene desaturase catalyzes conversion of phytoene to lycopene.
This part is the β-carotene hydroxylase (BHY) coding sequence from Haematococcus pluvialis. The Pea transit peptide is functionally fused with BHY in order to help BHY polypeptide folding correctly. BHY sequence is codon optimized for expression in rice. The BHY is part of astaxanthin biosynthesis pathway and it catalyzes conversion of β-carotene to zeaxanthin (an astaxanthin precursor).
This part contains Pea transit peptide and coding sequence of β-carotene ketolase (BKT). BKT catalyzes zeaxanthin to astaxanthin. This sequence is from Chlamydomonas reinhardti, and codon optimization has been made for stable expression in rice.
When the above four foreign genes were stacked into rice genome, under the control of endosperm-specific promoters, the functional astaxanthin biosynthetic pathway(see figure below) was reconstructed in rice endosperm.
The reconstruction of astaxanthin biosynthetic pathway in the endosperm. β-LYC (β-lycopene cyclase) is a endogenous gene of rice.
The genes described above were assembled in a transformation-competent artificial chromosome (TAC)-based binary plasmid and introduced into rice genome using Agrobacterium-mediated method.
The presences of these genes were confirmed by PCR analyses and the expression of the transgenes in rice endosperm was verified by RT-PCR and qRT-PCR.
Orange-red rice seeds could be easily observed by naked eyes and the products of astaxanthin was detected in the extraction of rice seeds by HPLC analyses. Furthermore, the HPLC profile shows that astaxanthin was predominant composition of the total carotenoids in rice endosperm. All of the experimental results proved that we successfully reconstructed the functional astaxanthin biosynthesis pathway in rice endosperm.
Our transgenic results fully proved that above parts are functional components for astaxanthin biosynthesis. These functional parts can be further used in synthetic biology of other plants.
For more details, please refer to our RESULTS section.