Part:BBa_K1959000
Modification of Phytoene synthase (PSY)
The BBa_K1959000 harbors a coding sequence of phytoene synthase (PSY) derived from maize (Zea mays). Codon-optimization has been made for rice (Oryza sativa). PSY catalyzes the conversion of geranylgenranyl diphosphate (GGDP) to phytoene, which is a key enzyme for carotenoid biosynthesis.
Usage and Biology
Phytoene synthase (PSY, EC 2.5.1.32) is an enzyme involved and responded for the first synthetic reaction in β-carotene biosynthetic pathway. In this reaction, two GGDP are converted to one molecule phytoene and two diphosphates (Figure. 1). PSY genes had been applied to the nutrition-improved crops such as Golden Rice. PSY gene of maize (ZmPSY) was introduced into rice genome to achieve the synthesis of β-carotene. In our project, the ZmPSY was used to reconstruct the pathway of astaxanthin biosynthesis. For maize is a monocots similar to rice, thus the ZmPSY was not fused with transit peptide for plastid sorting.
Transcriptional activity of PSY
Semi-quantitative RT-PCR was performed to detect the expression level of PSY involved in astaxanthin biosynthesis, total RNA of transgenic rice seeds were extracted and cDNA was synthesized from 1μg DNase-treated RNA. Expected bands of the PSY gene were observed on the gel, indicated that PSY gene was transcribed in endosperm.
aSTRice Phenotype
PSY is the key enzyme of astaxanthin biosynthesis. Rice without PSY fails to accumulate astaxanthin, appearing white in “wild type”. aSTARice contains astaxanthin and appears orange-red-color because of the coordinated expression of PSY gene and other key astaxanthin biosynthetic genes (Figure. 3). Therefore, the phenotype of aSTARice indicated that the PSY gene is a functional gene in rice.
Validation of astaxanthin by HPLC analysis
To further confirm the synthetic astaxanthin in aSTARice, HPLC was performed to analyze the pigment composition. Astaxanthin was identified on the basis of retention times related to standard sample. According to the retention time of standard astaxanthin sample, astaxanthin compound of extracts from aSTARice can be confirmed. In addition, astaxanthin possessed the biggest peak area in the carotenoids profile, indicated that astaxanthin was the predominant carotenoid in aSTARice.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 1045
Illegal NheI site found at 1164 - 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 105
Illegal AgeI site found at 956 - 1000COMPATIBLE WITH RFC[1000]
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