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.

Figure. 1 Enzymatic reaction catalyzed by PSY

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.

Figure.2　　RT-PCR analyses of expression levels of PSY genes in several transgenic rice.
Rice OsActin1 was served as an internal control. CK+, positive control (plasmid pYLTAC380MF-BBPC). WT, negative control (wild-type rice cultivar HG1).

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.

Figure. 3　　The polished rice phenotype of aSTARice.Wild type rice with transgenic PSY and CrtI produces β-carotene resulting in Golden Rice; wild type rice with transgenic PSY, CrtI, BHY and BKT produces astaxanthin resulting in aSRARice.

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.

Figure. 4　　Validation of astaxanthin by HPLC.
HPLC chromatogram of methanol extracts from the seeds of transgenic aSTARice (red line) and wild-type rice (blue line). HPLC analysis recorded at 480 nm of extracts.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal NheI site found at 1045
  Illegal NheI site found at 1164
  
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 105
  Illegal AgeI site found at 956
  
• 1000
  COMPATIBLE WITH RFC[1000]