Difference between revisions of "Part:BBa K1959003"

Latest revision as of 17:43, 19 October 2016

BKT coding sequence fused with Pea transit peptide

This part contains the coding sequence (CDS) of β-carotene ketolase (BKT, EC 1.14.11.B16) of algae (Chlamydomonas reinhardti), which catalyzes the conversion of zeaxanthin to astaxanthin. A Pea transit peptide of RUBISCO small subunit has been fused to BKT and the codon has been optimized for rice (Oryza sativa).

Usage and Biology

　As a strong antioxidant, astaxanthin synthesis, especially, its biosynthesis attracts much interest of scientists. BKT symmetrically introduces two keto groups to two β-ionone rings of zeaxanthin to generate astaxanthin in algae (Figure.1).
　In our reconstructed pathway, the CDS of BKY was codon-optimized for rice. In addition, a Pea transit peptide was fused to the BKY for proper sorting into the plastid.

Figure. 1　The reconstructed biosynthesis pathway of astaxanthin in the endosperm of aSTARice.
The dotted arrows indicate pathway is absent in rice endosperm. The solid arrows indicate the existence of carotenogenic reactions. The red arrows indicate the reactions catalyzed by four exogenous transgenic PSY, CrtI, BHYand BKT.

Transcriptional activity

　Semi-quantitative RT-PCR was performed to detect the expression level of BKT involved in astaxanthin biosynthesis, total RNA of transgenic rice seeds were extracted and cDNA was synthesized from 1μg DNase-treated RNA.

Figure. 2　　RT-PCR analyses of expression levels of BKT gene 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).

　Expected bands of the BKT gene were observed on the gel, indicated that BKT gene was transcribed in endosperm.

aSTRice Phenotype

　BKT is the key enzyme of astaxanthin biosynthesis. Rice without BKT fails to accumulate astaxanthin, appearing white or gold color in “wild type” and “Golden Rice”, respectively. aSTARice contains astaxanthin and appears orange-red-color because of the coordinated expression of BKT gene and other key astaxanthin biosynthetic genes (Figure. 3). Therefore, the phenotype of aSTARice indicated that the BKT 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. 5　　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