Difference between revisions of "Part:BBa K1959002"
(→Usage and Biology) |
|||
| Line 8: | Line 8: | ||
===Usage and Biology=== | ===Usage and Biology=== | ||
| − | + | Astaxanthin is a keto-carotenoid which processes a powerful antioxidant activity with board health implications. Given its high value, astaxanthin synthesis, especially, its biosynthesis attracts considerable interest of the scientists. ''Haematococcus pluvialis'' is a promising bioresource of astaxanthin. β-carotene hydroxylase (BHY, EC 1.14.13.129) is one key enzymes of the astaxanthin biosynthesis in ''Haematococcus pluvialis''. It catalyzes the conversion of β-carotene to zeaxanthin, the precursor of astaxanthin (Figure.1). In this reaction, β-ionone of β-carotene converts into a 3-hydroxy-β-ionone of zeaxanthin, with the concomitant oxidation of NADH and two oxygen to NAD+ and two H20 (Figure.2).<br> In our project, we used BHY to convert β-carotene into zeaxanthin and subsequently complete the overall astaxanthin biosynthetic reaction. The CDS of BHY was codon-optimized for a better expression in rice. In addition, a Pea transit peptide was fused to the BHY for correct sorting of BHY into the plastid. | |
| − | [[File:T--SCAU-China--BHY-P.jpg | | + | [[File:T--SCAU-China--BHY-P.jpg |550px|thumb|centre|<p> '''Figure.1 The reconstructed biosynthesis pathway of astaxanthin in the endosperm of aSTARice.'''<br> 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'', ''BHY'' and ''BKT''.</p>]] |
===Transciptional activity=== | ===Transciptional activity=== | ||
| − | + | Semi-quantitative RT-PCR was performed to detect the expression level of BHY 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 BHY gene were observed on the gel, indicated that BHY gene was transcribed in endosperm. | |
===aSTRice Phenotype=== | ===aSTRice Phenotype=== | ||
| − | + | BHY is the key enzyme of astaxanthin biosynthesis. Rice without BHY expression are unable to accumulate astaxanthin, in other words, rice would not appear in orange-red color (Rice marked as “Wild Type” and “Golden Rice” in Figure.3). Orange-red-color rice is the consequence of the cooperation of BHY gene and other astaxanthin-producing genes. Therefore, the phenotype of aSTARice demonstrates that the BHY gene is capable to express in the rice cell. | |
| + | |||
| + | |||
| + | |||
| + | |||
<!-- Uncomment this to enable Functional Parameter display | <!-- Uncomment this to enable Functional Parameter display | ||
Revision as of 07:36, 19 October 2016
Modification of β-carotene hydroxylase (BHY)
This part contains the coding sequence (CDS) of β-carotene hydroxylase of algae (BHY, EC 1.14.13.129), which catalyzes the conversion of β-carotene to zeaxanthin. A Peatransit peptide of RUBISCO small subunit has been fused to BHY and the codon has been optimized for rice.
Usage and Biology
Astaxanthin is a keto-carotenoid which processes a powerful antioxidant activity with board health implications. Given its high value, astaxanthin synthesis, especially, its biosynthesis attracts considerable interest of the scientists. Haematococcus pluvialis is a promising bioresource of astaxanthin. β-carotene hydroxylase (BHY, EC 1.14.13.129) is one key enzymes of the astaxanthin biosynthesis in Haematococcus pluvialis. It catalyzes the conversion of β-carotene to zeaxanthin, the precursor of astaxanthin (Figure.1). In this reaction, β-ionone of β-carotene converts into a 3-hydroxy-β-ionone of zeaxanthin, with the concomitant oxidation of NADH and two oxygen to NAD+ and two H20 (Figure.2).
In our project, we used BHY to convert β-carotene into zeaxanthin and subsequently complete the overall astaxanthin biosynthetic reaction. The CDS of BHY was codon-optimized for a better expression in rice. In addition, a Pea transit peptide was fused to the BHY for correct sorting of BHY 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, BHY and BKT.
Transciptional activity
Semi-quantitative RT-PCR was performed to detect the expression level of BHY 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 BHY gene were observed on the gel, indicated that BHY gene was transcribed in endosperm.
aSTRice Phenotype
BHY is the key enzyme of astaxanthin biosynthesis. Rice without BHY expression are unable to accumulate astaxanthin, in other words, rice would not appear in orange-red color (Rice marked as “Wild Type” and “Golden Rice” in Figure.3). Orange-red-color rice is the consequence of the cooperation of BHY gene and other astaxanthin-producing genes. Therefore, the phenotype of aSTARice demonstrates that the BHY gene is capable to express in the rice cell.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 334
Illegal NgoMIV site found at 726
Illegal NgoMIV site found at 793 - 1000COMPATIBLE WITH RFC[1000]