Beta-carotene hydroxylase and gene and application thereof
A carotene and hydroxylase-based technology, applied in the fields of application, genetic engineering, oxidoreductase, etc., can solve the problem of low catalytic synthesis efficiency of β-carotene hydroxylase, achieve high-efficiency transformation, increase yield, and be widely used foreground effect
- Summary
- Abstract
- Description
- Claims
- Application Information
AI Technical Summary
Problems solved by technology
Method used
Image
Examples
preparation example Construction
[0026] In this embodiment, the preparation method of the HpCrtR-b2 gene includes the following steps: subject Haematococcus pluvialis to high light and high salt treatment, collect algal cells of different treatment time periods, and extract total RNA; use TaKaRa's PrimeScript RTreagent Kit with gDNA Eraser (Perfect Real Time) kit. According to the kit instructions, using Oligo (dT18) as an anchor primer, the RNA was reverse-transcribed to obtain the first-strand cDNA; the first-strand cDNA was used as a template, and the designed specific primers were used to obtain high-resolution DNA using Invitrogen Platinum SuperFi DNA Ploymerase. The real enzyme was used to amplify the HpCrtR-b2 gene. The target fragment was cloned by T-A cloning method and verified by sequencing.
[0027] In this example, the application of the HpCrtR-b2 gene in converting β-carotene into zeaxanthin includes the following steps: link the codon-optimized artificially synthesized HpCrtR-b2 to the pET In...
Embodiment 1
[0032] Isolation, Cloning and Obtaining of HpCrtR-b2 Gene from Haematococcus Pluvialls
[0033] The material used in this embodiment is the algal cell of Haematococcus pluvialis strain 192.80 (purchased from The CultureCollection of the University of Germany). After culturing, the algal cells treated with high light and high salt for 0h, 1.5h, 3h, 6h, 9h, 12h, 24h, and 48h were collected, quickly frozen in liquid nitrogen, and stored in an ultra-low temperature refrigerator (-80°C). The collected algal cells were mixed for subsequent implementation.
[0034] 1) Extraction of total RNA from Haematococcus pluvialis
[0035] According to the instructions of the RNAfast200 total RNA rapid extraction kit (purchased from Shanghai Feijie Biotechnology Co., Ltd.), the specific operation is as follows: put the algae cells in a 1.5mL centrifuge tube, add 500 μL of RA2 solution, and immediately shake and mix for a few seconds; All the lysates are sucked into the inner cannula, centri...
Embodiment 2
[0051] Homology Analysis of HpCrtR-b2
[0052] By comparing with the reported Haematococcus pluvialis β-carotene hydroxylase gene (HpCrtR-b1, GeneBank accession number: AF162276), it was found that the nucleotide sequence similarity was only 79.83%, and the similarity of the coding region was 79.88%. Using the online analysis software SMART (http: / / smart.embl.de / smart / set_mode.cgi?NORMAL=1) to analyze the protein domain of the deduced amino acid sequence of the HpCrtR-b2 gene, it was found that the protein contained a 23-amino acid The transmembrane region and a 136-amino acid fatty acid / carotene hydroxylase domain (FA_hydroxylase domain) conform to the characteristics of the β-carotene hydroxylase gene.
PUM
| Property | Measurement | Unit |
|---|---|---|
| Gene length | aaaaa | aaaaa |
| Full length | aaaaa | aaaaa |
| Molecular weight | aaaaa | aaaaa |
Abstract
Description
Claims
Application Information
Login to View More