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Application of sll0147 gene in synthesizing synechocystis carotenoids

A technology of carotene and Synechocystis, applied in the application field of zeaxanthin production, to achieve the effect of enhancing biomass and increasing carotenoid content

Active Publication Date: 2015-11-25
BIOTECH RES CENT SHANDONG ACADEMY OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The amino acid similarity between the sll0147 gene in Synechocystis sp. PCC6803 and Synechococcus sp. CruA is 63.32%. Studies have shown that the mutation of sll0147 has no obvious effect on the cyclization process of lycopene, but there is no effect on the sll0147 gene in Synechocystis sp. PCC6803. Application Report on Improving β-Carotene Synthesis of Synechocystis sp. PCC6803

Method used

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  • Application of sll0147 gene in synthesizing synechocystis carotenoids
  • Application of sll0147 gene in synthesizing synechocystis carotenoids
  • Application of sll0147 gene in synthesizing synechocystis carotenoids

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Experimental program
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Effect test

Embodiment 1

[0049] The isolated and cloned sll0147 gene cDNA fragment, the psbA2 promoter fragment, the upstream fragment of homologous recombination slr1285UcDNA fragment and the upstream fragment of homologous recombination slr1285DcDNA fragment were used to construct the overexpression vector of Synechocystis sp. PCC6803sll0147 gene.

[0050] Cloning of Synechocystis sp. PCC6803 psbA2 promoter fragment: according to GenBank landing Synechocystis sp. PCC6803 (accession number: BA000022, AP012205), the first 500 bp of psbA2 ORF was used as the promoter sequence, and primers were designed:

[0051] Promoter-SalI-F: 5'-AATGTCGACTGCCCAGATGCAGGCCTTC-3';

[0052] Promotor-R: 5'-GTAGAGCAGTTCACGCATTTGGTTATAATTCCTTAT-3';

[0053] The amplification procedure is as follows:

[0054] Pre-denaturation at 94°C for 5min, denaturation at 94°C for 30s, renaturation at 55°C for 30s, extension at 72°C for 1min, after 35 cycles, 10min at 72°C, and storage at 4°C.

[0055] After the PCR reaction, the gel ...

Embodiment 2

[0081] Construction of Synechocystis sp. PCC6803sll0147 gene overexpression vector:

[0082] Preparation of plasmid pBluscriptSKplus-T1T2:

[0083] The plasmid pKK233-2 (purchased from Clontech Company) that amplifies the Escherichia coli T1T2 terminator, introduced a PstI restriction site at its 5' end, and introduced a BamHI restriction site at its 3' end, and the primers used were:

[0084] T1T2-F: 5'-ATACTGCAGCCAAGCTTGGCTGTTTTGGC-3';

[0085] T1T2-R: 5'-TTAGGATCCCCCATTATTGAAGCATTTAT-3';

[0086] The amplification procedure is as follows:

[0087] Pre-denaturation at 94°C for 5 minutes; denaturation at 94°C for 30s, renaturation at 60°C for 30s, extension at 72°C for 30s, after 35 cycles; extension at 72°C for 10 minutes, and storage at 4°C; the obtained gene fragments were digested with PstI and BamHI and the same The digested pBluescriptSK was ligated to obtain plasmid pBluescriptSKT1T2.

[0088] The homologous recombination upstream arm slr1285U gene fragment obtaine...

Embodiment 3

[0104] PCR detection of sll0147 gene overexpressed algal strain

[0105] Using transgenic Synechocystis PCC6803 and wild-type Synechocystis PCC6803 containing the expression vector p5S1285UDs110147 as materials, the total DNA was extracted for PCR detection and analysis. The specific method is as follows:

[0106] Neutral phenol reagent (purchased from Invitrogen) was used to extract DNA from sll0147 gene knockout and overexpression Synechocystis sp. and wild-type Synechocystis sp. The specific operation steps are as follows: take 50mlOD 730 =1.8 cyanobacteria, 4°C, 5000rpm centrifugation for 10min to collect algae cells, add 0.4mL neutral phenol to 0.4mL BG-11 liquid medium, then add an appropriate amount of glass beads (purchased from sigma company) with a diameter of about 0.17mm to the glass There is 0.5 mL of suspension above the bead interface. Vibrate with a vortex shaker at the maximum speed for 1min, centrifuge at 11900rpm for 10min at 4°C, take the supernatant to ...

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Abstract

The invention relates to an application of a sll0147 gene in synthesizing synechocystis carotenoids, wherein the nucleotide sequence of the sll0147 gene in the application is shown as SEQ ID NO.1. For the first time, the invention discloses the important effect of the sll0147 gene of synechocystis PCC6803 in synthetising carotenoids; by increasing the expression of the sll0147 gene in the synechocystis PCC 6803 through an overexpression method by the applicant, the percentage composition of the carotenoid component in a mutant strain is obviously changed, wherein the content of myxoxanthophyll is increased by 47.4%, the content of zeaxanthine is increased by 93.8%, while the content of echinenone is reduced by 60.9%, and the content of beta-carotene is reduced by 15.9%.

Description

technical field [0001] The invention relates to the application of a sll0147 gene in synthesizing Synechocystis carotenoids, in particular to the application of the sll0147 gene in Synechocystis PCC6803 to regulate the production of zeaxanthin in Synechocystis PCC6803, and belongs to the technical field of genetic engineering. Background technique [0002] Carotenoids (Carotenoids) is the general term for two major types of pigments, Carotene (Carotenes) and Xanthophylls (Xanthophylls), usually hydrocarbons and their oxidized derivatives, composed of 8 isoprenoid units, is a class of Important natural pigments, more than 700 carotenoids have been discovered so far, the common ones are α-Carotene, β-Carotene, Zeaxanthin, Lutein (Lutein) and astaxanthin (Astaxanthin), etc. [0003] Carotenoids are widely distributed in nature and are mainly synthesized by plants, algae, cyanobacteria, and some bacteria, fungi and other microorganisms. Animals cannot synthesize carotenoids the...

Claims

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Application Information

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IPC IPC(8): C12N15/31C12N15/74C12N1/21C12R1/01
Inventor 陈高何庆芳张燕于金慧郑玲边斐葛海涛宣宁王瑜刘园园
Owner BIOTECH RES CENT SHANDONG ACADEMY OF AGRI SCI
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