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Application of a sll0659 gene in synthesizing Synechocystis carotenoids

A technology of carotene and Synechocystis, applied in the field of genetic engineering, to achieve the effect of enhancing biomass and improving photosynthetic efficiency

Active Publication Date: 2018-12-25
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 sll0659 gene in Synechocystis sp. PCC6803 (Genbank accession number is WP_010873989.1) and Synechococcus CruP (Genbank accession number is WP_012305684.1) have an amino acid similarity of 51.12%. However, there is no report on the application of the sll0659 gene in Synechocystis sp. PCC6803 in increasing the synthesis of α-carotene and promoting photosynthesis in Synechocystis sp. PCC6803.

Method used

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  • Application of a sll0659 gene in synthesizing Synechocystis carotenoids
  • Application of a sll0659 gene in synthesizing Synechocystis carotenoids
  • Application of a sll0659 gene in synthesizing Synechocystis carotenoids

Examples

Experimental program
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Effect test

Embodiment 1

[0054] Isolate and clone the sll0659 gene cDNA fragment that is used to construct the knockout of the Synechocystis sp. PCC6803 sll0659 gene:

[0055] Access: http: / / blast.st-va.ncbi.nlm.nih.gov / Blast.cgi? PROGRAM=blastn&PAGE_TYPE=Blast

[0056] Search&LINK_LOC=blasthome, input Synechococcus sp. 7002CruP nucleotide sequence (Genbank accession number: EF529627.1), and obtain the sll0659 gene sequence in Synechocystis sp. PCC6803 with sequence number CP003265.1.

[0057] Amplification of upstream and downstream fragments of the sll0659 gene used to construct the Synechocystis sp. PCC6803 sll0659 gene knockout vector:

[0058] According to the Synechocystis PCC6803 sll0659 sequence published in the GenBank database (accession number: CP003265.1), the primers for amplifying the upstream cDNA fragment of Synechocystis PCC6803 sll0659 were designed:

[0059] sll0659-1-F: 5'-ATGAACTATGCAACTACACC-3';

[0060] sll0659-1-BamHI-R:5'-CGAGGATCCGGCAATGATTGGTAATT-3';

[0061] The amplifi...

Embodiment 2

[0073] Construction and transformation of Synechocystis sp. PCC6803 sll0659 gene knockout vector

[0074] The sll0659 gene fragment in Synechocystis sp. PCC6803 was mutated by insertion inactivation technology, and the application of the sll0659 gene in Synechocystis sp. PCC6803 sll0659 gene in carotenoid synthesis and photosynthesis was studied according to the phenotype and carotenoid composition of the sll0659 gene deletion mutant strain.

[0075] Synechocystis sp. PCC6803 sll0659 gene knockout vector construction:

[0076] The upstream fragment positive clone plasmid of the Synechocystis PCC6803 s110659 gene obtained in Example 1 is double-digested with restriction endonucleases BamHI and SalI, and the carrier fragment 1 is reclaimed; The downstream fragment positively cloned the plasmid, and fragment 2 was recovered. The recovered fragment 2 and vector fragment 1 were used for ligation reaction to transform Escherichia coli DH5α. By screening positive clones, obtain the...

Embodiment 3

[0080] PCR detection of sll0659 knockout algal strain

[0081] Using the transgenic Synechocystis sp. PCC6803 and the wild type Synechocystis sp. PCC6803 containing the knockout vector pKNsll0659 as materials, the total DNA was extracted for PCR detection and analysis. The specific method is as follows:

[0082] Neutral phenol reagent (purchased from Invitrogen) was used to extract DNA from sll0659 gene knockout and overexpression Synechocystis sp. and wild-type Synechocystis sp. The specific operation steps are as follows:

[0083] Take 50ml OD 730 =1.8 cyanobacteria, 4°C, 5000rpm centrifuge 10min to collect algae cells, add 0.4mL neutral phenol reagent (purchased from Invitrogen Company) to connect 0.4mL BG-11 liquid medium, and then add an appropriate amount of glass beads with a diameter of about 0.17mm (purchased from sigma company) to 0.5mL of suspension above the glass bead interface. Vibrate with a vortex shaker at the maximum speed for 1min, centrifuge at 11900rpm...

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Abstract

The invention relates to application of a sll0659 gene to synthesis of synechocystis carotenoids. The nucleotide sequence of the sll0659 gene is shown in SEQ ID No.1. The invention provides application of the sll0659 gene in synechocystis PCC6803, which has an important effect on synthesis of synechocystis PCC6803 beta-carotene. By knocking out the sll0659 gene in synechocystis PCC6803, the lutein content of the synechocystis PCC6803 is increased by 22.3%, zeaxanthin content is increased by 31.3%, the echinenone content is reduced by 34.3%, and the percentage content of beta-carotene is reduced by 1.3%. Meanwhile, HPLC detection results show when synechocystis PCC6803 involved in the invention is compared with wild synechocystis PCC6803, an obvious peak exists in the carotenoid components in the sll0659 gene knockout mutant, and the obvious peak is determined to be alpha-carotene by contrasting the obvious peak with a standard product and detecting.

Description

technical field [0001] The present invention relates to the application of a sll0659 gene in the synthesis of Synechocystis carotenoids, in particular to the application of the sll0659 gene in Synechocystis PCC6803 in the synthesis of Synechocystis carotenoids in the photosynthesis of Synechocystis PCC6803, belonging to the technical field of genetic engineering . Background technique [0002] Carotenoids (Carotenoids) is the general term for the two major pigments of carotene (Carotenes) and lutein (Xanthophylls), usually hydrocarbons and their oxidized derivatives, composed of 8 isoprenoid units, is An important class of natural pigments, more than 700 carotenoids have been discovered so far, the common ones are α-Carotene, β-Carotene, Zeaxanthin, leaf Lutein and 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 car...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/74C12N1/21C12R1/01
Inventor 陈高何庆芳杨连群王兴军范仲学岳寿松毕玉平张燕谢红艳马德源
Owner BIOTECH RES CENT SHANDONG ACADEMY OF AGRI SCI
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