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Application of gene spkD for regulating growth rate of Synechocystis

A technology of growth speed and Synechocystis, applied in the application of gene spkD, biological field, can solve problems such as unable to meet market demand, achieve the effect of improving growth speed and stress resistance performance, and improving feasibility

Active Publication Date: 2019-01-18
TIANJIN AGRICULTURE COLLEGE +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] Although this method can regulate the growth of Synechocystis, it still cannot meet the actual market demand.

Method used

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  • Application of gene spkD for regulating growth rate of Synechocystis
  • Application of gene spkD for regulating growth rate of Synechocystis
  • Application of gene spkD for regulating growth rate of Synechocystis

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0074] Construct the spkD gene cDNA fragment, psbA2 promoter fragment, homologous recombination upstream fragment s1r1285U cDNA fragment and homologous recombination downstream fragment s1r1285D cDNA fragment of the Microcystis aeruginosa PCC7806 spkD gene overexpression vector of Synechocystis sp. PCC6803.

[0075] Cloning of Synechocystis sp. PCC 6803 psbA2 promoter fragment: according to Synechocystis sp. PCC6803 registered in GenBank: (accession number: BA000022, AP012205), the first 500 bp of psbA2 ORF was used as the promoter sequence, and primers were designed:

[0076] Promoter-SalI-F: 5'-AATGTCGACTGCCCAGATGCAGGCCTTC-3';

[0077] Promotor-R: 5'-GTAGAGCAGTTCACGCATTTGGTTATAATTCCTTAT-3';

[0078] After the PCR reaction, the gel was recovered by electrophoresis, connected to the T3 vector cloning vector, and then transformed into Escherichia coli DH5α, and the positive clones were screened and sequenced. The promoter fragment of Synechocystis PCC 6803psbA2 was obtained, t...

Embodiment 2

[0103] Preparation of plasmid pBluscript SK plus-T1T2:

[0104] 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:

[0105] T1T2-F:5'-ATACTGCAGCCAAGCTTGGCTGTTTTGGC-3';

[0106] T1T2-R:5'-TTAGGATCCCCCATTATTGAAGCATTTAT-3';

[0107] The amplification procedure is as follows:

[0108] Pre-denaturation at 94°C for 5 min; denaturation at 94°C for 30 s, renaturation at 60°C for 30 s, extension at 72°C for 30 s, and 35 cycles; final extension at 72°C for 10 min, and storage at 4°C; The same digested pBluescriptSK was ligated to obtain plasmid pBluescript SK T1T2.

[0109] The homologous recombination upstream arm slrl285U gene fragment obtained in Example 1 was digested with KpnI to recover the s1r1285U gene fragment; the plasmid pBluescript SK T1T2 was digested with the same method to recover th...

Embodiment 3

[0125] PCR Detection of Microcystis aeruginosa SpkD Gene Overexpression

[0126] Using transgenic Synechocystis PCC6803 and wild-type Synechocystis PCC6803 containing the expression vector p5S1285UD spkD as materials, the total DNA was extracted for PCR detection and analysis. The specific method is as follows:

[0127] DNA was extracted from Synechocystis spkD gene overexpression and wild-type Synechocystis spkD gene by using neutral phenol reagent (purchased from Invitrogen Company) and glass bead shaking method. The specific operation steps are as follows: take 50m1OD 730 =1.8 cyanobacteria, 4 DEG C, 5000rpm centrifuge 10min to collect algal cells, add 0.4mL neutral phenol to connect 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 There is 0.5 mL of suspension above the glass bead interface. Vibrate with a vortex shaker at the maximum speed for 1min, centrifuge at 11900rpm at 4°C f...

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Abstract

The invention relates to the application of a gene spkD for regulating growth rate of Synechocystis. Application of the gene spkD regulating the growth rate of Synechocystis in increasing the growth rate of Synechocystis and / or restoring the growth rate after light stress; The nucleotide sequence of the gene spkD is shown in SEQ ID NO. 1. The invention discloses for the first time the important role of spkD gene of Microcystis aeruginosa PCC7806 in improving the growth rate of Synechocystis. The average specific growth rate of the transgenic Synechocystis sp. Increased by 13.97% compared withthe wild type in 6 days under normal culture conditions, However, both mutants overexpressing spkD gene and wild type exhibited overcompensatory growth performance in the light recovery stage (6 daysculture stage) after light limitation stress, and the specific growth rate of mutants increased by 7.13% compared with wild type in the overcompensatory growth stage.

Description

technical field [0001] The invention relates to the application of a gene spkD regulating the growth rate of Synechocystis sp., belonging to the technical field of biotechnology. Background technique [0002] Cyanobacteria are a class of prokaryotes with photoautotrophic ability. After more than 3 billion years of long evolution, the signal transduction system of cyanobacteria can quickly sense changes in the external environment, and through precise regulation of the expression of functional genes such as division and differentiation of algal cells, the cells can actively respond to changes in the external environment. Quick response to survive in various adversities. Serine / threonine kinases system (STKs) is one of the two main signal transduction systems in prokaryotes. STKs kinases can directly phosphorylate or activate soluble protein kinases in the cytoplasm through phosphorylation to indirectly phosphorylate downstream target proteins in the signaling pathway, there...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/79C12N15/66C12N15/31
CPCC07K14/195C12N15/66C12N15/79
Inventor 毕相东陈高吕东路晓媛戴伟曹月蕾董少杰张树林张达娟
Owner TIANJIN AGRICULTURE COLLEGE
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