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
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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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