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Method for transforming schizochytrium limacinum by agrobacterium and application

A technique for transformation of Agrobacterium and Schizochytrium, applied in the biological field, achieves the effects of simple experimental operation, simplification of genetic operation steps, and saving time and cost

Pending Publication Date: 2022-05-03
NANJING NORMAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Schizochytriumsp.HX-308 reported in the prior art is a Schizochytrium strain isolated from seawater. The biomass of this bacterial strain can reach 134.5g / L, and the oil output can reach 80.14g / L. It has huge commercial applications value, there is no complete set of genetic operating system and its targeted knockout in Schizochytrium

Method used

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  • Method for transforming schizochytrium limacinum by agrobacterium and application
  • Method for transforming schizochytrium limacinum by agrobacterium and application
  • Method for transforming schizochytrium limacinum by agrobacterium and application

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Experimental program
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Effect test

Embodiment 1

[0044] 1. Preparation of target gene

[0045] According to the genome annotation, the constitutive expression gene glyceraldehyde triphosphate dehydrogenase was found, its number is 2845, the 1 kb upstream of the start codon of glyceraldehyde triphosphate dehydrogenase is the promoter P2845 (SEQ ID NO.2), and the stop codon The downstream 500bp is the terminator T2845 (SEQ ID NO.4).

[0046] According to the sequence obtained by genome sequencing, corresponding primers were designed, P2845-F / R and T2845-F / R, and the sequences are shown in Table 1. Using the Schizochytrium HX-308 genome as a template, PCR amplification was performed using the above primers to obtain the corresponding promoter P2845 and terminator T2845 fragments.

[0047] The sequence of the G418 resistance gene NeoR is shown in SEQ ID NO.3, and the corresponding primer NeoR-F / R was designed according to the sequence of NeoR, and the pZPK plasmid was used as a template (Sun W, Yang X, Wang X, et al. Homologous...

Embodiment 2

[0061] Construction of recombinant Agrobacterium AGL-1

[0062] The original Agrobacterium AGL-1 was streak-inoculated on a YEB solid plate containing 50 μg / mL carbenicillin and 50 μg / mL kanamycin, and cultured at 28°C for 2 days. After 2 days, single clones were picked and inoculated in 5 mL of YEB liquid medium containing 50 μg / mL carbenicillin (containing beef extract 5 g / L, yeast extract 1 g / L, peptone 5 g / L, sucrose 5 g / L, MgSO 4 -7H 2 (00.5g / L), 28 ℃, 220rpm cultured overnight. Pipette 2 mL of the overnight cultured bacterial liquid into a new 5 mL YEB liquid medium containing 50 μg / mL carbenicillin, and cultivate to OD at 28°C and 220 rpm 600 = 0.5. The bacterial liquid was taken out, and after 30 minutes of ice bathing, the bacterial cells were collected by centrifugation at 5000 rpm at 4°C for 5 minutes. With 10mL 20mM CaCl 2 Resuspended and ice bathed for 15 minutes. Centrifuge at 5000 rpm for 5 min at 4°C to collect the bacteria again. Discard the supernatant...

Embodiment 3

[0065] Construction of Recombinant Schizochytrium HX-308

[0066] Take the Schizochytrium HX-308 stored in the refrigerator at -80°C and put it on the solid plate of GPYS (peptone 10g / L, yeast extract 5g / L, glucose 50g / L, sea salt 20g / L) for activation by streaking and single clone Inoculate in 50mL liquid seed medium (MSG 20g / L; Na 2 SO 4 10g / L; KH 2 PO 4 4g / L; MgSO 4 2g / L; (NH 4 ) 2 SO 4 0.8g / L; KCl 0.2g / L; CaCl 2 0.1g / L; trace elements (g / L): Na 2 EDTA 6g / L; MnCl 2 4H 2 O 0.86g / L; ZnSO 4 0.8g / L; FeSO 4 0.29g / L; CoCl 2 ·6H 2 O 0.01g / L; CuSO 4 5H2O 0.6g / L; NiSO 4 ·6H 2 O 0.06g / L; Na 2 MoO 4 2H 2 (00.01g / L), 28 DEG C, 170rpm overnight activation, 200g, remove supernatant after 5min centrifugation, use sterile water to resuspend thalline, the concentration of Schizochytrium HX-308 is modulated 10 6 individual / mL.

[0067] Take the recombinant Agrobacterium AGL-1 bacterial liquid containing the recombinant plasmid prepared in Example 2, which was stor...

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Abstract

The invention discloses a method for transforming schizochytrium limacinum by agrobacterium tumefaciens and application, and the transformation method comprises the following steps: introducing an exogenous gene into the agrobacterium tumefaciens, and introducing the exogenous gene into the schizochytrium limacinum by utilizing the agrobacterium tumefaciens, so that the schizochytrium limacinum obtains the function of the related exogenous gene. According to the invention, the agrobacterium AGL-1 transferred into a G418 resistance gene expression cassette and the schizochytrium limacinum HX-308 are co-cultured, so that an exogenous gene is successfully transformed into the schizochytrium limacinum HX-308, and fixed-point knockout of the gene is realized. Experimental operation is very simple, time and cost are effectively saved, Agrobacterium tumefaciens can be converted into schizochytrium limacinum, more importantly, gene knockout can be achieved, and a set of complete genetic operating system can be constructed in the schizochytrium limacinum through gene knockout. According to the invention, site-specific integration can be completed by using a short homologous arm, and a 2-3kb homologous arm is possibly needed in general fungal transformation, so that a foundation is laid for simplifying genetic manipulation of schizochytrium limacinum.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a method and application of Agrobacterium transforming Schizochytrium. Background technique [0002] At present, exogenous DNA is mainly transformed into Schizochytrium by electric shock, but the conditions of electric shock transformation vary widely. The reported electric shock voltage, resistance and capacitance are different, and electric shock transformation requires high concentration of linearized exogenous DNA. , the workload is huge. In addition, the homologous recombination efficiency of electroporation transformation is very low, which is not conducive to gene knockout. [0003] The Agrobacterium transformation system is a naturally occurring gene transformation system that transforms T-DNA on tumor-inducing (Ti) plasmids into plant cells. There are two 25bp repeat sequences at both ends of the T-DNA, which are the left border and the right border respectivel...

Claims

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Application Information

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IPC IPC(8): C12N15/80C12N15/74C12N1/15C12N1/21C12R1/01C12R1/645
CPCC12N15/80C12N15/743C12N2830/34
Inventor 黄和黄鹏伟孙小曼马旺贾雨雷李进
Owner NANJING NORMAL UNIVERSITY
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