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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  • Abstract
  • Description
  • Claims
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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 oi

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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

Examples

Experimental program
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Example Embodiment

[0043] Example 1

[0044] 1. Preparation of target gene

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

[0046] According to the sequence obtained by genome sequencing, the corresponding primers, P2845-F / R and T2845-F / R, were designed, and the sequences are shown in Table 1. Using the genome of Schizochytrium HX-308 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. The corresponding primer NeoR-F / R was designed according to the sequence of NeoR, and the pZPK plasmid was used as the template (Sun W, Yang X, W...

Example Embodiment

[0060] Example 2

[0061] Construction of Recombinant Agrobacterium AGL-1

[0062] Agrobacterium primitiveum AGL-1 was streaked on YEB solid plates containing 50 μg / mL carbenicillin and 50 μg / mL kanamycin, and cultured at 28°C for 2 days. After 2 days, pick a single clone and inoculate it into 5mL YEB liquid medium containing 50μg / mL carbenicillin (containing beef extract 5g / L, yeast extract 1g / L, peptone 5g / L, sucrose 5g / L, MgSO 4 -7H 2 (00.5g / L), 28°C, 220rpm overnight culture. Inoculate 2mL of the overnight cultured bacterial solution into a new 5mL YEB liquid medium containing 50μg / mL carbenicillin, and cultivate to OD at 28°C and 220rpm. 600 = 0.5. The bacterial liquid was taken out, and after 30 min of ice bath, the cells were collected by centrifugation at 4°C and 5000 rpm for 5 min. with 10mL of 20mM CaCl 2 Resuspend in ice bath for 15min. The cells were collected by centrifugation at 5000 rpm for 5 min at 4°C. Discard the supernatant and add 2 mL of 20 mM CaCl...

Example Embodiment

[0064] Example 3

[0065] Construction of Recombinant Schizochytrium HX-308

[0066] Take the Schizochytrium HX-308 stored in the refrigerator at -80°C on the GPYS (10g / L peptone, 5g / L yeast extract, 50g / L glucose, 20g / L sea salt) solid plate for activation, and pick a single clone. Inoculated in 50mL liquid seed medium (monosodium glutamate 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°C, activated at 170rpm overnight, centrifuged at 200g for 5min, removed the supernatant, resuspended the cells with sterile water, and adjusted the concentration of Schizochytrium HX-308 to 10 6 pcs / mL.

[0067] Take the recombinant Agrobacterium AGL-1 bacterial solution containing the recombinant plasmid prepared in Ex...

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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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