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Universal expression vector for large transgenic green alga and transformation expression method thereof

An expression method and expression vector technology, which are applied to the general expression vector of transgenic macrogreen algae and the field of stable transformation and expression, can solve the problems of low efficiency and backward research on seaweed transgenic, achieve widespread consumption, increase cell regeneration rate, and increase growth rate. quick effect

Active Publication Date: 2010-06-02
SHANGHAI OCEAN UNIV
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AI Technical Summary

Problems solved by technology

However, compared with higher plants, seaweed transgenic research is still very backward
At present, the vast majority of studies have completely borrowed from the technology of higher plants, using macroalgae tissue sections or protoplasts as transformation recipients, only achieved transient expression of foreign genes, and no foreign genes were detected in new plants regenerated from protoplasts. Stable expression of the source gene, due to the use of only a higher plant-derived promoter (CaMV35S), is therefore less efficient, and the susceptibility test of macroalgae to antibiotics has not been involved

Method used

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  • Universal expression vector for large transgenic green alga and transformation expression method thereof
  • Universal expression vector for large transgenic green alga and transformation expression method thereof
  • Universal expression vector for large transgenic green alga and transformation expression method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Example 1 Construction of the universal expression vector Psv-bar of transgenic macroalgae

[0037] The initial vector is psv-β-Galactosidase Control purchased from Promega, USA. The vector is about 6820bp in length, and the 710-3755 of the vector is the coding region of lacZ. Use the restriction enzyme sites HindIII and EcoR I at both ends of the gene to cut the gene and delete it for later use.

[0038]Utilize the existing plasmid vector PUC-bar in the laboratory, and its properties are the same as those commercially available. [Wherein, the PUC plasmid was purchased from Promega Company, and the synthetic bar gene (sequence number FJ858786.1 or FJ826509.1) was used to construct the plasmid vector PUC-bar by conventional molecular experiment methods].

[0039] The bar gene was cloned from the above-mentioned plasmid vector PUC-bar, and the full length of the bar gene was 555 bp. The primers used for cloning are: upstream primer 5'-3'GCACCATCGTCAACCACTA;

[0040] Do...

Embodiment 2

[0042] Example 2 Screening of Transformation Selectable Markers of Macroalgae

[0043] Through screening, the herbicide glufosinate Basta has a strong killing effect on Enteromorpha spores and seedlings, and Basta at a concentration of 5 μg / ml can kill all Enteromorpha spores within 3 days, and Basta at a concentration of 12.5 μg / ml Basta can kill all Enteromorpha seedlings in about a week. Subsequent experiments showed that the results were also applicable to other types of Enteromorpha, such as Yuanguan.

Embodiment 3

[0044] Example 3 Optimizing the conditions for the separation and purification of protoplasts from Enteromorpha spp.

[0045] It was found that when protoplasts of Enteromorpha barbadensis and Enteromorpha spp. were obtained by enzymatic method, the optimal enzymatic hydrolysis formula was 2% cellulase mixed with 2% isolated enzyme, the optimum enzymatic hydrolysis pH value was 6.5, and the concentration of osmotic agent mannitol was 0.6 M. The protoplasts of Enteromorpha protoplasts transformed by protoplasts mainly have three different development modes: single-cell seedlings, cell clusters, and sporangia / gametes.

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Abstract

The invention discloses a universal expression vector for a large transgenic green alga and a transformation expression method thereof. Through studying the universality of a resistant screening marker and a resistant screening marker gene of large transgenic green algae, such as Enteromorpha prolifera, long Ulva, sea lettuce, and the like, corresponding resistant screening marker gene is cloned,the traditional carrier of the higher plant is adequately used on the basis or a homologous recombination technology is utilized to reconstruct the vector, and a universal carrier pSVB suitable for the large transgenic green alga is constructed. In the invention, on the basis of a cell regeneration system and a gametic parthenogenesis system, the efficiently transformed and stably expressed universal carrier is utilized to finally establish a large alga transformation and expression system so that the exogenous gene can be efficiently and stably expressed in the large algae, and the foundation for establishing an alga reactor and producing an important medicament and the alga energy sources in future is established.

Description

technical field [0001] The invention relates to the field of genetic bioengineering, in particular to a general expression vector of transgenic large green algae (enteromorpha, Ulva ulvae, etc.) in the field of genes and a stable transformation expression method thereof. Background technique [0002] Since the production of the first transgenic tobacco in 1983, the research on various transgenic plants in the world has made great progress, and has produced huge economic benefits. However, the transgenic research of algae has progressed relatively slowly, because seaweed is different from higher plants, and it is more difficult to transgenic algae. The main reasons are as follows: 1) For a long time, the development of seaweed transgenes has been greatly restricted due to the absence of ideal resistance selection markers; 2) Stable expression systems have not been established, and most of them can only achieve instantaneous expression levels. [0003] Large green algae grow ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/82A01H13/00
Inventor 何建华汤文仲陈群芳何培民秦松叶静
Owner SHANGHAI OCEAN UNIV
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