Expression vector for large green alga bioreactor and transformation method thereof

A technology of bioreactors and expression vectors, which is applied in the construction and transformation of expression vectors, can solve problems such as untrackable detection, and achieve the effect of avoiding false positives

Inactive Publication Date: 2011-06-22
SHANGHAI OCEAN UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In algae genetic engineering, GUS and lacZ are detected by chemical staining as reporter genes. Although the technology is mature and the sensitivity is high, neither of them can track and detect the expression level in vivo. Due to its inherent advantages, green fluorescent protein is very difficult. ok solved the problem
At present, the transient expression of green fluorescent protein in Porphyra and Laminaria gametophytes has been obtained; the atpA promoter of Chlamydomonas reinhardtii chloroplast is expressed in Dunaliella salina, but it has not been successfully used as a reporter gene in large green algae

Method used

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  • Expression vector for large green alga bioreactor and transformation method thereof
  • Expression vector for large green alga bioreactor and transformation method thereof
  • Expression vector for large green alga bioreactor and transformation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] Example 1 Transformation plasmid vector construction and amplification

[0044] 1.1 According to the pZsGeenl-N1 expression vector (purchased from Clontech, about 4.7kb), such as figure 1 As indicated, a pair of specific primers was designed. Add Xba I restriction site at the 5' end of upstream primer pZsGeen1 P1 and downstream primer pZsGeen2 P2, its sequence includes CMV immediate earlypromoter, enhancer region, TATA box, start point, start codon, stop codon (about 1500bp).

[0045] The primer sequences are as follows:

[0046] pZsGeen1 P1: 5'-GTTATTAATAGTAATCAATTACGG-3'

[0047] pZsGeen1 P2: 5'-TCTAGATCAGGGCAAGGCGGAGCCGG-3'

[0048] 1.2 The PCR reaction system and conditions for amplification are:

[0049] PCR reaction system (25μl):

[0050] Sterile ultrapure water 17.1μl

[0051] 10×Buffer (including Mg 2+ ) 2.5 μl

[0052] dNTP 1μl

[0053] Primer (10ppm) each 1μl

[0054] Template (0.8μg / μl) 2μl

[0055] Taq enzyme (TAKARA) 0.4μl (2.5U)

[0056] Pre-...

Embodiment 2

[0075] Example 2 Screening of Transformation Selectable Markers of Macroalgae

[0076] Through screening, the herbicide Basta (glufosinate) has a strong killing effect on Enteromorpha spores and seedlings, and Basta at a concentration of 5 μg / ml can completely kill Enteromorpha spores within 3 days, and 12.5 μg / ml It takes about a week under the concentration to kill all the seedlings of Enteromorpha. Subsequent experiments showed that the results were also applicable to other types of Enteromorpha, such as Yuanguan.

Embodiment 3

[0077] Example 3 Optimizing the conditions for the separation and purification of protoplasts from Enteromorpha spp.

[0078] 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. Through protoplast culture, it was observed that Enteromorpha cells mainly had three different development modes: single-cell seedlings, cell clusters, and sporangia / gametes.

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Abstract

The invention provides a vector capable of making an exogenous gene expressed stably and efficiently in a large green alga bioreactor and a method thereof for transformation and expression in a large green alga. In the invention, a protoplast of a large green alga is used as a receptor, the conventional vector of higher plants or a vector modified by a homologous recombination technique are fully utilized to construct a green fluorescent tracing and fusion protein gene-containing vector PSB-bar-ZsGeen for use in a large green alga bioreactor, and the vector PSB-bar-ZsGee is connected with a SV40 gene promoter, a resistance gene, a cytomegalovirus (CMV) promoter and the like in turn to realize the high-efficiency and stable expression of the exogenous gene in the large green alga.

Description

technical field [0001] The invention relates to the field of genetic bioengineering, in particular to the construction of an expression vector for a bioreactor of transgenic large green algae (enteromorpha, Ulva ulvae, etc.) and a transformation method thereof. Background technique [0002] Large green algae grow very fast, have a particularly good de-eutrophication effect, and can effectively inhibit the occurrence of red tides. They are currently ideal marine ecological restorers. Large green algae are also an ideal "seaweed reactor", which can be used to produce important biologically active substances and develop seaweed energy. my country is one of the countries with the most developed algae farming industry in the world, among which the production of kelp and seaweed farming ranks first and third in the world respectively. Since the mid-1990s, the discovery of seaweed bioactive substances and the rise of seaweed pharmaceutical technology have opened up new ways for th...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/82A01H13/00
Inventor 何培民何建华汤文仲张婷时旭
Owner SHANGHAI OCEAN UNIV
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