Method for constructing transgene kelp

A transgenic, large-scale technology, applied in the field of genetic engineering, can solve the problems of low efficiency, transformation system without Agrobacterium, large differences in cell and tissue culture research, etc., and achieve the effect of expanding the scope and stabilizing expression

Inactive Publication Date: 2004-09-01
INST OF OCEANOLOGY - CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] Macroalgae are lower plants that live in the marine environment. They are different from the above-mentioned higher plants in the following aspects: 1. Transformation method: no transformation system similar to Agrobacterium; 2. Vector element: no self-promoter has been cloned so far; 3. Transformation of receptors and plant regeneration: Different types of cell and tissue culture research are quite different. Some species (such as laver) can achieve protoplast regeneration of plants, and many types of protoplasts (such as kelp, wakame) are difficult to regenerate plants and tissues. The efficiency of cultivating regenerated plants is also very low; 4. Screening markers: the types and sensitivity of macroalgae to antibiotics are different from those of higher plants
[0006] At present, the vast majority of studies have completely borrowed the technology of higher plants, using the tissue cuts of macroalgae or protoplasts as the transformation recipients, and only realized the transient expression of exogenous genes, and no exogenous genes were detected in the new plants regenerated from protoplasts. Stable expression of the gene; low efficiency due to the use of only one promoter of higher plant origin (CaMV35S); susceptibility experiments of macroalgae to antibiotics have not been covered

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0017] Example 1: Demonstrating effectiveness on kelp female gametophytes with reporter genes

[0018] Using fcp promoter (fucus xanthophyll chlorophyll a / c binding protein gene from marine diatoms)-GUS reporter gene plasmid, kelp female gametophytes were transformed by biolistic method, and GUS gene was detected in parthenogenetic kelp sporophytes. stable expression. The specific process flow is as follows:

[0019] 1. Preparation of Gene Gun Microparticles:

[0020] Weigh 60 mg of gold powder (Bio-Rad company's gene gun consumables, diameter 1.0 μm), add 1 ml of absolute ethanol, shake vigorously for 1 minute; centrifuge at 10,000 rpm for 10 seconds, remove the supernatant; add 1 ml of sterile water Suspended and centrifuged to remove the supernatant, and repeated three times in total. Finally, the gold powder was suspended in 1 ml of sterile water, divided into 50 μl portions, and stored at 4°C for later use.

[0021] Transfer 1 aliquot (1 aliquot is 50 μl) to a 1.5 ml c...

Embodiment 2

[0031] Example 2: Use reporter gene to prove that both male and female gametophytes of kelp are effective (Examples 1 and 2 are methodological proofs mediated by spores)

[0032] Using the SV40 promoter-lacZ reporter gene plasmid, the female and male gametophytes of kelp were transformed by biolistic method, and the stable expression of lacZ gene was detected in the fertilized diploid sporophytes of kelp.

[0033] The preparation and transformation operation of the transformed microparticles are the same as in Example 1, and the preparation process of the female and male gametophytes of kelp is the same, and the specific reference is to step 2 in Example 1. The other steps are:

[0034] 3. Post-transformation culture

[0035] After the transformation, the kelp female gametophytes of the transformation group and the control group are transferred into the iron-containing seawater culture medium, and the nutrient salt formula and the culturing conditions are the same as in Examp...

Embodiment 3

[0038] Example 3: Extension to application using both foreign genes and selectable marker genes

[0039] The SV40 promoter-cat selectable marker gene-HBsAg gene (encoding hepatitis B virus surface antigen) plasmid was used to transform the male gametophytes of kelp with a gene gun. After screening with chloramphenicol, the HBsAg gene was detected in the fertilized and developed kelp diploid sporophytes. stable expression.

[0040] Wherein: the preparation of transformed microparticles, the preparation of kelp male gametophytes, and the transformation operations are the same as those of Example 1, and the culture after transformation is the same as that of Example 2. The other steps are:

[0041] 3. Screening of Chloramphenicol

[0042] After transformation, chloramphenicol selection was performed when the fertilized and developed kelp diploid sporophytes reached about 2 cm in length. Add kelp, N-P nutrient seawater and chloramphenicol to the sterilized beaker. The concentra...

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PUM

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Abstract

The invention relates to a method for preparing transgenic large scale sea weed, wherein the reportedly exogenesis gene or functional exogenesis gene and antibiotic or weedicide genes are recombined to the promotor downstream originated from advanced plant or algae for constructing carrier for transportation, the large-scale sea weed spores are used as transportation receptor, and gene guns are used to direct the recombined plasmid DNA into the spores of large-scale sea weed, new plants can be grown through the approach of natural growth. The invention can realize the stabilized expression of exogenesis genes.

Description

technical field [0001] The invention relates to the field of genetic engineering, in particular to a method for constructing transgenic macroalgae. Background technique [0002] Macro seaweed is the main source for the production of alginate (agar, carrageenan, alginic acid, etc.), and large-scale seaweeds that have achieved large-scale artificial cultivation include red algae (pork, gracilaria, stone lily, unicorn), green algae (stone Ulva) and brown algae (kelp, wakame), the global cultivation area reaches 3 million mu, and the annual output reaches 6.35 million tons of fresh weight. Except for some of them as nutritious food, it is mainly used for the extraction of algin. In recent years, due to the rapidly increasing demand for algin production and quality improvement, genetic engineering technology has been introduced into the research field of macroalgae for targeted transformation of seaweed germplasm. Transgenic macroalgae can also be used for the expression and pro...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/82
CPCC12N15/8207C12N15/8242
Inventor 秦松姜鹏于道展李富超孙国琼
Owner INST OF OCEANOLOGY - CHINESE ACAD OF SCI
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