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A kind of seaweed endogenous constitutive promoter and its application

A constitutive promoter and promoter technology, applied in the direction of peptide source, unicellular algae, algae/bryopeptide, etc., can solve the problems of low efficiency of exogenous gene expression, low-efficiency transgenic expression of large seaweed and potential safety hazards, etc., to achieve accelerated The effect of large screening reagent concentration, reducing the generation of resistant clones (false positives), and increasing the ratio

Inactive Publication Date: 2019-12-10
INST OF OCEANOLOGY - CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, since most of the promoter elements used are from terrestrial organisms, there are also many deficiencies
For example, the highly efficient CaMV35S promoter (from cauliflower mosaic virus), which is widely used in higher plants, is generally less efficient in driving exogenous gene expression in seaweed; in addition, the most widely used SV40 promoter line in seaweed comes from primate Simian vacuolar virus, therefore, the use of exogenous promoters brings about two outstanding problems of low-efficiency expression of transgenes in macroalgae and potential safety hazards

Method used

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  • A kind of seaweed endogenous constitutive promoter and its application
  • A kind of seaweed endogenous constitutive promoter and its application
  • A kind of seaweed endogenous constitutive promoter and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] Example 1: Cloning, sequencing and bioinformatics analysis of the endogenous constitutive actin gene promoter pUpActin of U.prolifera

[0034] 1. Cloning and sequencing of the 5' upstream sequence of Enteromorpha actin gene

[0035] The total DNA template of Enteromorpha was prepared in a conventional way by using the Plant Genomic DNA Extraction Kit of Tiangen Company. After detection by 0.8% agarose electrophoresis, the extracted band of Enteromorpha genomic DNA was clear and complete, which could meet the needs of PCR amplification. Using the (Genome Walking Kit) chromosome walking kit from TaKaRa Company, three specific primers (SP1: 5'-CAAGGCAATCAGTAATGGACACG-3'; SP2: 5'-CAAGGCAATCAGTAATGGACACG-3'; SP2: 5'-GTGCCGAGGTCTGCCAACGATT-3'; SP3:5'-CCCGCAACAATCATCCTTCAAAA-3'), chromosome walking was performed according to the kit instructions, and the amplified fragments were recovered by agarose gel, TA cloned and ligated for nucleotide sequence It was determined that the...

Embodiment 2

[0038] Example 2: Enteromorpha actin gene promoter pUpActin drives reporter gene GUS expression in seaweed

[0039] 1. Cloning of the promoter fragment of Enteromorpha actin gene and construction of pUPA1-GUS vector

[0040] Enteromorpha actin gene 5' upstream sequence that obtains according to embodiment 1, has promoter activity region for bioinformatics prediction, design enteromorpha actin gene promoter pUpActin specific primer (actin1-F13B-HindIII: 5'- TGATTACGCCAAGCTT tgttagggagggtgtctatgcg-3', wherein the italic letters represent the recognition site of restriction endonuclease Hind III; actin1-R13B-SmaI:5'- AGGGACTGACCACCCGGG tctccaggtttagacgtctac-3', where the italic letters represent the recognition site of the restriction endonuclease Sma I. Capital letters represent subsequent use HD Cloning Kit seamless connection cloning kit (seamless connection cloning kit required for constructing the vector fragments homologous to the end of the vector), PCR amplification, ...

Embodiment 3

[0064] Example 3: Enteromorpha actin gene promoter pUpActin drives reporter gene EGFP expression in Enteromorpha

[0065] 1. Cloning of the promoter fragment of Enteromorpha actin gene and construction of pUPA1-EGFP vector

[0066] According to the Enteromorpha actin gene 5' upstream sequence obtained in Example 1, the region with promoter activity predicted by bioinformatics was designed to design the Enteromorpha actin gene promoter pUpActin specific primer (actin1-F13B-Hind III : 5'-TGATTACGCCAAGCTTtgttagggagggtgtc tatgcg-3', wherein the italic letters represent the recognition site of the restriction enzyme HindⅢ; actin1-R14B-Sma I: 5'-ACCATGGTGGCGCCCGGGtctccaggtttagacgtctac-3', wherein the italic letters represent the restriction enzyme Sma Recognition site for I; capital letters indicate subsequent use HD Cloning Kit seamless connection cloning kit (seamless connection cloning kit) for PCR amplification, the amplified fragments were recovered by agarose gel, TA cloned ...

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Abstract

The invention belongs to the technical field of seaweed genetic engineering, and relates to a seaweed endogenesis constructive promoter and application thereof. The promoter is a nucleotide sequence shown in SEQ ID NO:1, one or more nucleotide substituting, or deficiency or addition is carried out on the nucleotide sequence shown in SEQ ID NO:1 to obtain the promoter, or the promoter is a nucleotide sequence having 90% or more homology with the nucleotide sequence defined in SEQ ID NO:1 and having a transcription initiation function. Enteromorpha actin promoter is obtained through chromosome walking, gene cloning and DNA sequencing and has the sequence shown in SEQ ID NO:1; on the basis, the cloned actin promoter is inserted into reported gene GUS upstream, and a transformation vecter is constructed and guided into an Enteromorpha thallus to detect high-efficiency GUS gene expression. The technology is capable of improving Enteromorpha transgenosis efficiency and biosecurity, and has important significance in algae seed variety improvement and genetic engineering product development.

Description

technical field [0001] The invention belongs to the technical field of seaweed genetic engineering, and relates to a seaweed endogenous constitutive promoter and its application. Background technique [0002] Macroalgae belong to lower plants, mainly including green algae, brown algae and red algae. Many of these economic species have achieved large-scale artificial cultivation, such as the green algae Enteromorpha, Ulva and reef membranes, the brown algae kelp, wakame, hijiki, and the red algae laver, red cabbage, Gracilaria, Cauliflower and eucalyptus. At present, the total area of ​​large-scale seaweed cultivation in the world exceeds 3 million mu, and the annual output exceeds 6 million tons of fresh weight. In addition to being used as nutritious food, it is also used as important chemical raw materials such as iodine, mannitol, and alginate, as well as marine drugs and protein reagents. The extracted raw material has high economic value. [0003] In recent years, th...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/113C12N15/79C12N1/13
CPCC07K14/405C12N15/113C12N2830/00
Inventor 姜鹏吴春辉赵瑾陈华新
Owner INST OF OCEANOLOGY - CHINESE ACAD OF SCI