A promoter pro-biutnt that efficiently drives the stable expression of target genes in apple protoplast cells

A target gene and protoplast technology, applied in the field of plant biotechnology and biochemistry, can solve the problems of weak expression of some genes, inability to express genes, and obstacles.

Active Publication Date: 2022-07-19
SHANDONG AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, in apple protoplast cells, some genes cannot be expressed and some genes are weakly expressed by using the conventionally used CaMV 35S promoter. This characteristic of the CaMV 35S promoter hinders the application of apple protoplast cell target protein expression technology. Study on gene function in apple

Method used

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  • A promoter pro-biutnt that efficiently drives the stable expression of target genes in apple protoplast cells
  • A promoter pro-biutnt that efficiently drives the stable expression of target genes in apple protoplast cells
  • A promoter pro-biutnt that efficiently drives the stable expression of target genes in apple protoplast cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0051] Embodiment 1, the separation method of apple protoplast cell

[0052] Using apple callus cells as the test material, apple protoplast cells were separated and prepared by the mixed hydrolysis method of cellulase, pectinase and isozyme. Specific steps are as follows:

[0053] 1. Culture and preservation of apple callus cells

[0054] There are two kinds of apple callus cells: 1 is Orin apple callus cells ( figure 1 a); 2 is purple red 2-4 apple callus cells ( figure 1 b).

[0055] 1. Source, culture and preservation of Orin apple callus cells

[0056] 1) Source of Orin apple callus cells

[0057] For the source of Orin apple callus cells, see reference "AN JP, Yao JF, Xu RR, You CX, WangXF, Hao YJ. Apple bZIP transcription factor MdbZIP44 regulates abscisic acid-promoted anthocyanin accumulation. Plant, Cell & Environment, 2018, DOI: 10.1111 / pce.13393.”.

[0058] 2) Culture and preservation of Orin apple callus cells

[0059] Orin apple callus cells were cultured...

Embodiment 2

[0078] Example 2. Amplification of Pro-BIUTNT promoter and construction of target gene expression vector using it as promoter

[0079] 1. Cloning of Pro-BIUTNT promoter

[0080] The genomic DNA of Arabidopsis thaliana was extracted, and the genomic DNA of Arabidopsis thaliana was used as a template to PCR-amplify the polyubiquitin-encoding gene (At4g05320. 2) A nucleotide sequence of 1307 bp upward from A in the translation initiation site ATG (excluding A), this nucleotide fragment is named Pro-BIUTNT in this patent application.

[0081] Table 2. Names, genes, source species, amplification primers and promoter fragment lengths of 7 promoters

[0082]

[0083]

[0084] The amplification system is: total volume 100 μL, including DNA template 4 μL, dNTP 8 μL, forward (F) and reverse (R) primers 5 μL each, 5×HF Buffer 20 μL, ddH 2 O 57 μL, Phusion High-Fidelity Enzyme (Thermo SCIENTIFIC, #F-530L) 1 μL.

[0085] The amplification procedure was: ①98℃ for 3min; ②98℃ for 30s...

Embodiment 3

[0126] Example 3. Amplification of Pro-MdBIUTNT and Pro-MdBIUTNT-2 promoters in apples and construction of expression vectors using them as promoters

[0127] 1. Amplification of 6 nucleotide fragments in apple

[0128] Using apple genomic DNA as the template, the primers corresponding to the Pro-MdBIUTNT, Pro-MdBIUTNT-2, Pro-MdRP-1, Pro-MdRP-2, Pro-MdRC-1, and Pro-MdRC-2 fragments in Table 2 were used respectively. Amplification was performed to obtain Pro-MdBIUTNT, Pro-MdBIUTNT-2, Pro-MdRP-1, Pro-MdRP-2, Pro-MdRC-1, and Pro-MdRC-2 nucleotide fragments, respectively.

[0129]The 6 nucleotide fragments are derived from the following 3 genes: MdBIUTNT (Pro-MdBIUTNT, Pro-MdBIUTNT-2), MdRP (Pro-MdRP-1, Pro-MdRP-2), MdRC (Pro-MdRC-1, Pro-MdRC-2). MdBIUTNT is the homologous gene of Arabidopsis polyubiquitin gene in apple; MdRP is 1,5-ribulose diphosphate carboxylase small subunit gene; MdRC is apple 1,5-ribulose diphosphate carboxylase enzyme activating enzyme gene; the numbers ...

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Abstract

The invention discloses a promoter Pro-BIUTNT that efficiently drives the stable expression of a target gene in apple protoplast cells. The promoter is a polyubiquitin encoding gene that starts from A in the translation initiation site ATG (excluding A) Upward 1307bp nucleotide fragment. Experiments have shown that the Pro‑BIUTNT promoter can efficiently and stably drive and regulate the expression of 12 representative target genes involved in apple fruit ripening and immune response in apple protoplast cells, a breakthrough in apple protoplast cells. Using CaMV 35S promoter, some genes are not expressed, and some genes are weakly expressed, which lays a foundation for the application of apple protoplast cell protein expression technology to apple signal transduction research.

Description

technical field [0001] The invention relates to the field of plant biotechnology and biochemistry, in particular to a promoter Pro-BIUTNT that efficiently drives the stable expression of a target gene in apple protoplast cells. Background technique [0002] Apple (Malus x domestica Borkh.) is a fruit tree species of the Rosaceae family with great economic value. The molecular mechanism research on the regulation of apple tree growth, development and fruit quality formation makes this tree species expected to become a model plant for studying the growth, development, fruit quality formation, disease resistance and immunity of higher perennial woody plants. The disclosure of important functional genes and specific signal transduction pathways in apples is inseparable from the efficient and stable expression technology of target genes in apple cells. Although callus transformation can be used to obtain the target protein, but the technical method has a long cycle, and the rese...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/113C12N5/10C12N15/82C12N15/11
CPCC12N15/8222C12N5/04C12N15/8251
Inventor 王宪璞张芮刘秀霞张彬彬辛丽王立双冀晓昊王楠姜生辉张艳敏陈学森吴树敬
Owner SHANDONG AGRICULTURAL UNIVERSITY
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