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Promoter WY195 and application thereof

A WY195, promoter technology, applied in the field of genetic engineering, can solve the problem of molecular biology research lag and other problems

Inactive Publication Date: 2018-03-06
HAINAN UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In terms of obligate parasites, due to the inability to carry out in vitro culture, the research on their genetic transformation and molecular biology is seriously lagging behind, and the promoters of obligate parasites have not been reported so far.

Method used

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  • Promoter WY195 and application thereof
  • Promoter WY195 and application thereof
  • Promoter WY195 and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Embodiment 1: PCR amplification of WY195 promoter fragment

[0037]Use the fungal genome DNA extraction kit (OMEGA, D3390-01) to extract the genome DNA of powdery mildew fungi, according to the sequence of WY195 promoter, design a pair of specific amplification primers (upstream primer WY195F, plus restriction enzyme cutting site HindⅢ and protection bases, downstream primer WY195R, plus restriction site BamHI and protection bases). Using the above-mentioned extracted Genomic DNA of Powdery mildew Hevea as a template, high-fidelity Ex Taq polymerase (TRANSGEN, `AP122) was used for PCR amplification. As shown in Table 1.

[0038] Table 1 PCR system for gene promoter amplification

[0039]

[0040] The PCR amplification program was as follows: pre-denaturation at 94°C for 5 min, then denaturation at 94°C for 60 s, annealing at 55°C for 50 s, extension at 72°C for 60 s, 35 reaction cycles, and finally extension at 72°C for 5 min.

[0041] Among them, the upstream pri...

Embodiment 2

[0044] Embodiment 2: Construction of pGEM-T easy-WY195 recombinant vector

[0045] The PCR amplification product obtained above was transformed into Escherichia coli by T / A cloning (pGEM-T easy plasmid, PROMEGA, A1360), and positive clones were picked and sequenced, which proved to be accurate.

[0046] Among them, the connection conditions of T / A clone are as follows:

[0047] T / A connection system: 10ul

[0048] pGEM-T EasyVector (PROMEGA, A137A): 1ul

[0049] 2×Rapid ligation Buffer: 5ul

[0050] PCR amplification product (recovered insert): 2ul

[0051] T4DNAligase: 1ul

[0052] wxya 2 O: 1ul

[0053] First place at room temperature for 1 hour, then ligate overnight at 4°C to obtain the pGEM-T easy-WY195 recombinant vector. The product after the above connection was transformed into Escherichia coli as follows:

[0054] Take out 100 μl of DH5α (Transgene, CD201) competent cells prepared according to the calcium chloride method shown in "Molecular Cloning Experiment...

Embodiment 3

[0056] Embodiment 3: Construction of PBI121-WY195 recombinant vector

[0057] Pick a single colony of the DH5α-WY195 strain obtained from the above construction and shake it overnight at 220 rpm at 37°C. Extract the plasmid with OMEGA plasmid mini-extraction kit (D6943-01), and then use Hind Ⅲ (NEB, R0104S) and BamHI (NEB, R0136V) restriction endonuclease was used for double digestion, and the digested product was recovered with the OMEGA recovery kit (D2500-01) to recover the WY195 promoter fragment.

[0058] The recovered product obtained above was transformed into Escherichia coli by T / A cloning (PBI121 plasmid, TIANNZ, 60908-750y), and positive clones were picked and sequenced, which proved to be accurate.

[0059] Among them, the connection conditions of T / A clone are as follows:

[0060] T / A connection system: 10ul

[0061] PBI121Vector: 1ul

[0062] 10×T4 DNA Ligase Buffer: 1ul

[0063] Recovered product (WY195 promoter fragment): 6ul

[0064] T4DNALigase (TaKaRa, ...

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Abstract

The invention relates to a promoter derived from powdery mildew of rubber trees and application of the promoter. The invention provides a novel promoter derived from powdery mildew of rubber trees, and the promoter has a nucleotide sequence as shown in SEQ ID NO:1, or a variant with functions of the promoter. The invention further relates to a nucleic acid structure, a vector, a recombinant cell,a transgenic plant, explants and calluses with the promoter. The invention further relates to application of the promoter to regulation and control of target gene expression in fungi. The promoter canbe used for regulating and controlling expression of exogenous target genes in dicotyledon and monocotyledon, and a completely novel tool and option are provided for gene expression of transgenic plants.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and in particular relates to a promoter WY195 derived from powdery mildew of rubber tree and its application. Background technique [0002] A promoter is a DNA sequence in a gene that can bind to RNA polymerase and other transcription factors to accurately initiate transcription, and is usually located in the upstream region of the 5' end of the structural gene. The promoter can guide the RNA polymerase to bind correctly to the DNA template, activate the RNA polymerase, and form a specific transcription initiation complex with the corresponding transcription factors, thereby determining the direction and efficiency of transcription. It is the key to understanding the transcriptional regulation mechanism and expression mode The essential. It can be said that the promoter is the regulation center of transcription. [0003] According to the transcription mode and function, promoters ca...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/113C12N15/70C12N15/74C12N15/85A01H5/00
CPCC07K14/37C12N15/8205
Inventor 缪卫国王义刘文波郑服丛
Owner HAINAN UNIVERSITY
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