Oidium heveae Steinmann in-vivo promoter WY51 and application thereof

A technology of rubber tree powdery mildew and endogenous promoter is applied in the field of genetic engineering and can solve problems such as strengthening

Active Publication Date: 2018-08-07
HAINAN UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Powdery mildew of rubber tree is an obligate parasitic fungus, and its genetic transformation system has not yet been established. A strong endogenous promoter has been found to strengthen the related research on the

Method used

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  • Oidium heveae Steinmann in-vivo promoter WY51 and application thereof
  • Oidium heveae Steinmann in-vivo promoter WY51 and application thereof
  • Oidium heveae Steinmann in-vivo promoter WY51 and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] Embodiment 1, PCR amplification of WY51 promoter fragment

[0035] Use the fungal genomic DNA extraction kit (OMEGA, D3390-01) to extract the genomic DNA of Powdery mildew Hevea (provided by the Key Laboratory of Sustainable Utilization of Tropical Biological Resources in Hainan Province), and design a pair of specific amplification according to the sequence of the WY51 promoter Primers (upstream primer WY51F, plus restriction site HindⅢ and protective bases, downstream primer WY51R, plus restriction site BamHI and protective bases). Using the extracted genomic DNA of Erysiphaea hevea as a template, high-fidelity Ex Taq polymerase (TRANSGEN, AP122) was used for PCR amplification. As shown in Table 1.

[0036] Table 1 PCR system for gene promoter amplification

[0037]

[0038] 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 cycle...

Embodiment 2

[0041] Embodiment two, the construction of pGEM-T easy-WY51 recombinant vector

[0042] The PCR amplification product obtained above was transformed into Escherichia coli (TRANSGEN, CD201) by T / A cloning (pGEM-T easy plasmid, PROMEGA, A1360), and positive clones were picked and sequenced, which proved to be accurate.

[0043] Among them, the connection conditions of T / A clone are as follows:

[0044]T / A connection system: 10ul

[0045] pGEM-T Easy Vector (PROMEGA, A137A): 1ul

[0046] 2×Rapid ligation Buffer: 5ul

[0047] PCR amplification product (recovered insert): 2ul

[0048] T4DNA ligase: 1ul

[0049] wxya 2 O: 1ul

[0050] First place at room temperature for 1 hour, then ligate overnight at 4°C to obtain pGEM-T easy-WY51 recombinant vector. The product after the above connection was transformed into Escherichia coli as follows:

[0051] Take out 100 μl of DH5α (Transgene, CD201) competent cells prepared according to the calcium chloride method shown in "Molecular...

Embodiment 3

[0053] Example 3, Construction of PBI121-WY51 recombinant vector

[0054] Pick a single colony of the DH5α-WY51 strain obtained from the above construction and shake the bacteria overnight at 37°C at 220 rpm. The plasmid is extracted with the OMEGA plasmid mini-extraction kit (D6943-01), and then HindⅢ (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 WY51 promoter fragment.

[0055] The recovered product obtained above was ligated with the PBI121 plasmid (TIANNZ, 60908-750y), and then transformed into Escherichia coli, and positive clones were picked and sequenced, which proved to be accurate.

[0056] Among them, the connection conditions of T / A clone are as follows:

[0057] T / A connection system: 10ul

[0058] PBI121Vector: 1ul

[0059] 10×T4DNA Ligase Buffer: 1ul

[0060] Recovered product (WY51 promoter fragment): 6ul

[0061] T4D...

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Abstract

The invention discloses an oidium heveae Steinmann in-vivo promoter WY51 and application thereof. The promoter WY51 has a nucleotide sequence as shown in SEQ ID NO: 1, or is provided with a variant with functions of the promoter. The invention further relates to a nucleic acid builder, a carrier, reconstitution cells, a transgenic plant, an explant and a callus which comprises the promoter. The promoter can be used for regulating and controlling expression of exogenous genes in dicotyledon and monocotyledon, and a brand-new tool and selection are provided for expression of genes of the transgenic plant.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and in particular relates to endogenous promoter WY51 of powdery mildew of rubber tree and its application. Background technique [0002] A promoter is a DNA sequence that initiates gene transcription and can be recognized by RNA polymerase, binds and effectively initiates the transcription of downstream sequences, and is a key point in the regulation of gene expression. According to the characteristics of the promoter regulation mode, it can be roughly divided into three categories: constitutive promoters, tissue or organ-specific promoters, and inducible promoters. In some cases, one type of promoter often has the characteristics of other types of promoters. [0003] The expression of constitutive promoters is not regulated by the external environment and has no tissue specificity. It is expressed in almost all organs and tissues, and the expression is generally continuous and does...

Claims

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Application Information

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IPC IPC(8): C12N15/113C12N15/11C12N1/21C12N5/04A01H5/00A01H6/82
CPCC07K14/37C12N15/8222
Inventor 缪卫国王义刘文波郑服丛
Owner HAINAN UNIVERSITY
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