Endogenous promoter WY7 of powdery mildew of rubber tree and application of endogenous promoter WY7

A technology of rubber tree powdery mildew and endogenous promoter, which is applied in the field of genetic engineering and can solve the problems such as the genetic transformation system has not been established yet.

Inactive Publication Date: 2018-05-01
HAINAN UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Its genetic transformation system has not yet been established

Method used

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  • Endogenous promoter WY7 of powdery mildew of rubber tree and application of endogenous promoter WY7
  • Endogenous promoter WY7 of powdery mildew of rubber tree and application of endogenous promoter WY7
  • Endogenous promoter WY7 of powdery mildew of rubber tree and application of endogenous promoter WY7

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Embodiment 1, PCR amplification of WY7 promoter fragment

[0037] Using 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 amplifications according to the sequence of the WY7 promoter Primers (upstream primer WY7F, plus restriction enzyme site HindⅢ and protection base, downstream primer WY7R, plus restriction enzyme site BamHI and protection base). Using the above-mentioned extracted Genomic DNA of Erysipha spp. 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...

Embodiment 2

[0043] Embodiment two, the construction of pGEM-T easy-WY7 recombinant vector

[0044] 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.

[0045] Among them, the connection conditions of T / A clone are as follows:

[0046] T / A connection system: 10ul

[0047] pGEM-T Easy Vector (PROMEGA, A137A): 1ul

[0048] 2×Rapid ligation Buffer: 5ul

[0049] PCR amplification product (recovered insert): 2ul

[0050] T4DNA ligase: 1ul

[0051] wxya 2 O: 1ul

[0052] First place at room temperature for 1 hour, then ligate overnight at 4°C to obtain pGEM-T easy-WY7 recombinant vector. The product after the above connection was transformed into Escherichia coli as follows:

[0053] Take out 100 μl of DH5α (Transgene, CD201) competent cells prepared according to the calcium chloride method shown in "Molecular ...

Embodiment 3

[0055] Example 3, Construction of PBI121-WY7 recombinant vector

[0056] Pick a single colony from the DH5α-WY7 strain obtained above and shake it overnight at 37°C at 220 rpm. Use the OMEGA plasmid mini-extraction kit (D6943-01) to extract the plasmid, and then use 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 WY7 promoter fragment.

[0057] 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.

[0058] Among them, the connection conditions of T / A clone are as follows:

[0059] T / A connection system: 10ul

[0060] PBI121Vector: 1ul

[0061] 10×T4DNA Ligase Buffer: 1ul

[0062] Recovered product (WY7 promoter fragment): 6ul

[0063] T4DNA Ligase (Ta Ka Ra, D2011A): 0.5...

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Abstract

The invention discloses an endogenous promoter WY7 of powdery mildew of a rubber tree and application of the endogenous promoter WY7. The promoter WY7 has a nucleotide sequence as shown in the SEQ IDNO:1, or the promoter has a variant having the function of the promoter. The invention further relates to a nucleic acid construction body comprising the promoter, a carrier, a reconstitution cell, atransgenic plant, an explant and a callus. The promoter is used for regulating the expression of exogenous target genes in the dicotyledon and the monocotyledon, and provides a brand new tool and choice for the 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 WY7 of powdery mildew of rubber tree and its application. Background technique [0002] The promoter is an important cis-acting element of gene expression, located upstream of the 5' end of the structural gene, it can activate RNA polymerase and make it accurately combine with the template DNA, and has the specificity of transcription initiation. The promoter can provide a binding site specifically recognized by RNA polymerase, and can control gene transcription initiation and gene expression. It is usually located in the nucleotide sequence proximal to the gene transcription initiation site, mainly composed of the core promoter region, upstream components and response components. [0003] According to the products encoded by eukaryotic genes and the type of RNA polymerase, promoters can be divided into three types, type I promoters ar...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/113C12N15/11C12N1/21C12N5/04A01H5/00A01H6/82
CPCC07K14/37C12N15/8222
Inventor 缪卫国王义徐良向殷金瑶刘文波郑服丛
Owner HAINAN UNIVERSITY
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