Transcription factor zinc finger protein gene ZxZF and application thereof

A technology of zinc finger proteins and transcription factors, which is used in applications, genetic engineering, plant genetic improvement, etc.

Inactive Publication Date: 2008-01-09
INST OF FORESTRY CHINESE ACAD OF FORESTRY +2
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Some of these domains can be divided into several subclasses according to the number and position of conserved amino acid residues in their characteristic regions, for example, zinc finger domains [viii] It generally consists of 20-30 amino acid residues, including two conservative cysteine ​​(C) and histidine (H) residues, which bind to zinc ions in the quaternary structure, according to cysteine ​​( C) and the number and position of histidine (H) residues, the transcription factors containing zinc finger domains can be divided into C2H2, C3H, C2C2, C3HC4, C2HC5 subclasses, the zinc finger protein genes involved in the present invention belong to Subclass C2H2, genes encoding this zinc finger protein from other plants have not yet been retrieved

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0012] Example 1. Cloning and sequence structure analysis of Bawang ZxZF gene

[0013] The 3-year-old Bawang was used as the material, and after being induced by drought, the leaves were taken and RNA was extracted. The RNA extraction was carried out according to the method of RNeasyPlant Mini Kit (Cat. NO: 74904. Qiagen, Hilden, Germany), and the total RNA was added to DNaseI to digest the DNA , and then add chloroform to extract twice, take the supernatant, precipitate the total RNA with isopropanol, air dry, and dissolve in DEPC water.

[0014] Gene isolation using BD SMART TM The method provided by RACE cDNA Amplification Kit (Cat. No. 634914) was carried out with slight modifications.

[0015] 1. Reverse transcription to generate first strand

[0016] reverse transcription primer sequence

[0017] CDS5 (for 5′RACE): 5′-(T)25 V N-3′

[0018] CDS3 (for 3′RACE): 5′-AAGCAGTGGTATCAACGCAGAGTAC(T) 30 V N-3′

[0019] (N=A, C, G, or T; V=A, G, or C)

[0020] The reverse t...

Embodiment 2

[0060] Embodiment 2, the construction of the plant expression vector that is used for transformation

[0061] The construction of the ZxZF plant expression vector was carried out by conventional molecular biology methods (see "Refined Molecular Biology Experiment Guide" 2001, Science Press, translated by Yan Ziying and Wang Hailin). Using the cDNA of the ZxZF coding region as a template, use the above forward and reverse primers for PCR amplification, and insert it into the pBl121 binary expression vector by introducing the XbaI site in the forward primer and the Sac I site in the reverse primer [ix] Behind the CaMV 35S promoter, a binary expression vector was obtained. The map is shown in Figure 6. After digestion with XbaI and SacI and PCR identification of the inserted fragment, it was transformed into Agrobacterium LBA4404 [x] , and then extract the plasmid, digest it, and confirm it by PCR. The expression vector can be directly used for plant transformation.

Embodiment 3

[0062] Example 3, transformation of Arabidopsis and functional verification

[0063] 1. Arabidopsis Culture

[0064] 1) Seeds of Arabidopsis thaliana were sown in organic culture soil, and cultured in a 22°C / 18°C, 16hr / 8hr light / dark growth chamber.

[0065] 2) After one week, the first pair of true leaves grow, and they are transplanted into large pots.

[0066] 3) After 3 to 4 weeks, top off the heart.

[0067] 2. Transformation of Arabidopsis thaliana with ZxZF gene

[0068] 1) Select a single colony of Agrobacterium with the target gene that grows well, raise it in 5ml of YEP containing appropriate antibiotics, and culture it with shaking at 28°C and 220r / min

[0069] 2) The next day, grow the bacteria to 250ml.

[0070] 3) Take an Arabidopsis plant with an inflorescence of about 15 cm, and cut off the fruit pods and bloomed flowers.

[0071] 4) Cut out a suitable size of parafilm and set it on the soil to prevent the soil from falling.

[0072] 5) Centrifuge the bac...

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PUM

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Abstract

A drought-resisting ZxZF gene, its carrier, host cell and use in conversion of plant are disclosed. The gene comes from a kind of plant in desert-Zygophyllumxanthoxylum(Bunge)Maxim.

Description

technical field [0001] The present invention relates to a transcription factor zinc finger protein gene, in particular, the present invention relates to a zinc finger protein gene derived from Zygophyllum xanthoxylum (Bunge) Maxim., a shrub plant that can grow in the desert, named as ZxZF. The invention also relates to the protein encoded by the gene-zinc finger protein, the plant expression vector containing ZxZF, and the plant transformed with the gene. Background technique [0002] Transcription factor, also known as trans-acting factor, is a DNA-binding protein that can specifically interact with cis-acting elements in the promoter region of eukaryotic genes, through the interaction between them and other related proteins function to activate or repress transcription. Because of its ability to regulate the expression of a series of downstream functional genes, the isolation of transcription factor genes is receiving increasing attention [i] . In recent years, a serie...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/29C07K14/415C12N15/74C12N15/82C12N15/70
Inventor 苏晓华吴斌黄秦军张冰玉张香华
Owner INST OF FORESTRY CHINESE ACAD OF FORESTRY
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