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Plant resistance-related protein ATWRKY46 as well as encoding gene and application thereof

A related protein and gene technology, applied in the direction of plant genetic improvement, botanical equipment and methods, application, etc., can solve the problems of increasing salicylic acid, not yet found, not found, etc., to achieve the effect of enhancing disease resistance

Active Publication Date: 2012-02-08
FUJIAN SANAN SINO SCI PHOTOBIOTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

So far, it has only been found that ETHYLENEINSENSITIVE3 (EIN3) and EIN3-LIKE1 can directly bind to the ICS1 promoter of the key SA biosynthesis gene, and negatively regulate the synthesis level of SA. It has not been found that ICS1 gene expression can be promoted by positively inducing the ICS1 promoter. proteins that increase the level of salicylic acid synthesis
In addition, studies have found that SAR Deficient 1 (SARD1) and CBP60g proteins are important factors in inducing ICS1 gene expression and SA synthesis, but failed to find more positive induction of ICS1 promoter to promote ICS1 gene expression, thereby increasing the level of salicylic acid synthesis protein

Method used

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  • Plant resistance-related protein ATWRKY46 as well as encoding gene and application thereof
  • Plant resistance-related protein ATWRKY46 as well as encoding gene and application thereof
  • Plant resistance-related protein ATWRKY46 as well as encoding gene and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0061] Example 1. Obtaining ATWRKY46 protein and its coding gene

[0062] 1. According to the existing NCBI database and literature, set a pair of primers as follows:

[0063] F1 (forward primer): 5’- TCTAGA CACATCTCCCACCAATCTCA-3' (underlined XbaI restriction recognition sequence);

[0064] R1 (reverse primer): 5'- CTCGAG TCGACCACAACCAATCCTGTC-3' (XhoI digestion recognition sequence is underlined).

[0065] 2. Extract the genomic DNA of Colombian ecotype Arabidopsis leaves.

[0066] 3. Using genomic DNA as a template, using the primer pair designed in step 1, PCR amplification was performed with TaKaRa's PrimeSTAR HS high-fidelity enzyme.

[0067] 4. Sequencing the PCR amplified products, as shown in sequence 2 of the sequence list.

[0068] The protein shown in sequence 1 of the sequence listing was named ATWRKY46 protein. The gene encoding the ATWRKY46 protein is named ATWRKY46 gene, and its genomic DNA is shown in sequence 2 of the sequence table (from the 73rd to 75th nucleotides ...

Embodiment 2

[0069] Example 2. Cloning of each gene and construction of its recombinant expression vector

[0070] 1. Acquisition of ATWRKY46 gene and construction of recombinant plasmid 326-ATWRKY46-FLAG

[0071] 1. Extract the genomic DNA of Colombian ecotype Arabidopsis leaves.

[0072] 2. Using the genomic DNA of step 1 as a template, use a primer pair composed of F1 and R1 to perform PCR amplification under the action of PrimeSTAR HS high-fidelity enzyme from TaKaRa Company to obtain PCR amplification products. See the agarose gel electrophoresis diagram of PCR amplified products figure 1 (M stands for the marker of nucleotides, DL2000 from TaKaRa).

[0073] 3. Double-cut the PCR amplified product of step 2 with restriction enzymes XbaI and XhoI, and recover the digested product.

[0074] 4. Double digestion of the 326-FLAG expression vector with restriction enzymes XbaI and XhoI to recover the vector backbone of about 3827bp.

[0075] 5. Connect the digested product of step 3 and the vector b...

Embodiment 3

[0096] Example 3. Application of ATWRKY46 gene in inducing ICS1 gene promoter (ProAtICS1) to initiate gene expression

[0097] 1. Transient expression of recombinant plasmid in Arabidopsis leaf protoplasts

[0098] The recombinant plasmid 326-ATWRKY46-FLAG constructed in Example 2 and the recombinant plasmid 326-Pro AtICS1 ::GFP co-transforms Arabidopsis thaliana protoplasts (the recombinant plasmid 326-T 7 -FLC is used as a negative control of recombinant plasmid 326-ATWRKY46-FLAG; the 326-FLAG expression vector is used as another negative control of recombinant plasmid 326-ATWRKY46-FLAG), the specific steps are as follows:

[0099] 1. Germinate Colombian ecotype Arabidopsis seeds on MS medium, transplant them into the soil when the roots grow to 1-3 cm, and cultivate them in a greenhouse at 23°C (12h light per day, 150μE light intensity).

[0100] 2. Add 20ml of double distilled water to a 90mm petri dish, then add 1.82g of D-mannitol and dissolve it.

[0101] 3. Take the leaves (appr...

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Abstract

The invention discloses a plant resistance-related protein ATWRKY46 as well as an encoding gene and application thereof. The protein provided by the invention is named ATWRKY46 protein, which is from Arabidopsis thaliana and is any one of the following: (a) the protein formed by amino acid sequences as shown in a sequence 1; and (b) the protein which is derived from the sequence 1 in a way that the amino acid sequences as shown in the sequence 1 are subjected to replacement and / or deletion and / or addition of one or a plurality of amino acid residues, and has any function of the following: inducing a starting function of an ICS1 gene promoter, and inducing a starting function of a PR1 (pathogenesis-related protein) gene promoter, and the protein which is related to synthesis of salicylic acid in a plant and plant resistance and is derived from the sequence 1. The ATWRKY46 protein provided by the invention can increase disease resistance of the plant through regulating and controlling the synthesis of the salicylic acid in the plant. The invention is of great value to cultivate disease-resistant plants.

Description

Technical field [0001] The invention relates to a plant resistance-related protein ATWRKY46 and its coding gene and application. Background technique [0002] Various diseases often occur during the cultivation and production of plants. Some diseases pose a great threat to plant growth and severely damage the yield and quality of crops. For plant diseases, drug measures are currently used for prevention and control. Recently, people began to use non-biological inducers to induce plant disease resistance, and they have been widely used in tobacco, potatoes, tomatoes, cucumbers, kidney beans, rice and other important crops. Although the effect of this measure is ideal, it is costly and pollutes the environment. Therefore, there is an urgent need to develop new biological methods to improve plant disease resistance. [0003] Systemic acquired resistance is a defensive response of plants. When the plant is attacked by pathogens and pests, this response will be induced and can quickl...

Claims

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Application Information

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IPC IPC(8): C07K14/415C12N15/29C12N15/63C12N5/10C12N1/21C12N15/82A01H5/00
Inventor 金京波蔡斌张强
Owner FUJIAN SANAN SINO SCI PHOTOBIOTECH CO LTD
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