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Application of protein and encoding gene of protein in regulation of verticillium wilt resistance of plants

A technology for transgenic plants and proteins is applied in the application field of proteins and their encoded genes in regulating the resistance of plants to Verticillium wilt, and can solve the problems of lack of effective resistance sources and difficulty in achieving ideal effects.

Active Publication Date: 2017-02-15
INST OF BOTANY CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Due to the lack of effective resistance sources and other problems, it is difficult to achieve the desired effect only by a single conventional traditional breeding method

Method used

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  • Application of protein and encoding gene of protein in regulation of verticillium wilt resistance of plants
  • Application of protein and encoding gene of protein in regulation of verticillium wilt resistance of plants
  • Application of protein and encoding gene of protein in regulation of verticillium wilt resistance of plants

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0100] Embodiment 1, the cloning of the coding gene of protein GhBZR1 and the coding gene of GhBZR1 mutein

[0101] 1. Cloning of the gene encoding the protein GhBZR1

[0102] The inventors of the present invention cloned the gene encoding the protein GhBZR1, namely the GhBZR1 gene, from upland cotton TM-1.

[0103] 1. The total RNA of the leaves of upland cotton TM-1 seedlings grown to 14 days was extracted by the Trizo1 method, and then the first-strand cDNA was reverse-transcribed by reverse transcriptase AMV.

[0104] 2. Artificially synthesized primers F: 5'-CACCATGACGTCAGATGGGGCGACGT-3' and R: 5'-ACATCGAGCTTTCCCACTTCCG-3'.

[0105] 3. After completing steps 1 and 2, use the cDNA extracted in step 1 as a template and use F and R as primers for PCR amplification to obtain a double-stranded DNA molecule of about 940 bp.

[0106] 4. Ligate the double-stranded DNA molecule with the vector pENTR / SD / D-TOPO to obtain the recombinant plasmid pGW-GhBZR1.

[0107] According to t...

Embodiment 2

[0128] Embodiment 2, the acquisition and identification of transgenic Arabidopsis

[0129] 1. Construction of recombinant vector and recombinant Agrobacterium

[0130] 1. Acquisition of recombinant plasmids 35S::GhBZR1-GFP and GV3101 / 35S::GhBZR1-GFP

[0131] The recombinant plasmid pGW-GhBZR1 constructed in Step 1 of Example 1 and the vector pMDC83 were subjected to LR recombination reaction to obtain an LR reaction product.

[0132] The LR reaction product was added to TOP10 competent cells for transformation, and the clone obtained was the target clone, and the plasmid in the target clone was the target plasmid, and the target plasmid was named recombinant plasmid 35S::GhBZR1-GFP. Sequencing results showed that the recombinant plasmid 35S::GhBZR1-GFP contained the DNA molecule shown in the 1st to 939th positions from the 5' end of the sequence 1 in the sequence listing, and expressed the protein GhBZR1 shown in the sequence 2 in the sequence listing.

[0133] The recombina...

Embodiment 3

[0187] Embodiment 3, acquisition and identification of cotton silent strain

[0188] 1. Construction of recombinant plasmid PTRV2-GhBZR1 and acquisition of recombinant Agrobacterium

[0189] 1. The total RNA of the leaves of upland cotton TM-1 seedlings grown to 14 days was extracted by the Trizo1 method, and then the first-strand cDNA was reverse-transcribed by reverse transcriptase AMV.

[0190] 2. Artificially synthesized primers F1: 5'-CGACGACAAGACCCTCACCACTTATCGAAAGGG-3' and R1: 5'-GAGGAGAAGAGCCCTATTTCTGGAGGTTGGAG-3'.

[0191] 3. After completing steps 1 and 2, use the cDNA extracted in step 1 as a template, and use F1 and R1 as primers to carry out PCR amplification, and then purify and recover to obtain a double-stranded DNA molecule of about 300 bp.

[0192] 4. Place the double-stranded DNA molecule obtained in step 3 in T4 DNA synthetase buffer containing dATP, treat at 22°C for 30 minutes, and then place it at 70°C for 20 minutes to obtain Fragment A.

[0193] 5. T...

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Abstract

The invention discloses an application of protein and an encoding gene of the protein in regulation of verticillium wilt resistance of plants. The protein comprises an N-terminal component and a C-terminal component from an N terminal to a C terminal sequentially, wherein the amino acid sequence of the N-terminal component is shown as the first position to the 158th position from the N terminal in the sequence 2 in a sequence table; the amino acid sequence of the C-terminal component is shown as the 166th position to the 313th position from the N terminal in the sequence 2 in the sequence table. Experiments prove that transgenic plants with increased petiole length and increased verticillium wilt resistance are obtained by introducing the encoding gene of the protein into arabidopsis thaliana; transgenic plants with reduced plant height, compact plant type, reduced cotton fiber length and reduced verticillium wilt resistance are obtained by introducing protein expression inhibited substances into Xuzhou142 or Gossypium hirsutum TM-1. Therefore, the protein has great theoretical significance and practical value in culture of the plants with verticillium wilt resistance.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to the application of the protein and its coding gene in regulating the resistance of plants to verticillium wilt. Background technique [0002] Verticillium wilt is one of the important diseases that endanger cotton production at present. It is called the "cancer" of cotton and spreads all over the cotton-producing areas in the world. Its pathogen is Verticillium dahliae. Cotton verticillium wilt is a soil-borne fungal disease that infects along the vascular system, seriously endangering the growth and development of cotton, resulting in greatly reduced cotton fiber yield and quality, causing huge economic losses to cotton production, and seriously affecting the cotton industry. sustainable development. [0003] Cotton is an important fiber crop in the world, and it is also an important oil and bioenergy crop, which plays an important role in national economic and social development. ...

Claims

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Application Information

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IPC IPC(8): C07K14/415C12N15/29C12N15/82A01H5/00
CPCC07K14/415C12N15/8261C12N15/8282
Inventor 朱生伟蒙福宁罗小敏吴金霞张志国
Owner INST OF BOTANY CHINESE ACAD OF SCI
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