Wheat salt-tolerant and drought-resistant gene TaWRKY80 and application thereof

A technology of salt tolerance and genetics, applied in the field of genetic engineering, to achieve the effect of improving drought resistance

Inactive Publication Date: 2012-10-03
UNIV OF JINAN
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the research on wheat WRKY transcription factors in salt tolerance is still very limited. Niu et al showed that overexp

Method used

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  • Wheat salt-tolerant and drought-resistant gene TaWRKY80 and application thereof
  • Wheat salt-tolerant and drought-resistant gene TaWRKY80 and application thereof
  • Wheat salt-tolerant and drought-resistant gene TaWRKY80 and application thereof

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0044] Embodiment 1, TaWRKY80 Cloning of gene cDNA sequence

[0045] 1. Primer design

[0046] The gene-specific downstream primer Wrky2: 5′-CTTAGGGAATTTTGCCACACTCTTTG-3′ was designed according to the sequence of the chip probe, and paired with the 5′-end anchor primer NT3: 5′-ACTAAAGggaACAAAAGCTGG-3′ of the library to obtain the whole wheat seedling root The long cDNA library is the full-length cDNA of the template amplified gene.

[0047] 2. PCR reaction system (50μL) and program

[0048] 2×GC bufferⅠ 25μl

[0049] Template cDNA library 1ul

[0050] dNTPs (10mM each) 1μl

[0051] Primer1 (10μM) 1μl

[0052] Primer2 (10μM) 1μl

[0053] LA Taq (TaKaRa) 0.5ul

[0054] wxya 2 O was added to a final volume of 50 μl

[0055] Pre-denaturation at 94°C for 3min; denaturation at 94°C for 45sec, renaturation at 58°C for 1min, extension at 72°C for 2min, cycle 35 times; extension at 72°C for 5min.

[0056] 3.1% agarose gel electrophoresis

[0057] After the PCR amplificati...

Embodiment 2

[0070] Embodiment 2, under different treatment conditions TaWRKY80 Gene Expression Analysis

[0071] 1. material handling

[0072] The seeds of Wheat Shanrong 3 germinated at room temperature, removed the endosperm after 1 week, and continued to culture in Hangload liquid medium for 1 week for stress treatment.

[0073] Salt stress: Add NaCl to the Hangload liquid medium to a final concentration of 200mM;

[0074] ABA treatment: add ABA to the medium to a final concentration of 100 μM;

[0075] After 0, 0.5, 3, 12, 24, and 48 hours of treatment under different conditions, the leaves and roots of the seedlings were taken respectively, and the total RNA of each material was extracted.

[0076] 2. Wheat Total RNA Extraction

[0077] RNA was extracted by Trizol method. The quality of the extracted RNA was detected by 1% Agrose gel electrophoresis, and the concentration of the extracted RNA was detected by an ultraviolet spectrophotometer.

[0078] 3. Synthesis of first...

Embodiment 3

[0098] Embodiment 3, TaWRKY80 Construction of overexpression plant expression vector

[0099] The plant expression vector pCAMBIA-super1300 is a binary vector containing 35S promoter and NPTⅡ gene, and contains restriction enzymes in its multiple cloning site Xba I site. According to genes TaWRKY80 The gene-specific primers containing the complete ORF were designed W2ORF-1: 5'-TCTAGAATGGATCCATGGGTCAGCAGC-3' (xbaI), W2ORF-2: 5'-GAGCTCCCTACCTAACTTGATCAAACTTGC-3' (SacI). Amplified with the pair of primers TaWRKY80 cDNA sequence. then restriction endonuclease Xba I, SacI digestion vector pCAMBIA-super1300 and amplified target gene TaWRKY80 sequence. The completely digested vector and target gene were separated by 1% agarose gel electrophoresis, recovered, ligated, transformed into Escherichia coli DH5a, positive clones were detected by PCR, plasmids of positive clones were extracted, verified by enzyme digestion, and the plant expression vector pCAMBIA-super1300 was con...

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Abstract

The invention belongs to the technical field of genetic engineering, and relates to the cloning of a wheat salt-tolerant and drought-resistant WRKY transcription factor gene TaWRKY80 and application thereof. The invention discloses a wheat salt-tolerant WRKY transcription factor gene TaWRKY80 and application thereof in cultivating salt-tolerant plants. Experiments prove that: the salt tolerance of the transgenic plants is improved. The gene provided by the invention plays an important role in cultivating salt-tolerant plants (especially crops).

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and relates to a wheat salt-tolerant and drought-resistant WRKY transcription factor gene TaWRKY80 cloning and its application. Background technique [0002] In recent years, soil salinization and water shortage have become a global problem restricting agricultural production. Salt damage and drought not only seriously affect the growth and development of plants and cause crop yield reduction, but also worsen the ecological environment (Saibo et al., Annals of Botany, 2009, 103 (4):609-623). Therefore, improving the drought resistance and / or salt tolerance of crops has become one of the key issues to be urgently solved in modern crop breeding. Isolating and cloning salt-tolerant genes and cultivating new salt-tolerant varieties have become one of the research hotspots all over the world. Wheat is an important crop, cloning salt-tolerant and drought-resistant genes, and breeding new...

Claims

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Application Information

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IPC IPC(8): C12N15/29C07K14/415C12N15/84A01H5/00
Inventor 秦余香田延臣
Owner UNIV OF JINAN
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