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cDNA sequence of dehydrolysis responding transcription factor DREB gene of wheal

A technology of transcription factors and genes, applied in the field of agricultural biological genetic engineering, can solve problems such as difficulty in improving plant stress resistance and difficulty in researching the stress resistance of cultivated wheat

Inactive Publication Date: 2007-10-24
TIANJIN NORMAL UNIVERSITY
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] The stress resistance trait of plants to abiotic stress is a quantitative trait, which is a comprehensive response controlled by multiple genes. It is difficult to improve the stress resistance of plants through single-gene improvement, especially for the resistance of cultivated wheat with complex genome sources. Reverse research is more difficult

Method used

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  • cDNA sequence of dehydrolysis responding transcription factor DREB gene of wheal
  • cDNA sequence of dehydrolysis responding transcription factor DREB gene of wheal
  • cDNA sequence of dehydrolysis responding transcription factor DREB gene of wheal

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] (1) Plant material and treatment:

[0037] The seeds of cultivated wheat Jing 411 were sterilized with 10% antifumin, soaked in deionized water, and then cultured at 23°C in the dark for 6-10 days. The leaves were harvested for DNA extraction. The wheat seedlings of cold-treated samples were subjected to cold stress at 4°C for 2 hours before RNA extraction.

[0038] (2) Extraction of Wheat Jing 411 Genomic DNA

[0039]Grind 0.2g of etiolated seedlings into powder in liquid nitrogen; add 2ml of DNA extraction buffer (500mM NaCl, 100mM Tris HCl, 50mM EDTA), bathe in 65°C water for 15 minutes, ice bath for 10 minutes, centrifuge at 3,000rpm 4°C for 15 minutes , remove tissue pieces; take the supernatant, add an equal volume of phenol and centrifuge at 3,000rpm 4°C for 10 minutes; take the supernatant, add an equal volume of phenol and centrifuge at 3,000rpm 4°C for 10 minutes; Centrifuge for 10 minutes; take the supernatant, add 2 times the volume of cold absolute ethano...

Embodiment 2

[0041] (1) Wheat Jing 411 DREB gene fragment (WtJ 292 ) PCR amplification

[0042] 1. WtJ 292 Fragment PCR amplification

[0043] 1) Design of primers

[0044] Three DREB gene sequences (gi|17148646, gi|17148648, gi|17148650) of published rye were queried on GenBank, and multiple sequences were compared, and the primer design software Primer 5.0 was used to find and synthesize ( All the following primers were entrusted to Shanghai Sangong to synthesize) The primers are as follows:

[0045] Forward1: 5'TGC CTC AAC TTC GCC GAC TCC3' (SEQ ID NO.3)

[0046] Reverse1: 5'CGA GCA TCC CCT GCG CCA AG3' (SEQ ID NO.4)

[0047] 2) PCR amplification reaction system: 10×Buffer 2.5μl, MgCl 2 (25mM) 1.5μl, dNTP (10mM) 0.5μl, Forward1 (10μM) 0.25μl, Reverse1 (10μM) 0.25μl, DNA Template 100ng, Taq (5U / μl) 0.25μl, ddH 2 O make up to 25 μl. PCR parameters: pre-denaturation at 95°C for 2 minutes; denaturation at 95°C for 1 minute, annealing at 60°C for 30 seconds, extension at 72°C for 30 ...

Embodiment 3

[0078] Embodiment 3: the extraction of whole wheat RNA

[0079] The total RNA of wheat was extracted by acidic guanidine isothiocyanate method: Weigh 0.2 g of yellowed wheat seedlings, wash them with sterilized double distilled water, dry them with sterilized filter paper, cut them into pieces in a pre-cooled mortar; add liquid nitrogen and grind until In powder form, add 2ml of Solution D, grind with the powder until clear; (the following steps are operated on ice) pour into a 7ml centrifuge tube, add 200μl 2mol / L NaAC (PH4.0) and invert several times to mix well and place on ice ; Add an equal volume of water-saturated phenol, shake and mix vigorously; add 400 μl chloroform-isoamyl alcohol (49:1), shake and mix vigorously; bathe in ice for 30 minutes, invert several times during the period, it is advisable to avoid stratification; Centrifuge at 11,000rpm at 4°C for 15 minutes; take the supernatant, add an equal volume of phenolform, extract again, and centrifuge at 11,000rpm...

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Abstract

The invention discloses wheat dehydrating response transcription factor DREB gene cDNA sequence. It belongs to agriculture biology gene engineering field. The gene is gained by wheat genome DNA and isogeny cloning method, belongs to wheat dehydrating response factor CBF4. The gene cDNA sequence span is 936bp, has 130bp 5'UTR, 89bp 3'UTR, and 717bp opened reading frame. Its coding frame is the 131-847 nucleotides. One of the coding is long as 238aa polypeptide chain. The invention utilizes Agrobactrium Rhizogenes mediated gene conversion method to form the cloned gene coding sequence in plant expression vector pROK II, and convert to quasi south mustard. The invention starts from cloning transcription factor gene, further practices transgene study, and offers effective method and approach for increasing wheat backward resistance.

Description

Technical field: [0001] The invention belongs to the field of agricultural biological genetic engineering, and relates to a cDNA coding sequence of a dehydration-responsive transcription factor DREB gene in cultivated wheat. technical background: [0002] The discovery of DREB (Dehydration Responsive Element Binding) gene element is the most breakthrough progress in plant stress resistance research in recent years. Liu Qiang et al. (Plant Cell, 1998, 10:1391~1406) isolated five AP2 / EREBP transcription factor genes from Arabidopsis thaliana, belonging to two subfamilies, named DREB1A, DREB1B, DREB1C and DREB2A, respectively. DREB2B. Later, it was found that there is a corresponding relationship with the CBF (C-repeat / DRE-Binding Factor) family isolated by Stockinger et al. (1997). DREB1A is CBF3, DREB1B is CBF1, and DREB1C is CBF2. There is evidence that CBF1, CBF2 and CFB3 are located on chromosome IV of Arabidopsis as a gene cluster, and CBF2 and CBF3 exist at 3Kb and 7Kb...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/29C07K14/415
Inventor 王振英孔照胜王少峡薛志娟彭永康
Owner TIANJIN NORMAL UNIVERSITY
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