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Wheat salt-resistant and oxidation-resistant gene TaFLS and application thereof

An anti-oxidation and genetic technology, applied in the fields of application, genetic engineering, plant genetic improvement, etc., can solve the problems of poor knowledge of secondary metabolism and achieve the effect of improving antioxidant capacity

Inactive Publication Date: 2012-11-07
SHANDONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Over the years, a large number of studies have clarified the response mechanism of plants to salt stress at the level of DNA, RNA and protein, but still know little about the level of secondary metabolism, especially the relationship between flavonoid metabolism and plant stress tolerance is still unclear. Therefore, it is necessary to explore the mechanism of action of flavonoids in plant salt stress response

Method used

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  • Wheat salt-resistant and oxidation-resistant gene TaFLS and application thereof
  • Wheat salt-resistant and oxidation-resistant gene TaFLS and application thereof
  • Wheat salt-resistant and oxidation-resistant gene TaFLS and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] Cloning of embodiment 1, TaFLS

[0042] 1.1 Extraction of wheat Total RNA

[0043] 1. Put the tissue material into a liquid nitrogen pre-cooled mortar, and fully grind it into powder in liquid nitrogen;

[0044] 2. After the liquid nitrogen is evaporated to dryness, transfer it to a 2ml centrifuge tube immediately, add about 1ml of Invitrogen’s TRIzol extract for every 100mg of material, after melting, repeatedly suck and blow with a sample gun, shake and mix the sample vigorously, and make the sample Fully lyse and place at room temperature for 5 minutes;

[0045] 3. Add 0.2ml of chloroform (chloroform), vigorously shake and mix for 15 seconds, and place at room temperature for 10 minutes;

[0046] 4.4°C, centrifuge at 12000rpm for 15 minutes;

[0047]5. Use a pipette to carefully suck out the upper aqueous phase, add it to a new 1.5ml centrifuge tube, add 500 μl of isopropanol (1:1 volume), mix well, and settling at -20°C for 30 minutes or overnight;

[0048] 6.4°...

Embodiment 2

[0121] Embodiment 2, prokaryotic expression analysis

[0122] 2.1 Construction of prokaryotic expression vector

[0123] The expression vector used was Pet32a, and the recipient strain was DE3. Hind III and EcoR I were used to perform double enzyme digestion on pET32a and the pMD18-T vector containing the target gene, respectively, and recover the large fragment of the vector and the small fragment of the target gene, and transform Escherichia coli DH10B competent cells after ligation with T4 DNA ligase. After the recombination, the prokaryotic expression vector with the target gene is obtained, and then the recipient strain DE3 is transformed to be competent for protein expression.

[0124] 1. Enzyme digestion of PET32a and pMD18-T

[0125] The plasmids of pET32a and pMD18-T were extracted by alkaline lysis method, and 10ul of each was taken for HindIII and EcoR I double enzyme digestion. The plasmid extraction method is as above, and the plasmid double enzyme digestion sy...

Embodiment 3

[0194] Embodiment 3, the construction of vector and transformation Agrobacterium

[0195] 3.1 Construction of 35S promoter plant expression vector

[0196] Plant expression vector pSTART is a binary vector containing 35S promoter and NPT II gene, and contains restriction endonuclease XbaI and BamHI sites in its multiple cloning site. Respectively use restriction endonucleases XbaI and BamHI to double digest the vector pSTART and the target gene fragment. The fully digested vector is separated by electrophoresis on 1% agarose gel, recovered by gel, and then connected with the double digested cDNA fragment to construct a plant expression vector. Enzyme digestion system, transform Escherichia coli DH10B competent, and identify recombinants. The connection, recovery, conversion and identification methods are the same as above. see results Figure 4 .

[0197] 3.2 Preparation of Competent Agrobacterium AGL1 / EHA105

[0198] (1) Pick a single colony of Agrobacterium tumefaciens...

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Abstract

The invention discloses a wheat salt-resistant gene, i.e., a wheat secondary metabolism flavone synthetic route gene TaFLS and a plant expression vector pSTART-TaFLS containing the gene TaFLS. The invention further discloses application of an expression vector of the gene to the cultivation of salt-resistant and oxidation-resistant plants. As proved by an experiment, the salt resistance and the oxidation resistance of a transgenic plant are remarkably enhanced.

Description

technical field [0001] The invention belongs to the technical field of biological genetic engineering, and in particular relates to a wheat salt-tolerant and anti-oxidation gene, that is, a wheat secondary metabolism flavonoid synthesis pathway gene TaFLS and an application thereof. Background technique [0002] Soil salinization seriously affects crop yields. The development of industry has made soil salinization more and more serious, which has become a global problem. my country's soil salinization is more serious, the population is large, and the food crisis is particularly prominent. Therefore, cultivating new varieties of salt-tolerant crops has become a very urgent task in our country. [0003] It is a technology with broad application prospects to use transgenic improved plant technology to transfer new traits into crops, so as to develop new high-efficiency transgenic plant varieties and use them for planting in saline-alkali land. [0004] At present, great prog...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/29
Inventor 夏光敏李伟张凌霄
Owner SHANDONG UNIV
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