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Novel wheat grain lipoxygenase gene and application thereof

A technology of lipoxygenase and wheat grain, applied in the field of plant biology, can solve the problem of less research on wheat, and achieve the effect of simple method, saving time and resources

Active Publication Date: 2018-10-09
HENAN AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] So far, researchers at home and abroad have done a lot of research on Lox activity. The main research crops are rice, soybean and barley, and there are relatively few related studies on wheat.

Method used

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  • Novel wheat grain lipoxygenase gene and application thereof
  • Novel wheat grain lipoxygenase gene and application thereof
  • Novel wheat grain lipoxygenase gene and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] The screening of the specific primer pair of embodiment 1 wheat grain lipoxygenase new gene

[0039]Referring to the NCBI China Spring 3B chromosome sequence (HG670306.1), starting from the nucleotide sequence with the LH2 functional domain (Lipoxygenase homology 2 (beta barrel) domain) on the 3B chromosome, search for the closest start codon and A stop codon defines the open reading frame. Then the new lipoxygenase gene was divided into 6 sections, primers were designed and amplified by primer 3.0.

[0040] Experimental materials: Klein Proteo and Klein Chaja mature grains;

[0041] Primers: as shown in Table 1.

[0042] The specific method for extracting wheat Klein Proteo and Klein Chaja full-maturity grain genomic DNA is as follows:

[0043] (1) Smash it with a hammer and put it into a 1.5mL centrifuge tube, add 1mL sample extract, shake for 30 minutes, and make it fully mixed; the sample extract contains 288mM NaCl, 200mM Tris-HCl, 25mM EDTA, 0.5 % SDS.

[004...

Embodiment 2

[0054] Example 2 Using specific primer pairs (BJF, BJR) to detect lipoxygenase allelic types of wheat varieties / lines 11 wheat varieties / lines to be detected: Yumai 47 (Fengyou 3)a, Jinmai 54a, Jinmai Mai 50b, Yuanfeng 898b, Gaoyou 503 (Xiaoyan 503)a, 04zhong 70b, Ji 923235a, Neixiang 1822 (Yumai 17)b, Han 4589b, Xinmai 19b and Xinmai 9987b.

[0055] The grain genomic DNA of the above-mentioned 11 wheat varieties / lines was extracted by using the method for extracting genomic DNA from wheat matured grains in Example 1.

[0056] Using the genomic DNA of the grains of the above 11 wheat varieties / lines as templates, amplification was performed using specific primer pairs (BJF, BJR): the PCR reaction system contained 1 μL of genomic DNA (200 ng / μL), 0.4 μL of 10 mmol / L dNTP, 0.5 μL of 10 pmol upstream and downstream primers, 2 μL 10×Taq Buffer [200 mM Tris-HCl (pH 9.0), 200 mM KCl, 100 mM (NH 4 ) 2 SO 4 , 15mM MgCl 2 ], 0.4 μL of Taq DNA polymerase (2.5 U / μL), 0.6 μL of DMSO a...

Embodiment 3

[0060] Example 3 Detection of lipoxygenase alleles of 163 wheat varieties / lines

[0061] The grain genomic DNA of 163 wheat varieties / lines in Table 3 was extracted by using the extraction method of wheat full-maturity grain genomic DNA in Example 1.

[0062] The genomic DNA of the grains of 163 wheat varieties / lines in Table 3 was used as a template to amplify using specific primer pairs (BJF, BJR). The PCR reaction system and PCR reaction procedure were the same as in Example 2.

[0063] After PCR amplification, 5 μL of the above PCR amplification products were taken for 1% agarose gel electrophoresis (using 1×TAE buffer; ethidium bromide (EB) staining, 120V, 25min), after electrophoresis, gel electrophoresis The gel imaging system (MultiGeniusGel Documentation and Analysis System) scans the image and analyzes it with a computer. Then the amplified fragments were subjected to gel recovery monoclonal sequencing.

[0064] The lipoxygenase allelic types of the 163 wheat varie...

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Abstract

The invention belongs to the technical field of plant biology, and particularly relates to a novel wheat grain lipoxygenase gene and the application thereof. The invention provides the novel wheat grain lipoxygenase gene, protein encoded thereby and a specific primer pair for identifying allelotypes thereof. Genomic DNA of wheat to be detected is taken as a template, the specific primer pair is used for performing PCR amplification, and amplified products with 2159 bp stripes are detected as Lox-3B2a allelotype wheat, and amplified products with 393 bp stripes are detected as Lox-3B2b allelotype wheat. The novel wheat grain lipoxygenase gene can be applied to improvement on wheat quality traits.

Description

technical field [0001] The invention belongs to the field of plant biotechnology, and in particular relates to a new gene of wheat grain lipoxygenase and its application. Background technique [0002] Lipoxygenase (Lox) in wheat plays an important role in wheat quality, and the level of Lox activity is one of the main factors affecting the color of wheat flour and its storage characteristics. On the one hand, in wheat varieties / lines with high Lox activity, because Lox will couple carotenoids in oxidized wheat, it can replace chemical bleaching agents to whiten wheat flour, thereby increasing its commercial value; on the other hand, Lox activity is too high It will accelerate the oxidation reaction of lipids, oxidize and deteriorate the grains, shorten the storage time and reduce the nutritional value. Therefore, from the perspective of suitable storage, reducing the activity of Lox is considered to be one of the important methods to preserve seeds. Therefore, the phenotyp...

Claims

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Application Information

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IPC IPC(8): C12N9/02C12N15/53C12N15/11C12Q1/6876
CPCC12N9/0069C12Q1/6876C12Q2600/13C12Y113/11012
Inventor 陈锋田秋珍吕国国张宁赵磊任妍
Owner HENAN AGRICULTURAL UNIVERSITY
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