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Cultivation method of common wheat capable of stably expressing six HMW-GS (High Molecular Weight-Glutenin Subunits)

A technology of HMW-GS and stable expression, applied in the field of common wheat cultivation, can solve the problems of lack of molecular structure analysis, lack of stable expression, etc.

Inactive Publication Date: 2014-04-02
SICHUAN AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The common wheat involved in the only two reports belongs to spring wheat, and there is a lack of analysis of the molecular structure characteristics of the corresponding genes in the common wheat genetic background
In 2008, Ma Jianhua and others in my country used transgenic engineering to transfer the 1Ay gene of T. urartu into the common wheat Lankao 4, but only in T. urartu 1Ay subunit was detected in a seed of 0 generation, and there was no report of its stable expression
However, there has been no report about the introduction of the gene encoding the y-type subunit at the Glu-A1 locus of wild emmer wheat to hexaploid common wheat with a wider cultivation area, greater production and consumption

Method used

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  • Cultivation method of common wheat capable of stably expressing six HMW-GS (High Molecular Weight-Glutenin Subunits)
  • Cultivation method of common wheat capable of stably expressing six HMW-GS (High Molecular Weight-Glutenin Subunits)
  • Cultivation method of common wheat capable of stably expressing six HMW-GS (High Molecular Weight-Glutenin Subunits)

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] Example 1 SDS-PAGE detection to obtain HMW-GS results

[0039] Extraction solution composition: 62.5 mM Tris-HCl (pH=6.8), 10% (v / v) glycerol, 2% (w / v) SDS, 0.002% (w / v) bromophenol blue, 3.0% (v / v) β-Mercaptoethanol;

[0040] Resolving gel buffer (2.5 L): dissolve 378g Tris, 25g SDS, 95g boric acid, add water to make up to 2.5L, pH 8.9.

[0041] Stacking gel buffer: 1.0 M Tris-HCl, pH=6.8, 10% (W / V) SDS.

[0042] Running Buffer: Dilute Separating Gel Buffer 10-fold

[0043] Polyacrylamide gel composition:

[0044] Stacking Gel (3%) Separating Gel (10%)

[0045] 40% Acrylamide, ml 0.375 2.5

[0046] 2% Methylenebisacrylamide, ml 0.2 0.52

[0047] Resolving gel buffer, ml 0 1

[0048] Stacking gel buffer, ml 0.62 0

[0049] 10% SDS (W / V), ml 0.04 0

[0050] 10% (W / V) amine persulfate, ml 0.05 0.09

[0051] TEMED, μl 5 4.2

[0052] H 2 O, ml 3.7 5.9

[0053] Using the amphiphilic glutelin subunit as a control, electrophoresis at a constant current of 20 m...

Embodiment 2

[0057] Example 2 Somatic chromosome number detection

[0058] The seeds of the hybrid progeny were germinated at a constant temperature of 25°C to take the roots, and the root tips were frozen in an ice-water mixture for 24 hours. Carnot's I fixative solution (alcohol: glacial acetic acid = 3:1) was fixed for 24 h and then transferred to 70% ethanol for preservation. The root tips were placed in 1 mol / L hydrochloric acid, dissociated in a constant temperature water bath at 60 °C for 6-8 min, stained with Schiff reagent, and routinely pressed with modified phenol fuchsin for somatic chromosome observation. Observe 50 cells, statistics.

Embodiment 3

[0059] Example 3 Extraction of genomic DNA

[0060] The genomic DNA of common wheat, wild emmer and hybrid progeny was extracted by 2×CTAB method. The extraction steps were as follows:

[0061] (1) Take 2 g of fresh young leaves, grind them into fine powder in liquid nitrogen, and add 2 × CTAB extract solution (2% CTAB, 1.4 M NaCl, 0.1 M Tris-HCl pH=8.0, 0.1 M preheated to 65 °C) EDTA pH=8.0, add 2% β-mercaptoethanol) 15 ml and mix well before use;

[0062] (2) 65 ℃ water bath for 30-45 min, shake gently during the mixing. After cooling to room temperature, add an equal volume of chloroform:isoamyl alcohol (24:1), mix gently until the supernatant becomes milky, and centrifuge at 4000 rpm for 10 min.

[0063] (3) Take the supernatant, add an equal volume of isopropanol, and ice-bath for 2 h to precipitate DNA;

[0064] (4) Tick out the DNA, wash it twice with 70% alcohol, wash it once with absolute ethanol and air dry the DNA, and dissolve it in an appropriate amount of 1×...

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Abstract

The invention relates to a cultivation method of common wheat capable of stably expressing six HMW-GS (High Molecular Weight-Glutenin Subunits) and belongs to the field of plant chromosome engineering technologies. The method comprises the following steps of: taking common wheat as a female parent and wild emmer wheat as a male parent; pollinating pollen of the wild emmer wheat with four HMW-GS to sextuploid common wheat; and carrying out inter-specific crossing distant hybridization, multi-generation selfing and identification to breed the common wheat capable of stably expressing the six HMW-GS. The novel common wheat with the six HMW-GS, which is cultivated by the method, not only has a strain leave type which is similar with the male parent, but also has a purple stalk property which does not exist in the female parent, and has better grain setting property, output property and excellent quality property when being compared with the female parent; and HMW-GS detection is continuously carried out on grains of F10, F11 and F12 generations of hybrid seeds so as to testify that 1Ay genes can be stably transmitted and expressed generation by generation.

Description

technical field [0001] The invention belongs to the technical field of plant chromosome engineering, in particular to a method of introducing the functional Ay-type high molecular weight glutenin subunit (HMW-GS) coding gene of tetraploid wild emmer wheat into common wheat to realize all six HMW-GS A method for cultivating common wheat with stable expression. Background technique [0002] Wheat, as the main staple food crop in the world, contributes about 30% of the global total grain production and has become one of the main crops for human nutrition sources. It is estimated that by 2020, the global demand for wheat will increase by about 40%, so wheat plays a decisive role in human health and survival. [0003] A large number of studies have shown that the nutritional quality of wheat and the processing quality of flour are closely related to the content and composition of seed protein. Wheat high molecular weight glutenin subunits (High molecular weight glutenin subunit...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): A01H1/02
Inventor 伍碧华王真真胡喜贵胡继良郭孝辉王栋郑有良刘登才蒲至恩陈国跃代寿芬
Owner SICHUAN AGRI UNIV
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