Method for identifying high molecular weight glutenin subunit of wheat through flux

A high-molecular-weight, glutenin-based technology, applied in biochemical equipment and methods, microbiological determination/inspection, etc., can solve problems that have not been reported yet

Inactive Publication Date: 2010-06-30
CHENGDU INST OF BIOLOGY CHINESE ACAD OF S
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the multiplex PCR technology used in wheat research mainly involves high (low) molecular glutenin subunit genes, grain hardness genes and amylase genes related to quality [Ma W, Zhang W, Gale K R. Multiplex-PCR typing of high molecular weight glutenin alleles in wheat. Euphytica, 2003, 134:51-60; Zhang XK, Liu L, HeZH, Sun DJ, He XY, Xu ZH, Zhang PP, Chen F and Xia XC. Development of two multiplexPCR assays targeting Improvement of bread-making and noodle qualities in common wheat. Plant Breeding, 127(2): 109-115; Zhang Xiaoke, Xia Xianchun, Wang Zhongwei, Wan Yingxiu, Zhang Pingzhi, He Xinyao, Yang Yan, He Zhonghu. Multiplex PCR of molecular markers for wheat quality traits Establishment of the system. Acta Crops Sinica, 2007, 33(10): 1703-1710], the multiplex PCR technology that can simultaneously identify the important high-quality subunit genes on the three Glu-1 sites has not been reported

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  • Method for identifying high molecular weight glutenin subunit of wheat through flux
  • Method for identifying high molecular weight glutenin subunit of wheat through flux
  • Method for identifying high molecular weight glutenin subunit of wheat through flux

Examples

Experimental program
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Effect test

Embodiment 1

[0011] Example 1: Extraction and detection of required material DNA and selection of PCR primers

[0012] 1. DNA extraction and detection

[0013] The total DNA of wheat was extracted from seeds by CTAB method, and the quality and concentration of total DNA were detected by ultraviolet spectrophotometer.

[0014] PCR primer sequence

[0015] The primer sequences and target fragment sizes of each molecular marker are shown in the table below:

[0016] subunit name

Embodiment 2

[0017] Embodiment two: the establishment of multiplex PCR system

[0018] The total volume of the multiplex PCR reaction system is 25ul, including PCR Buffer (Mg 2+ Free), 1.5mM MgCI 2 , 200uMdNTP, 1.5U of Taq enzyme and 150ng of template DNA, subunit Bx7 OE , Dx5 and AxNull specific molecular markers of the upstream and downstream primers were 5 / 5 / 8pmol. Due to the high annealing temperature of one of the three pairs of primers, a method similar to touchdown PCR was used to optimize the amplification conditions. The optimal thermal cycle conditions were: 95°C pre-denaturation for 5 minutes, 94°C denaturation for 30S, and 62°C annealing for 30S , 72°C extension for 1min, 8 cycles; then denaturation at 94°C for 30S, annealing at 58°C for 30S, extension at 72°C for 1min, 27 cycles; finally, extension at 72°C for 10min. Using this amplification program can enrich the products of correct pairing of primers and templates, so that each pair of primers can synthesize specific ampl...

Embodiment 3

[0020] Example 3: Carrying out Multiplex PCR Identification of Tibetan Wheat

[0021] Using the multiplex PCR system established in this experiment to identify the composition of 89 Tibetan wheat HMW high-quality subunits, the results showed that: at the Glu-A1 locus, the distribution frequency of Ax1 or Ax2* subunits was 12.36%, and the occurrence frequency of Null low-quality subunits was higher High; the distribution frequency of the Dx5 subunit was 12.36%; no Bx7 was detected at the Glu-B1 locus OE Subunit. 10.11% of the tested varieties (lines) have both Ax1 / Ax2* and Dx5 subunits (see figure 2 ).

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Abstract

The invention belongs to the technical field of molecular biology, which relates to a PCR-based practical flux detection method for molecular markers of the quality characteristics of wheat. In the method, specific molecular markers of NullAx (Glu-A1 locus), Bx7OE(Glu-B1 locus) and Dx5(Glu-D1 locus) subunit genes are selected and multiple PCR systems for simultaneously identifying an important high-quality subunit on Glu-1 locus through one-step reaction are established by optimizing a PCR reaction system and thermal circulation conditions; and in addition, specific amplified products can be effectively separated by common agarose electrophoresis. Compared with single PCR reaction, the method greatly improves the detection efficiency and has reliable identification results and reproducibility through breed and group verification. The invention provides a simple, accurate, rapid and practical method for evaluating breeding materials as well as effectively identifying and selecting high molecular weight glutenin high-quality subunits of filial generation.

Description

technical field [0001] The invention belongs to the technical field of molecular biology, and relates to a practical detection method for molecular markers of wheat quality traits, in particular to the rapid identification of a plurality of wheat high-quality high-molecular-weight glutenin subunits (HMW-GS) based on PCR technology. Background technique [0002] The quantity and quality of protein are important factors affecting the quality of wheat flour. Wheat seed storage proteins are mainly composed of gliadin (Gliadin) and glutenin (Glutenin). Glutenin is divided into high molecular weight glutenin subunit (HMW-GS) and low molecular weight glutenin subunit (LMW-GS). Glutenin macropolymers are formed through disulfide bonds between substrates, which endow dough with unique viscoelasticity and extensibility [Payne P I, Law C N, Mudd E E. Control by homologous group 1 chromosomes of the high-molecular-weight subunits of glutenin, a majorprotein of wheat endosperm. Theor Ap...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68
Inventor 陈静郑寒付体华
Owner CHENGDU INST OF BIOLOGY CHINESE ACAD OF S
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