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Method for detecting activity of wheat polyphenol oxidase and special primer therefor

A technology of polyphenol oxidase and wheat, applied in biochemical equipment and methods, measurement/testing of microorganisms, DNA/RNA fragments, etc., can solve the problems of unfavorable breeding, complicated operation, high cost, etc.

Inactive Publication Date: 2011-08-24
ANHUI AGRICULTURAL UNIVERSITY
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  • Abstract
  • Description
  • Claims
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Problems solved by technology

[0004] The types of PPO molecular markers found so far are mainly RFLP and SSR markers. These markers are complicated to operate and costly, which is not conducive to the application in breeding.

Method used

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  • Method for detecting activity of wheat polyphenol oxidase and special primer therefor

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0016] Embodiment 1, the mensuration of wheat polyphenol oxidase (PPO) active character

[0017] 170 wheat varieties from the China National Germplasm Bank were selected and planted in the autumn of 2004-2005 in the farms and experimental bases (Hefei and Fengyang) of Anhui Agricultural University. See Table 1. Random block design was adopted, repeated twice, row length 2m, row spacing 25cm, 100 seeds were sown in each row, harvested and threshed in rows, stored in safe water (moisture content ≤ 13%) for 3 months after the seeds were harvested and then pressed Described method measures PPO activity, concrete steps are:

[0018] 1. Use a cyclone mill to prepare whole wheat flour (through a 0.5mm sieve), weigh 0.3g of whole wheat flour and put it into a 50mL small beaker, add 7.5mL L-DOPA / MOPS solution (50mM MOPS buffer at pH6.5, 10mM L- DOPA).

[0019] 2. Place the above sample on a warm water bath shaker for 5 minutes (so that the sample is fully exposed to the air). The os...

Embodiment 2

[0030] Example 2, the acquisition of the STS molecular marker PPO05 of the wheat polyphenol oxidase gene

[0031] 1. Acquisition of molecular marker PPO05 of wheat polyphenol oxidase gene

[0032] The wheat PPO gene sequence was searched (BLAST) on the NCBI website, and an mRNA sequence (GenBank accession number: AY515506) encoded by the PPO gene expressed in wheat grain was obtained, and primers were designed at different positions of the sequence to target the above 8 Wheat varieties were subjected to PCR amplification, and the results showed that a pair of primers (PPO05) composed of sequence 1 and sequence 2 in the sequence table had differences in PCR amplification results in wheat varieties with different PPO activities, such as figure 1 shown.

[0033] Among them, the reaction system for PCR amplification is: 20 μL total volume contains 1×PCR buffer, MgCl 2 1.5mM, dNTP 0.2mM each, Taq DNA polymerase 1.5U, upstream and downstream primers 10pmol each, template DNA 100n...

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Abstract

The invention discloses a method and specific primers of detecting the character of the activity of wheat polyphenol oxidase. The primers provided by the invention for detecting the character of the activity of wheat polyphenol oxidase is a pair of primers consisting of the nucleotide sequences of the sequence 1 and the sequence 2 in the sequence table. The method of detecting the character of the activity of wheat polyphenol oxidase is that PCR amplification can be conducted by taking the DNA of a to-be-detected gene group as a templet and taking the nucleotide sequences of the sequence 1 and the sequence 2 in the sequence table as primers; and then the amplified outcomes can be detected to see whether 750 to 1000 bp of bands exist. The method and specific primers can play an important role both in the detection of the character of the activity of wheat polyphenol oxidase and in the breeding.

Description

technical field [0001] The invention relates to a method for detecting the activity traits of wheat polyphenol oxidase and special primers thereof. Background technique [0002] Noodles, steamed buns, and dumplings are traditional foods in my country, and their quality problems have attracted more and more attention from manufacturers and consumers. Polyphenol oxidase (PPO) in wheat grains is a key factor affecting dough browning. It can generate brown substances by oxidizing phenols during the process of adding water to flour and placing dough, so that the color of noodles and dumpling skins changes. dark. Therefore, reducing the PPO activity in wheat grains can effectively improve the appearance quality of pasta. [0003] Studies at home and abroad have shown that wheat PPO activity is a genetic trait controlled by multiple genes, and the genes related to PPO activity may be located in the second homologous chromosome group. For the identification and screening of breed...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/26C12Q1/68C12N15/11
Inventor 王晓波马传喜司红起
Owner ANHUI AGRICULTURAL UNIVERSITY
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