DNA numerator of hairpin RNA for expressing inhibit wheat kernel polyphenol oxidase and uses thereof

A technology of DNA molecules and polyphenol oxidase, applied in the field of DNA molecules, can solve the problems of natural mutants and artificial mutants that are difficult to screen recessive mutants, traditional hybrid breeding lacks breeding materials, etc., and achieve the effect of improving whiteness

Inactive Publication Date: 2009-08-19
CHINA AGRI UNIV
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Problems solved by technology

[0004] Common wheat is an allohexaploid, and there are many members of the PPO multi-gene family. It is difficult to screen suitable recessive mutants in natural mutants and artificial mutagenesis (such as EMS mutagenesis), which makes traditional hybrid breeding fall into a shortage. The Dilemma of Corresponding Breeding Materials

Method used

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  • DNA numerator of hairpin RNA for expressing inhibit wheat kernel polyphenol oxidase and uses thereof
  • DNA numerator of hairpin RNA for expressing inhibit wheat kernel polyphenol oxidase and uses thereof
  • DNA numerator of hairpin RNA for expressing inhibit wheat kernel polyphenol oxidase and uses thereof

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Embodiment 1

[0024] Embodiment 1, cultivating the wheat that polyphenol oxidase activity reduces

[0025] 1. Construction of RNA interference vector pBAC47P+ppoIR

[0026] By comparing the mRNA homology between the gene encoding grain PPO and the gene encoding leaf PPO, a 0.8Kb target gene fragment of grain PPO was selected. A pair of primers TaPPO-s1 and TaPPO-a1 were designed;

[0027] TaPPO-s1: 5'-CACCACGGCAACATCGACAGCCT-3',

[0028] TaPPO-a1: 5'-ATACCACGGCGAGCCACCGACGC-3'.

[0029] Trizol RNA Extraction Kit (Tiangen Company) was used to extract total RNA, and M-MLV Reverse Transcription Kit (Promega Company) was used to carry out reverse transcription-PCR (RT-PCR) according to the two-step method of the instruction manual. The PCR products were subjected to 1 After % agarose gel electrophoresis, the gel scanner (model fti-500, pharmacia biotech company) detects, and uses the agarose gel DNA recovery kit (spin column type, Tiangen Company) to reclaim the 0.8Kb fragment to obtain the ...

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Abstract

The invention discloses a DNA molecule expressing a hairpin RNA which inhibits polyphenol oxidase, and application thereof. The DNA molecule expressing the hairpin RNA which inhibits the polyphenol oxidase consists of an A fragment, a B fragment and a C fragment which are connected in sequence, wherein a nucleotide sequence of the A fragment is sequence 1 in a sequence table; the C fragment and the A fragment are reversely compensated; and a nucleotide sequence of the B fragment is sequence 2 in the sequence table. The DNA expressing the hairpin RNA which inhibits the polyphenol oxidase is started by a strong promoter of HMW glutelin subunit 1D*5 expressed by specific of wheat grain, expression of ppo gene of grain filling in transgenic wheat is obviously inhibited, PPO isoenzyme activity is reduced, and whiteness of wheat flour can be improved on the premise that vegetative growth and resistance of wheat are not affected.

Description

technical field [0001] The invention relates to a DNA molecule expressing hairpin RNA inhibiting wheat grain polyphenol oxidase and application thereof. Background technique [0002] Flour whiteness is one of the important traits to measure flour quality, which is controlled by many genetic and environmental factors. Affecting the appearance and color of noodles, steamed bread, biscuits and other noodle foods mainly depends on the content of carotenoids and polyphenol oxidase (Polyphenol Oxidase, PPO, EC 1.10.3.1). Among them, polyphenol oxidase (PPO) is directly related to the browning of wheat flour and its pasta during processing and storage (Baik, 1995; Kruger, 1994; Hatcher, 1993). At the same time, the genetic variation and expression difference of PPO species also determine the total activity of PPO in wheat grain, which directly or indirectly affects the whiteness of flour. [0003] Although most of the PPO in the grain is removed with the aleurone layer (bran) dur...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/11C12N15/63C12N5/10C12N1/00C12N15/82C12N15/113
Inventor 梁荣奇孙其信倪中福姚颖垠解超杰
Owner CHINA AGRI UNIV
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