Method for identifying whether wheat to be tested is Pina-D1b deficient wheat and applications thereof

A deletion-type, wheat-based technology, applied in biochemical equipment and methods, recombinant DNA technology, and microbial assay/inspection, etc., can solve problems such as unqualified DNA quality or human error, and achieve faster screening efficiency and accuracy. The effect of increased reliability and credibility

Inactive Publication Date: 2010-09-29
HENAN AGRICULTURAL UNIVERSITY
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  • Abstract
  • Description
  • Claims
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Problems solved by technology

However, the above detection methods have certain limitations, that is, protein electrophoresis technology can only detect the type of Pina gene that is not expressed, and there are many reasons for the non-expression, and the Pina-D1b type is only one of them. The full-length primer is used as a marker to detect negative results. If there is no amplification product, it may be caused by various factors such as unqualified DNA quality or human error, and the Pina-D1b type is only one of the cases.

Method used

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  • Method for identifying whether wheat to be tested is Pina-D1b deficient wheat and applications thereof
  • Method for identifying whether wheat to be tested is Pina-D1b deficient wheat and applications thereof
  • Method for identifying whether wheat to be tested is Pina-D1b deficient wheat and applications thereof

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Embodiment 1

[0032] Embodiment 1, the mensuration of grain hardness of different wheat varieties

[0033]Detect the grain hardness of 12 wheat varieties (Durum Langdon, China Spring, Yunong 202, Shan 253, Gaocheng 8901, CMH82A.1294 / 2*KAUZ / / MUNIA / CHT O / 3 / MILAN, Mo 299, Mo 293, ink 215, ink 191, ink 181, ink 272, RDWG / MILAN). Grain hardness measurement method: measure the hardness value of 300 grains of wheat samples with a single-grain grain hardness instrument (SKC84100, Perten, Sweden), determine the hardness level of the kernels according to the measurement results, and record the thousand-grain weight and kernel diameter at the same time. The experiment was repeated three times, and the results were averaged.

[0034] The results of SKCS analysis showed that among the 12 wheat varieties, China Spring belongs to soft wheat, and the remaining 11 wheat varieties are all hard wheat (hardness above 60). The average hardness values ​​of SKCS are: China Spring is 12, Yunong 202 is 60, Shaanx...

Embodiment 2

[0035] Embodiment 2, the discovery of specific primer pair

[0036] Taking Shan 253 and Gaocheng 8901 as examples, the target fragments were amplified by the primer walking method. Primer design as figure 1 shown. Among them, the 1st, 2nd, 3rd, 4th, 5th and 19th pair of primers can amplify the target fragment, and the 6th, 7th, 8th, 9th, 10th, 11th, 12th, 13th, 14th, 15th, 16th, 17th and 18th pair The primers cannot amplify the target fragment, indicating that the missing fragment occurs between the downstream of the fifth pair of primers and the upstream sequence of the 19th pair of primers, and then redesign specific primer pairs between the fifth pair of primers and the 19th pair of primers Amplification was performed to obtain a 776bp fragment. After the fragment was sequenced, compared with the same sequence of the wild-type Ha site (NCBI number: CT009735), it was found that the Pina-D1b type Ha site sequence lacked 15380bp, and the missing fragment was from the +23rd ...

Embodiment 3

[0040] Embodiment 3, application-specific primer pair identification Pina-D1b deletion type wheat

[0041] 12 wheat varieties: China Spring (soft wheat), Yunong 202 (hard wheat), Shan 253 (hard wheat), Gaocheng 8901 (hard wheat), Mo 299 (hard wheat), Mo 293 (hard wheat), Mo 215 (hard wheat), ink 191 (hard wheat), ink 181 (hard wheat), ink 272 (hard wheat), RDWG / MILAN, CMH82A.1294 / 2*KAUZ / / MUNIA / CHTO / 3 / MILAN (hard wheat ).

[0042] The 12 wheat varieties were identified as follows:

[0043] 1. SDS-PAGE to identify whether each wheat variety belongs to the type of PINA protein deficiency

[0044] 1. Extract grain protein

[0045] Take one seed of each variety of wheat, and extract the grain protein according to the following steps: 1) put it into a 2mL centrifuge tube after grinding, add 1mL pre-cooled TBS solution and 0.15mL 12% Triton-X114 (Sigma company) After mixing, place it at 4°C for 12-24h; 2) Centrifuge at 12000rpm for 3 minutes, transfer the supernatant to a 1.5mL c...

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Abstract

The invention discloses a method for identifying whether wheat to be tested is Pina-D1b deficient wheat and applications thereof. The method according to the invention comprises the following steps: 1) using a specific primer pair for PCR amplification by taking genome DNA of the wheat to be tested as a template, wherein the specific primer pair is the primer pair composed of nucleotide sequences, which are shown in sequence 1 and sequence 2 of sequence table; and 2) detecting PCR-amplified product, wherein the wheat to be tested with 776bp strips in the amplified product is candidate Pina-D1b deficient wheat, and the Pina-D1b deficient wheat is the wheat having nucleotides, which are shown in sequence 3 of the deficient sequence table of the genome DNA. The specific primer pair according to the invention can take the place of traditional SDS-PAGE and two-dimensional electrophoresis technology and the detection for Pina-D1b type is directly carried out at molecular level, so accuracy and reliability are greatly enhanced, screening efficiency and accuracy for grain hardness genotype is remarkably quickened; in addition, the method plays an important role in improving gene engineering of wheat grain hardness, simultaneously aids in further research on genetics and molecular biology of wheat grain hardness and is also significant to the cultivation of high-quality wheat.

Description

technical field [0001] The invention relates to a method for identifying whether the wheat to be tested is Pina-D1b deletion type wheat and its application. Background technique [0002] Grain hardness is an important basis for grading and pricing in the international wheat market. It affects the amount of water added to wheat moistening, flour extraction rate, flour particle size, the number of damaged starch particles, and food processing quality. It is one of the important breeding goals. According to the endosperm texture, common wheat can be divided into hard wheat, mixed wheat and soft wheat. Grain hardness is mainly controlled by a pair of major genes Pina and Pinb (collectively called puroindoline) located on the short arm of chromosome 5D (5DS). Since a group of protein polypeptides with a molecular weight of about 13kD was discovered and named (friabilin) ​​in common wheat, the research progress of grain hardness at the molecular level has been greatly accelerated...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/11
Inventor 陈锋崔党群董中东詹克慧许海霞程西永
Owner HENAN AGRICULTURAL UNIVERSITY
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