Transgenic rice PA110-15 foreign insert flanking sequence and application

A technology of exogenous insertion and flanking sequences, which can be used in DNA/RNA fragments, recombinant DNA technology, determination/inspection of microorganisms, etc., and can solve problems such as lack of

Active Publication Date: 2014-08-06
RICE RES ISTITUTE ANHUI ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

So far, there is a lack of a specific and sensitive PCR detectio...

Method used

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  • Transgenic rice PA110-15 foreign insert flanking sequence and application
  • Transgenic rice PA110-15 foreign insert flanking sequence and application
  • Transgenic rice PA110-15 foreign insert flanking sequence and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] Example 1 Acquisition of the Flanking Sequence of the Transgenic Rice PA110-15 Exogenously Inserted Gene

[0038] The target gene for exogenous insertion of transgenic rice PA110-15 with high lysine storage protein gene is LRP gene, and its base sequence is shown in SEQ ID No.19.

[0039] The junction region sequence (SEQ ID No.1) of the exogenous inserted gene and the 5' end rice flank sequence (SEQ ID No.1) and the junction region sequence (SEQ ID No.2) of the exogenous inserted gene and the 3' end rice flank sequence were obtained by Hi-TAIL PCR ).

[0040] The basic principle of TAIL-PCR technology is to use the known sequence next to the target sequence to design three nested specific primers (special prime, referred to as spl, sp2, sp3, about 20bp), and use them respectively with one primer with a low T value. Short (14bp) random degenerate primers (Arbitrary degenerate primers, referred to as AD) combined, using genomic DNA as a template, according to the length...

experiment example 1

[0048] Experimental example 1 Screening of specific primers for qualitative detection of transgenic rice PA110-15 with high lysine storage protein gene and optimization of amplification conditions

[0049] 1. Screening of 5' end transformant-specific detection primers

[0050] According to the side sequence information of the 5' end, 4 pairs of detection primers (see Table 3) were designed to amplify and detect PA110-15, and 2 pairs of primers PA110-5-F1 / R1 and PA110-5-F2 / R2 ( figure 2 ), further through system optimization, design the orthogonal experiment of primer final concentration and annealing temperature, and finally found that the primer combination PA110-5-F1 / R1 as the 5' end transformant-specific detection primer has the highest amplification efficiency and the least primer dimer ( image 3 ).

[0051]The final concentration of primers in the PCR reaction system and the annealing temperature have a certain impact on the specificity and sensitivity of the detect...

experiment example 2

[0058] Experimental example 2 Establishment of specific detection system and reaction program for transgenic rice PA110-15 with high lysine storage protein gene

[0059] 1. Extraction and detection of plant genomic DNA

[0060] 1 Plant DNA Extraction

[0061] 1.1 Preparation of CTAB extraction buffer

[0062] Add 81.7g NaCl and 20g CTAB to 600mL water, after fully dissolved, add 100mL of 1mol / L Tris-HCl (pH7.5) solution, 100mL of 0.5mol / L EDTA (pH8.0) solution, and finally adjust the pH to 8.0 with HCl or NaOH solution , add water to make up to 1000mL. Use after being sterilized under high temperature and high pressure (103.4kPa / 121°C) conditions for 20 minutes.

[0063] 1.2 Extraction method

[0064] a. 100mg sample, fully ground into powder and transferred to a 2mL centrifuge tube.

[0065] b. 1 mL of CTAB extraction buffer preheated to 65°C, mix thoroughly, suspend the sample, and mix gently.

[0066] c. Water bath at 65°C for 40 minutes, during which time mix by inve...

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PUM

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Abstract

The invention discloses a transgenic rice PA110-15 foreign insert flanking sequence and an application. According to the transgenic rice PA110-15 foreign insert flanking sequence, firstly, a 5' end sequence and a 3'flanking sequence of a transgenic rice PA110-15 strain foreign inserted gene are provided, and nucleotide sequences of the 5' end sequence and the 3'flanking sequence are represented by SEQID NO.1 and SEQID NO.2.The invention provides aPCR (polymerase chain reaction) detection primer for transgenic ricePA110-15(expressing lysine-rich storage protein gene)strain specificity and a strain specificityqualitative detection method with the flanking sequence used as a target gene. Specificityexperiments indicate that the establishedtransgenic rice PA110-15 strain specificityqualitative PCR detection method has the high specificity. Sensitivity test results indicate thatthe sensitivity of the established detection method reaches 0.1%.

Description

technical field [0001] The present invention relates to the flanking sequence of the exogenous insert fragment of the transgenic rice line, in particular to the junction region sequence of the exogenous fragment of transgenic rice PA110-15 storage protein gene and the flanking rice sequence, and the present invention further relates to transgenic high lysine The invention relates to specific PCR detection primers for rice PA110-15 strain with storage protein gene, a qualitative detection method and a detection kit, which belong to the specific detection field of rice PA110-15 strain with high lysine storage protein gene. Background technique [0002] With the widespread cultivation of genetically modified plants, the safety of genetically modified foods has also attracted great attention. More than 50 countries and regions have implemented genetically modified product labeling systems, including China. The implementation of the genetically modified labeling system relies on ...

Claims

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Application Information

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IPC IPC(8): C12N15/11C12Q1/68
Inventor 杨剑波马卉李莉凃巨民沈平汪秀峰宋贵文倪大虎魏鹏程段武德陆徐忠宋丰顺李浩王淑云
Owner RICE RES ISTITUTE ANHUI ACAD OF AGRI SCI
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