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Connecting region sequence of exogenous inserting gene and flanking rice sequence of transgenic rice kefeng No.2 and application thereof

A transgenic rice and exogenous insertion technology, applied in biochemical equipment and methods, microbe determination/inspection, DNA/RNA fragments, etc., can solve problems such as strain-specific PCR detection methods that have not yet been found

Active Publication Date: 2014-06-04
THE INST OF BIOTECHNOLOGY OF THE CHINESE ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, so far, no line-specific PCR detection method for transgenic rice Kefeng 2 has been found

Method used

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  • Connecting region sequence of exogenous inserting gene and flanking rice sequence of transgenic rice kefeng No.2 and application thereof
  • Connecting region sequence of exogenous inserting gene and flanking rice sequence of transgenic rice kefeng No.2 and application thereof
  • Connecting region sequence of exogenous inserting gene and flanking rice sequence of transgenic rice kefeng No.2 and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] Example 1 Acquisition of flanking sequences inserted exogenously into the SCK gene in the transgenic rice Kefeng No. 2 strain

[0041] The exogenously inserted target gene of the transgenic rice Kefeng No. 2 line is the SCK gene, with a total of 415 bp, and its base sequence is shown in SEQ ID No.1.

[0042] Obtain the integration structure (exogenous insertion gene flanking sequence) by Hi-TAIL PCR

[0043] 1. Step 1: Design nested primers for Hi-TAIL PCR on the target gene SCK

[0044] ① Downstream primers:

[0045] Sck-sp1 CTTTCTCATCATCTTCATCCCTGGACTTG

[0046] Sck-sp2 ACGATGGACTCCAGTCCGGCCGATTTGCAAGCCGAGTGACACGAATT

[0047] Sck-sp3 TTGATTTAGTGCAGATGCATGAATCGC

[0048] Upstream primers:

[0049] Sck-Ap1GCACCATCTTCTTTGCTCTCTTTTCTCTGT

[0050] Sck-Ap2 ACGATGGACTCCAGTCCGGCCTTTCGTTTTAAAGGTGTGTGTGCTGGTAC

[0051] Sck-Ap3 TGATGACTCAAGCGATGAACCTTCTGAG

[0052] Random primers:

[0053] AD1-1 ACGATGGACTCCAGAGCGGCCGCVNVNNNGGAA

[0054] AD1-2 ACGATGGACTCCAGAGCGGCCGCBN...

experiment example 1

[0082] Experimental example 1 Screening of specific primers for line-specific qualitative detection of transgenic rice Kefeng 2 and optimization of amplification conditions

[0083] 1. Screening of specific primers for qualitative detection of 5' end detection

[0084] 1. Design and screening of primers

[0085] Five pairs of primers were designed at the flanking sequence of the 5' end, and the primer sequences were as follows:

[0086] Primer a: The size of the amplified product fragment is 202bp

[0087] F: GATTAACCATACTGGAAAATCTGGC

[0088] R: GTCCGCAATGTGTTATTAAGTTGTC

[0089] Primer b: the size of the amplified product fragment is 156bp

[0090] F: GATTAACCATACTGGAAAATCTGGC

[0091] R: CGCAGGATAGCTAGTAGGATCG

[0092] Primer c: the size of the amplified product fragment is 142bp

[0093] F: CTTCCTGGATTTACAGCCTAAAGAT

[0094] R: GTCCGCAATGTGTTATTAAGTTGTC

[0095] Primer d: the size of the amplified product fragment is 170bp

[0096] F: ACATCGTCAGTACCATAGTGATTGC

[...

experiment example 2

[0133] Experimental example 2 Establishment of strain-specific detection system and reaction program of transgenic rice Kefeng 2

[0134] 1. Extraction and detection of plant genomic DNA

[0135] 1 Plant DNA Extraction

[0136] 1.1 Preparation of CTAB extraction buffer

[0137] 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.

[0138] 1.2 Extraction method

[0139] a. 100mg sample, fully ground into powder and transferred to a 2ml centrifuge tube;

[0140] b. 1ml of CTAB extraction buffer preheated to 65°C, mix thoroughly, suspend the sample, and mix gently.

[0141] c. Water bath at 65°C for 40 minutes, and mix by inverting several times during the period;

[0142...

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Abstract

The invention discloses a connecting region sequence of an exogenous inserting gene and a flanking rice sequence of transgenic rice kefeng No.2 and an application thereof. The connecting region sequence of an exogenous inserting gene vector and 5'-end and 3'-end flanking rice sequences at two sides is obtained through Hi-TAIL PCR, and nucleotide is shown in SEQ ID No.2. The invention also discloses a connecting region sequence of the exogenous inserting vector and the 3'-end flanking rice sequence, and the nucleotide sequence is shown in SEQ ID No.13. The invention further discloses the connecting region sequence as a target gene applied to detection of the specificity of a transgenic rice kefeng No.2 strain. A foundation is laid for building of a method for detecting the specificity of the transgenic rice kefeng No.2 strain.

Description

technical field [0001] The invention relates to the flanking sequence of transgenic rice, in particular to the sequence of the junction region of the transgenic rice Kefeng 2 inserted into the gene carrier and the flanking rice sequence and its application, and belongs to the field of detection of the specificity of transgenic rice strains. Background technique [0002] With the widespread planting of genetically modified plants, the safety of genetically modified foods has also attracted great attention. More than 30 countries have successively implemented genetically modified product labeling systems, including China. The implementation of the genetically modified labeling system relies on the component testing of genetically modified plants and their processed products. PCR technology is currently the most widely used method in the detection of genetically modified products at home and abroad. When applying this technique for qualitative detection of genetically modified...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/11C12Q1/68
Inventor 宛煜嵩梁利霞张秀杰朱祯金芜军苗朝华李亮黄卫红
Owner THE INST OF BIOTECHNOLOGY OF THE CHINESE ACAD OF AGRI SCI