An internal standard gene suitable for the detection of the copy number of exogenous genes in sesame and its construction method and application

A technology of internal standard gene and exogenous gene, applied in application, genetic engineering, plant gene improvement and other directions, can solve the problems of limited application of transgenic technology, weak research strength, etc., to achieve reliable results, low price, and perfect inheritance of exogenous genes The effect of conversion characterization techniques

Active Publication Date: 2016-08-24
HENAN SESAME RES CENT HENAN ACADEMY OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, because sesame is a crop that is difficult to establish a regeneration system and a transgenic technology system, and the basic research strength is relatively weak, it was not until 2012 that my country successfully obtained the first transgenic sesame plant, and the sesame transgenic technology system was basically established
However, at present, analytical techniques for the genetic characteristics of exogenous genes introduced into sesame have not yet been established, and the application of transgenic technology in the study of sesame genetic basis and material creation is still largely restricted.

Method used

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  • An internal standard gene suitable for the detection of the copy number of exogenous genes in sesame and its construction method and application
  • An internal standard gene suitable for the detection of the copy number of exogenous genes in sesame and its construction method and application
  • An internal standard gene suitable for the detection of the copy number of exogenous genes in sesame and its construction method and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] Example 1: Analysis of the copy number of the GUS gene in the T1 generation of the wild sesame-transferred GUS gene line

[0031] The developed internal standard gene Seγ-tm and fluorescence real-time quantitative PCR method were used to analyze the copy number of GUS gene in wild sesame transgenic lines.

[0032] (1) Cloning of internal standard genes: Yuzhi 11 genome database constructed from the laboratory

[0033] (www.sesamum.org) to select low-copy γ-tocopherol methyltransferase gene (Seγ-tmt, γ-tocopherol methyltransferase gene, 2128bp), and select Seγ-tmt gene from the established sesame transcriptome database mRNA sequence (ESTsequence, GenBank accession no.JP645320); primers were designed according to the known non-homologous region sequences at both ends, and the primer sequences for Southern blot probes were:

[0034] Forward primer: 5'-CCTTTCAAGTTGCCGATGC-3', that is, SEQ ID No: 3; Reverse primer: 5'-CGCTCCCCTTATTGTTTTCC-3', that is, SEQ ID No: 4, respecti...

Embodiment 2

[0051] Example 2: Using the internal standard gene Seγ-tmt to conduct a study on the copy number distribution of transgenic sesame lines after GUS gene introduction

[0052] The internal standard gene Seγ-tm and fluorescent real-time quantitative PCR were used to analyze the copy number of GUS gene in transgenic sesame lines after introduction.

[0053] (1) Primers for real-time fluorescent quantitative PCR of internal standard gene and GUS exogenous gene:

[0054] Seγ-tm internal standard gene primer: Primer F: 5'-TCCACCGTTCTGATCGCA-3', that is, SEQ ID No: 5; Primer R: 5'-GAAGGAGAGAGATCCCCTATGACAC-3', that is, SEQ ID No: 6; PCR amplification, real-time The fluorescent quantitative PCR reaction system is: 300-500ng template DNA, 12.5μl Maxima SYBR Green / R0X qPCR Master Mix (2×), 0.3μM primer F, 0.3μM primer R, add sterile ultrapure water to a final volume of 25μl; real-time fluorescence Quantitative fluorescence real-time quantitative PCR reaction conditions: 95°C denaturatio...

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Abstract

The invention discloses an endogenous reference gene suitable for detection of sesame exogenous gene copy number and a building method and an application of the endogenous reference gene. The endogenous reference gene is gamma-tocopherol methytransferase gene Se gamma-tmt, wherein a nucleotide sequence is as shown in SEQ ID No:1 or SEQ ID No:2. The invention further discloses the building method of the endogenous reference gene and the application of the endogenous reference gene. The number of exogenous genes in the transgenic plant DNA is detected by a fluorescence real-time quantitative PCR technology, so as to determine the copy number of the endogenous genes in the transgenic sesame plant. The endogenous reference gene suitable for detection of the sesame exogenous gene copy number can be applied to evaluation of a sesame transgenic technology system, large-scale endogenous gene copy number analysis of transgenic sesame, unknown gene copy number evaluation and genetic characteristic analysis of the sesame; a technical foundation is laid for sesame functional genomics research in future and excellent sesame new material evaluation; meanwhile, the operation steps of the copy number detection of the sesame exogenous genes are simple; the result is reliable; and an important basis is provided for obtaining efficient and stable transgenic sesame plants.

Description

technical field [0001] The invention relates to an internal standard gene suitable for detecting the copy number of exogenous genes in transgenic plants and its construction method and application, in particular to an internal standard gene suitable for Sesamum raditum (Sesamumindicum L., 2n=26) and wild species (Sesamum raditum, 2n =64) Internal standard gene for detection of exogenous gene copy number and its construction method and application. Background technique [0002] The internal standard gene refers to the conserved DNA with plant species specificity, constant copy number, and no allelic variation. Species specificity includes inter-species specificity and intra-species non-specificity. Inter-species specificity means that there is very low homology in different species, and PCR amplification can only amplify the expected product in the target species; Intraspecific non-specificity refers to high homology and little allelic variation in different cultivars of the...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/68C12N15/54
CPCC12N9/1007C12Q1/6895C12Q2600/166C12Y201/01095
Inventor 苗红梅张海洋魏利斌段迎辉李春韩秀花
Owner HENAN SESAME RES CENT HENAN ACADEMY OF AGRI SCI
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