A method for detecting unbalanced expression of plant alleles

An allele and detection method technology, applied in biochemical equipment and methods, microbial determination/inspection, etc., can solve the problems of poor accuracy and high cost of allele unbalanced expression detection, to enhance specificity and reduce errors. The probability of matching occurrence and the effect of simplifying the experimental matching conditions

Active Publication Date: 2020-10-13
BEIJING FORESTRY UNIVERSITY
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  • Abstract
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Problems solved by technology

[0005] In view of this, the object of the present invention is to provide a method for detecting allelic imbalance expression to solve the problems of high cost and poor accuracy in the detection of allelic imbalance expression in the prior art

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  • A method for detecting unbalanced expression of plant alleles
  • A method for detecting unbalanced expression of plant alleles

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Experimental program
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Embodiment 1

[0038] According to the Phytozome database (https: / / phytozome.jgi.doe.gov / pz / portal.html#!

[0039] gene? search=1&crown=1&detail=1&method=0&searchText=transcriptid:27040781) Populus trichocarpa heat shock transcription factor gene 2 (HSF2) resequencing data screened the SNPs located in the unconserved region at the 3' end of the gene to identify alleles, The frequency of the SNPs in the population needs to be greater than 5%, and the site is in a heterozygous state in the experimental individual. The SNP8583997 locus meets the above requirements, and individuals who are heterozygous for this locus are selected as allele expression detection materials. The allelic expression pattern of heat shock transcription factor gene 2 (Potri.001G108100) was detected by qPCR using LNA modified primers, and SNP8583997 in Populus trichocarpa heat shock transcription factor gene 2 (HSF2) was used as the allele identification site, and its region The gene sequence is as follows:

[0040] A...

Embodiment 2

[0067] According to the Phytozome database (https: / / phytozome.jgi.doe.gov / pz / portal.html#!gene?search=1&crown=1&detail=1&method=0&searchText=transcriptid:32066262) Eucalyptus grandis MYB transcription factor gene (Eucgr.K02470 ) resequencing data to screen the SNPs located in the non-conserved region at the 3' end of the gene to identify alleles, the frequency of the SNPs in the population needs to be greater than 5%, and the site is heterozygous in the experimental individual state. The SNP8583997 locus meets the above requirements, and individuals who are heterozygous for this locus are selected as allele expression detection materials. Use LNA modified primers qPCR to detect the allelic expression pattern of the MYB transcription factor gene (Eucgr.K02470), using SNP32768853 in the Eucalyptus grandis MYB transcription factor gene (Eucgr.K02470) as the allele identification site, and the gene sequence of its region As follows: CTCTTTGTTTGTCTGTT GTGTTTATCG, italics is th...

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Abstract

The invention provides a method for detecting allelic expression imbalance of plants. The method comprises the following steps: 1) comparing candidate gene sequences, screening SNPs locus for labelling allele; 2) designing a qPCR specific primer and a degenerate primer according to the screened SNPs locus; 3) performing PCR amplification on cDNA of a sample to be detected with the qPCR specific primer and the degenerate primer, to obtain an amplified product; 4) if a melting curve of the amplified product is single peak, calculating expression specificity of the allele of the sample to be detected according to a Ct value of PCR amplification, wherein the qPCR specific primer comprises a forward primer and a reverse primer; LNA modification is performed on nucleotide of a 3' terminus of the primer complementary to the 3' terminus of the SNP locus. The method improves accuracy and reliability of detection results, simplifies experimental conditions and significantly reduces experimental cost.

Description

technical field [0001] The invention relates to the field of gene expression detection, in particular to a method for detecting unbalanced expression of plant alleles. Background technique [0002] Allele (also called allelomorph) generally refers to a pair of genes that control relative traits at the same position on a pair of homologous chromosomes. [0003] The unbalanced expression of alleles (allelic expression imbalance, AEI) is that in the same cell, each gene usually has 2 copies, and the expression ratio of the 2 copies of the gene deviates from 1:1 due to cis-action. The phenomenon of unbalanced expression of alleles is ubiquitous. In addition to the absolute unbalanced expression of genetically imprinted genes, a considerable number of genes have AEI in some individuals and in different time and space in the same individual. And associated with polymorphic sites in some specific regions of the genome. [0004] At present, the commonly used detection of allelic i...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/6858
CPCC12Q1/6858C12Q2531/113
Inventor 张德强宋跃朋次东
Owner BEIJING FORESTRY UNIVERSITY
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