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Reference gene for barley gene expression researches as well as application of reference gene

An internal reference gene and gene expression technology, which is applied in the field of plant molecular biology, can solve problems such as not being the most stable, stability research, internal reference gene instability, etc., and achieve the effect of saving dosage

Active Publication Date: 2017-08-18
SHANGHAI ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although some internal reference genes have been used in barley low-nitrogen tolerance quantitative PCR analysis, their stability has not been studied. If some so-called internal reference genes are used directly, they may not be effective in samples treated with low nitrogen. Therefore, it may not be accurate to use such an internal reference gene to study the expression of the target gene
[0005] Although there are also some studies on the screening of internal reference genes, due to the inconsistent barley genotypes, experimental conditions, and low nitrogen stress levels, the screened internal reference genes may also be unstable or not the most stable and optimal. Therefore, it is necessary to screen and identify stably expressed internal reference genes for the forthcoming barley low nitrogen tolerance gene expression research

Method used

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  • Reference gene for barley gene expression researches as well as application of reference gene
  • Reference gene for barley gene expression researches as well as application of reference gene
  • Reference gene for barley gene expression researches as well as application of reference gene

Examples

Experimental program
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Effect test

Embodiment 1

[0051] The acquisition of embodiment 1 barley internal reference gene

[0052]The present invention uses the results of sequencing analysis of barley low-nitrogen tolerance-related transcriptomes to select three barley internal reference genes E2, EF2 and β-tubulin 6 with stable expression, wherein the gene sequence source of the ubiquitin conjugating enzyme E2 gene fragment is MLOC_9934.1, its nucleotide sequence is shown in SEQ ID NO.1, the gene sequence source of the elongation factor EF2 gene fragment is MLOC_15661.3, its nucleotide sequence is shown in SEQ ID NO.2, the The gene sequence of the tubulin β-tubulin 6 gene fragment comes from MLOC_74587.1, and its nucleotide sequence is shown in SEQ ID NO.3.

[0053] Primers were designed by Primer 3 software (http: / / primer3.wi.mit.edu / ), and the primer information is as follows:

[0054] The primer sequence for amplifying the ubiquitin conjugating enzyme E2 gene fragment is:

[0055] F1: 5'-TTTTTGGCCCTGATGATAGC-3';

[0056...

Embodiment 2

[0065] Example 2 utilizes existing internal reference genes to verify stability and accuracy

[0066] 1. Plant growth and low nitrogen stress treatment

[0067] Several barley seeds of two genotypes (BI-04 and BI-45) were taken, sterilized with 1% NaClO for 30 minutes, then rinsed with water for 3 times, soaked in water for 4 hours, and then the seeds were taken out to germinate overnight. Take out the seeds with the same buds and sow them directly in the pot containing vermiculite. When the one leaf and one heart stage, wrap the seedlings with sponge strips and fix them on the foam board with holes drilled, and transfer the foam board to the incubator containing the nutrient solution Continue to grow inside, the nutrient solution contains 114.3mg / L (1.43mM) NH 4 NO 3 , 50.4mg / L of NaH 2 PO 4 2H 2 O, K at 89.3 mg / L 2 SO 4 , 110.8mg / L of CaCl 2 , 405.0mg / L of MgSO 4 , 1.6mg / L of MnSO 4 H2O, 18.8 μg / L Na 2 MoO 4 ·H 2 O, 1.2 mg / L of H 3 BO 3 , 43.8μg / L ZnSO 4 ·7H ...

Embodiment 3

[0087] Example 3 Using internal reference genes to detect low nitrogen stress genes in barley

[0088] A method for detecting the expression of the process-related protein gene PR1 (GenBank: Z21494.1) in barley under low nitrogen stress, comprising the following steps:

[0089] 1) Low nitrogen stress treatment and sampling

[0090] The barley seedlings of two barley genotypes (BI-04 and BI-45) were cultured to the three-leaf and one-heart stage, and placed in low-nitrogen nutrient solution for low-nitrogen stress treatment. and the stem and leaf parts of barley after 24 hours as samples;

[0091]Wherein, in the low-nitrogen nutrient solution, NH 4 NO 3 The concentration is 0.1 ~ 1.0mM;

[0092] 2) Extract RNA, reverse transcription and synthesize cDNA

[0093] Use Invitrogen's Trizol reagent to complete RNA extraction, and refer to the kit instructions for specific procedures.

[0094] Then use Promega's RQ1 DNase kit for DNA treatment, and then use TaKaRa's PrimeScript ...

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Abstract

The invention discloses a reference gene for barley gene expression researches as well as application of the reference gene. The reference gene is a gene segment of a ubiquitin-conjugating enzyme E2, an elongation factor EF2 and / or a tubulin beta-tubulin 6, wherein a nucleotide sequence of the gene segment of the ubiquitin-conjugating enzyme E2 is shown as SEQ ID NO.1, a nucleotide sequence of the gene segment of the elongation factor EF2 is shown as SEQ ID NO.2, and a nucleotide sequence of the gene segment of the tubulin beta-tubulin 6 is shown as SEQ ID NO.3. According to the reference gene, the detection accuracy of a barley related gene (such as low nitrogen stress and drought stress) expression can be improved, and the reference gene is large in expression quantity and stable in expression, and has a Ct value of 20 to 30.

Description

technical field [0001] The invention belongs to the field of plant molecular biology, and in particular relates to an internal reference gene for barley gene expression research and application thereof. Background technique [0002] Fluorescent quantitative PCR has been widely used in the study of gene expression changes in plants to reveal plant-related mechanisms. [0003] Especially in recent years, with the development of genome-wide RNA sequencing technology, it is possible to discover differentially expressed genes on a large scale by using this technology, and then serve for related mechanism research. For the verification of RNA sequencing, fluorescent quantitative PCR is basically used first. . The key to the accuracy of fluorescent quantitative PCR detection is to have one or a group of very reliable internal reference genes. [0004] Barley is the fourth largest cereal crop in the world, with wide distribution, good adaptability and strong stress tolerance, and ...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/6851C12Q1/6895C12Q2600/13C12Q2600/158C12Q2600/166C12Q2531/113C12Q2545/101C12Q2563/107
Inventor 陈志伟陆瑞菊黄剑华刘成洪何婷郭桂梅
Owner SHANGHAI ACAD OF AGRI SCI
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