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Method for studying populus diversifolia gene by using multi-reference-gene combination

An internal reference gene, Populus euphratica technology, applied in the field of plant molecular biology, can solve problems such as inaccurate deviation and wrong experimental results, achieve high reliability and promote improvement

Inactive Publication Date: 2014-07-02
BEIJING FORESTRY UNIVERSITY
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Problems solved by technology

A wide range of experimental data shows that the stability of internal reference genes may be affected by conditions such as different tissues, different developmental stages, or different environmental stresses, so a single internal reference may lead to biased, inaccurate or even wrong experimental results

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  • Method for studying populus diversifolia gene by using multi-reference-gene combination

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Embodiment 1

[0040] A method of using multiple internal reference gene combinations to study Populus euphratica genes, comprising the steps of:

[0041] Step 1, carry out stress resistance treatment to Populus euphratica, and extract the total RNA of Populus euphratica under each treatment respectively, reverse transcribe into cDNA, as the template of PCR;

[0042] 1. The stress resistance treatment comprises the steps of:

[0043] Take Populus euphratica seedlings from the Xinjiang Uygur Autonomous Region in April every year, and plant them in round pots with a diameter of 60 cm and a height of 50 cm in early April, with 3-5 plants per pot. The soil is mainly sandy soil, containing a small amount of vermiculite and flower nutrient soil. , pour water twice a week, and treat the seedlings for 3-4 months, that is, at the beginning of July or at the end of the month, when the height of the seedlings is 60-80cm, carry out stress-resistant treatment, and the stress-resistant treatment method is...

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Abstract

The invention discloses a method for studying a populus diversifolia gene by using a multi-reference-gene combination. The method comprises the following steps: (1) extracting total RNA of populus diversifolia, and reversely transcribing total RNA into cDNA as a template of PCR; (2) designing a specific primer to react on a real-time fluorescent quantitative PCR instrument through an SYBR Green method by using not less than 9 reference genes to obtain a CT value; (3) determining the number of reference genes by taking 0.15 as a threshold value through a formula and a PV value calculation function of geNorm software; (4) selecting the reference genes according to the required number, performing qRT-PCR reaction together with a target gene, and calculating the obtained CT value through the geNorm software to obtain change data of the target gene in a process of obtaining relative expression quantity by using the multi-reference-gene combination in each treatment. Compared with a conventional one-reference-gene method, the method has the characteristic that a relatively reliable conclusion can be obtained.

Description

technical field [0001] The invention relates to the field of plant molecular biology, in particular to a method for studying stress-resistant key genes of Populus euphratica by real-time fluorescence quantitative PCR using a combination of multiple internal reference genes. Background technique [0002] Populus euphratica Oliv is an ancient tree species remnant in the third century. It survived on the earth more than 60 million years ago. It is distributed in the desert areas of Asia and Africa. It is the only one that can grow in the world's largest mobile desert, the Taklamakan Desert. The arbor tree species that have been built together have long adapted to the extreme desert environment of drought, salinity and drastic temperature changes, and have evolved a complete mechanism to deal with adversity factors such as drought, salinity, and low temperature. Therefore, Populus euphratica contains rich stress resistance gene resources waiting Development, and with the complet...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68
CPCC12Q1/686C12Q2545/101C12Q2563/107
Inventor 尹伟伦夏新莉王厚领
Owner BEIJING FORESTRY UNIVERSITY
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