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Method for detecting expression quantity of miR156 of malus sieversii

A detection method and expression level technology, applied in the field of plant genes, can solve problems such as undiscovered patent publications, and achieve the effects of convenient operation and improved detection efficiency

Inactive Publication Date: 2017-02-15
TIANJIN AGRICULTURE COLLEGE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0003] Through the search, no patent publications related to the patent application of the present invention have been found

Method used

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  • Method for detecting expression quantity of miR156 of malus sieversii
  • Method for detecting expression quantity of miR156 of malus sieversii
  • Method for detecting expression quantity of miR156 of malus sieversii

Examples

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Effect test

Embodiment 1

[0063] A method for detecting the expression level of wild apple miR156 in Xinjiang, the steps are as follows:

[0064] Use CTAB method to extract the total RNA of leaves and callus of Xinjiang wild apple, PolyA tailing method and neck loop primer method to reverse RNA to obtain cDNA, after diluting the cDNA template by 2 times (it is best to take a total of 6 μL cDNA for each sample to do three techniques) Repeat), real-time fluorescent quantitative PCR was performed.

[0065] Specific steps are as follows:

[0066] (1) Extraction of total RNA

[0067] Total RNA was extracted by CTAB method, the specific steps are as follows:

[0068] 1. Take the leaves or callus of Xinjiang wild apples of different tree ages, and grind them into powder in liquid nitrogen.

[0069] 2. Take 0.1g of the powder and place it in a centrifuge tube added with 800μL lysate 1, vortex to mix, incubate at 65°C for 10min, and mix by inverting up and down 2-3 times during this period.

[0070] Wherein, ...

Embodiment 2

[0114] A method for detecting the expression level of wild apple miR156 in Xinjiang, the steps are as follows:

[0115] (1) Extraction of total RNA

[0116] Total RNA was extracted by CTAB method, the specific steps are as follows:

[0117] (1) Take the leaves or callus of Xinjiang wild apples of different ages, and grind them into powder in liquid nitrogen;

[0118] (2) Take the powder and place it in a centrifuge tube added with lysate 1, the ratio of powder to lysate 1 is 1:8000 in g:μL, vortex and mix, incubate at 60-70°C for 8-15min, and mix up and down during the period 2-3 times;

[0119] The composition and ratio of the lysate 1 are as follows:

[0120] 100mM pH 8.0 Tris-HCl;

[0121] 25m MEDTA;

[0122] 2M NaCl;

[0123] Mass final concentration is 2% CTAB;

[0124] The final mass concentration is 2% PVP;

[0125] Mass final concentration is 0.5% spermidine;

[0126] β-mercaptoethanol at a final concentration of 2% by volume;

[0127] (3) Then add chloroform...

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Abstract

The invention relates to a method for detecting expression quantity of miR156 of malus sieversii. The method comprises the following steps: (1) extracting total RNA; (2) inversely transcribing; (3) carrying out qRT-PCR detection; and (4) analyzing data obtained by qRT-PCR by using a method as shown in the specification, and making a histogram in an excel chart to obtain the relative expression quantity of targeted gene miR156 of the malus sieversii. The invention provides the novel method for extracting small RNA of the malus sieversii for the first time. The method can provide reference for detection of expression conditions of small RNA of ligneous plants, has reference significance for research of a phase transformation regulation and control net during vegetative growth and reproductive growth, and meanwhile, can also provide theoretical basis for crossbreeding and genetic improvement practice of the malus sieversii in the later period.

Description

technical field [0001] The invention belongs to the technical field of plant genes, in particular to a method for detecting the expression level of miR156 in Xinjiang wild apples. Background technique [0002] Xinjiang wild apple (Malus sieversii (Ledeb.) Roem.), also known as Seville's apple, is a perennial wild fruit tree of Rosaceae (Rosaceae) Malus, mainly distributed in Ili area of ​​Xinjiang in my country. Wild apples in Xinjiang are located in the western mountainous area. Affected by the environment, they often encounter adverse environmental effects such as drought, low temperature, and extreme heat during the flowering and fruiting stages. Reproduction and development, thus seriously affecting the population structure of the whole wild apple forest in Xinjiang. MiR156 is related to the growth and development of plants, especially plays a key role in the phase transition regulation of vegetative growth and reproductive growth. Therefore, using appropriate methods t...

Claims

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Application Information

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IPC IPC(8): C12Q1/68
CPCC12Q1/6851C12Q2521/107C12Q2531/113C12Q2545/101C12Q2563/107
Inventor 阎国荣龙鸿于玮玮胡宝全李慧李芳杨美玲张云秀
Owner TIANJIN AGRICULTURE COLLEGE
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