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Method for detecting salidroside synthetase gene expression activity by using dual reference genes

A technology of gene expression and salidroside, which is applied in biochemical equipment and methods, microbiological determination/testing, DNA/RNA fragments, etc., can solve the problem of detection of key enzyme activities in metabolic pathways, inaccurate quantification, and time-consuming And other problems, to achieve the effect of good application prospects

Inactive Publication Date: 2016-08-24
SHANXI UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, these methods have disadvantages such as time-consuming, insignificant effect, and inaccurate quantification.
In addition, the practice of promoting the accumulation of salidroside in Rhodiola rosea plants by using endophytic fungal resources to co-exist with hosts by rebuilding microorganisms or their communities is an emerging method to solve problems, and there is no key enzyme activity in the metabolic pathway detection report

Method used

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  • Method for detecting salidroside synthetase gene expression activity by using dual reference genes
  • Method for detecting salidroside synthetase gene expression activity by using dual reference genes
  • Method for detecting salidroside synthetase gene expression activity by using dual reference genes

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0023] 1) Tissue culture seedlings of Rhodiola grandiflora were obtained by tissue culture method. Inoculate fungus CGMCC No.9347, culture in greenhouse (light intensity 30 μmol m-2 s-1; light culture 14h, temperature 28°C; dark culture 10h, temperature 22°C; humidity 70%-75%), regularly (4d, 6d, 8d, 10d, 12d, 14d, 16d) collect Rhodiola rosea tissue-cultured seedlings; collect non-inoculated tissue-cultured seedlings as a control (ck).

[0024] 2) Extraction of total RNA: extract total RNA of Rhodiola grandiflora tissue culture seedlings by conventional methods, and synthesize the first strand of cDNA.

[0025] 3) Using the primers provided in Table 1 and using the first strand of cDNA as a template, 5 genes were amplified by quantitative PCR. The amplification reaction program was: pre-denaturation at 94°C for 30s, denaturation at 95°C for 5s, annealing at 56°C for 15s, extension at 72°C for 10s, 45 cycles.

[0026] 4) The expression activity of TyDC, the key enzyme gene of...

Embodiment 2

[0029] 1) Tissue culture seedlings of Rhodiola grandiflora were obtained by tissue culture method. Inoculate fungus CGMCC No.9347, culture in greenhouse (light intensity 30 μmol m-2 s-1; light culture 14h, temperature 28°C; dark culture 10h, temperature 22°C; humidity 70%-75%), regularly (4d, 6d, 8d, 10d, 12d, 14d, 16d) collect Rhodiola rosea tissue-cultured seedlings; collect non-inoculated tissue-cultured seedlings as a control (ck).

[0030] 2) Extraction of total RNA: extract total RNA of Rhodiola grandiflora tissue culture seedlings by conventional methods, and synthesize the first strand of cDNA.

[0031] 3) Using the primers provided in Table 1 and using the first strand of cDNA as a template, 5 genes were amplified by quantitative PCR. The amplification reaction program was: pre-denaturation at 94°C for 30s, denaturation at 95°C for 5s, annealing at 56°C for 15s, extension at 72°C for 10s, 45 cycles.

[0032] 4) The expression activity of TAT, the key enzyme gene of ...

Embodiment 3

[0035] 1) Tissue culture seedlings of Rhodiola grandiflora were obtained by tissue culture method. Inoculate fungus CGMCC No.9347, culture in greenhouse (light intensity 30 μmol m-2 s-1; light culture 14h, temperature 28°C; dark culture 10h, temperature 22°C; humidity 70%-75%), regularly (4d, 6d, 8d, 10d, 12d, 14d, 16d) collect Rhodiola rosea tissue-cultured seedlings; collect non-inoculated tissue-cultured seedlings as a control (ck).

[0036]2) Extraction of total RNA: extract total RNA of Rhodiola grandiflora tissue culture seedlings by conventional methods, and synthesize the first strand of cDNA.

[0037] 3) Using the primers provided in Table 1 and using the first strand of cDNA as a template, 5 genes were amplified by quantitative PCR. The amplification reaction program was: pre-denaturation at 94°C for 30s, denaturation at 95°C for 5s, annealing at 56°C for 15s, extension at 72°C for 10s, 45 cycles.

[0038] 4) GAPDH, PCS double internal reference gene combination wa...

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Abstract

The invention provides a method for detecting salidroside synthetase gene expression activity by using dual reference genes. Specifically, a primer which is obtained through design and screening is adopted to amplify to obtain glyceraldehydes phosphate dehydrogenase and phytochelatin synthetase genes, and correct and detect the gene expression activity of three key enzymes, namely, tyrosine decarboxylase, tyrosine transaminase and phenylalanine ammonialyase of synthesized salidroside in rhodiola crenulata tissue culture seedlings by using the gene as the dual reference genes. The method comprises the following steps: extracting total RNA of a rhodiola crenulata plant; synthesizing a cDNA first chain; and by using a fluorescent quantitative PCR technique, amplifying by using a given primer so as to obtain the dual reference genes of GAPDH and PCS, and correcting and detecting the expression activity of synthesized salidroside key enzyme genes TyDC, TAT and PAL in the rhodiola crenulata tissue culture seedlings. The method has the advantages of rapidness, accuracy, practicability, stability and the like in aspects such as production quality control and product activity detection on rhodiola crenulata tissue culture seedlings, cell suspension culture, hairy root culture and the like.

Description

technical field [0001] The invention belongs to the detection of key enzyme activities of active compounds in plants, and in particular relates to the correction detection of gene expression of phenylalanine ammonia lyase, tyrosine decarboxylase and tyrosine transaminase in Rhodiola daflora by using double internal reference gene combination active method. Background technique [0002] Rhodiola crenulata (Rhodiola crenulata) is a rare medicinal plant belonging to the genus Rhodiola (Crassulaceae) of Crassulaceae. It is a perennial succulent herb. Arid, cold, and hypoxic mountainous areas, it is the only source plant designated by the traditional Chinese medicine Rhodiola rosea included in the Pharmacopoeia of the People's Republic of China, and it is also a folk botanical medicine that has been used by mountain people in my country for nearly a thousand years. It is mainly used to treat altitude sickness and hypoxia, and is known as "high altitude ginseng". Its main active ...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12Q1/527C12Q1/52C12N15/11
CPCC12Q1/6851C12Q1/6895C12Q2600/158C12Q2600/166C12Q2531/113C12Q2545/101C12Q2563/107
Inventor 崔晋龙王雅楠王梦亮
Owner SHANXI UNIV
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