Method for screening real-time fluorescent quantitative PCR internal reference molecules of desert plant Eremosparton songoricum (Litv.) Vass.

A real-time fluorescence quantitative and internal reference technology, applied in biochemical equipment and methods, fluorescence/phosphorescence, microbial measurement/inspection, etc., can solve the problems of blank research on internal reference molecules and unreported internal reference gene sequence data

Inactive Publication Date: 2012-01-18
XINJIANG INST OF ECOLOGY & GEOGRAPHY CHINESE ACAD OF SCI
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Problems solved by technology

[0008] In recent years, some stress-resistant genes have been cloned from Junggar leafless bean, such as the dehydration response element binding protein (DREB) transcription factor gene (NCBI accession number: HQ687367). The charact

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  • Method for screening real-time fluorescent quantitative PCR internal reference molecules of desert plant Eremosparton songoricum (Litv.) Vass.
  • Method for screening real-time fluorescent quantitative PCR internal reference molecules of desert plant Eremosparton songoricum (Litv.) Vass.
  • Method for screening real-time fluorescent quantitative PCR internal reference molecules of desert plant Eremosparton songoricum (Litv.) Vass.

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Embodiment Construction

[0125] In the following examples, the experimental methods without specific conditions are usually carried out according to routine experimental operations. It was carried out according to the method described in "Refined Molecular Biology Experiment Guide" (Edited by F.M Osper, R.E. Kingston, J.G Seidman, translated by Ma Xuejun, Shu Yuelong, Beijing: Science Press, 2004).

[0126] Cloning of internal reference molecules:

[0127] Using the published GAPDH, Actin, 18SrRNA, UBQ, EF, α-tubulin, β-tubulin gene sequences in the NCBI database as reference, design specific primers for each internal reference gene, and Two-week-old leafless bean seedlings cultured in an incubator (temperature 25°C, time 12h / 12h) were used as materials, RNA was extracted, cDNA was reversed, and target genes were amplified with designed primers for each internal reference gene, and each amplified gene was recovered. A specific fragment of the gene is added, connected to the cloning vector, sequenced ...

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Abstract

The invention relates to a method for screening real-time fluorescence quantitative PCR internal reference molecules of a desert plant Eremosparton songoricum (Litv.) Vass.. The invention is characterized in that homologous clone is used for cloning 8 internal reference molecules in Eremosparton songoricum (Litv.) Vass., and a real-time fluorescent quantitative PCR internal reference gene specific primer is designed, 10 types of seedling grown for two weeks with abiotic stress and non stress (contrast) are taken as the experimental materials for carrying out a fluorescent quantitative PCR experiment to verify, analysis of fluorescent quantitative data is carried out by using geNorm software, so that the optimum and the stablest internal reference molecules screened for fluorescent quantitative researches under various abiotic stresses developed by the seedling of Eremosparton songoricum (Litv.) Vass. are EF and alpha-tubulin. The method of the invention is capable of avoiding the errors on RNA quality, output, inverse transcription efficiency and the like of the seeding material of Eremosparton songoricum (Litv.) Vass. under different abiotic stresses, better correcting and standardizing the data obtained by the real-time fluorescent quantitative determination and enhancing the accuracy and reliability of researches.

Description

technical field [0001] The invention relates to a method for screening internal reference molecules suitable for the real-time fluorescent quantitative PCR research of desert plant Junggar leafless bean seedlings under various abiotic stresses Background technique [0002] With global warming and the intensification of the greenhouse effect, agricultural production in arid areas is facing severe challenges. Facing the grim reality of my country's growing population, dwindling arable land and water and fertilizer resources, and increasingly sharp contradictions between food supply and demand, how to quickly increase crop yields while saving resources and improving the environment has become a major demand for the sustainable and efficient development of my country's agriculture. The drought-resistant gene resources accumulated in the long-term evolution process in arid areas are the source of germplasm innovation! [0003] The harsh natural environment in the arid region has...

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Application Information

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IPC IPC(8): C12Q1/68G01N21/64
Inventor 李小双张道远杨红兰裴金玲
Owner XINJIANG INST OF ECOLOGY & GEOGRAPHY CHINESE ACAD OF SCI
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