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Cryptomeria fortunei fluorescent quantitative internal reference gene under abiotic stress and special primer and application thereof

A technique of abiotic stress and fluorescent quantification, applied in the field of fluorescent quantitative internal reference genes and their special primers, to achieve strong specificity, improve detection efficiency, and improve reliability

Active Publication Date: 2021-05-14
NANJING FORESTRY UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, there have been no reports on the gene research of Cedar abiotic stress

Method used

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  • Cryptomeria fortunei fluorescent quantitative internal reference gene under abiotic stress and special primer and application thereof
  • Cryptomeria fortunei fluorescent quantitative internal reference gene under abiotic stress and special primer and application thereof
  • Cryptomeria fortunei fluorescent quantitative internal reference gene under abiotic stress and special primer and application thereof

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0058]1, total RNA extraction and cDNA synthesis

[0059]The total RNA of each sample was extracted by the total RNA extraction kit specification of Baortictor Biotechnology Co., Ltd.. Its integrity, purity, and concentrations were detected by 1% agarose gel electrophoresis and UV-lamination meter (NanoDrop2000, Thermo ScientiFC, Wilmington, DE, USA).

[0060]Template with total RNA, use RT-QPCR special premix kit (III RT SuperMix ForqPCR (+ GDNA WiPER) R323-01 Nanjing Novhashi Biotechnology Co., Ltd.) Synthesis cDNA, 0.8 μgRNA is used to synthesize the first chain cDNA, stored in the -20 ° C refrigerator.

[0061]2, the choice of internal parametrin gene and the design of its primer

[0062]By investigating an interferencer in other species with a local BLAST comparison (BLASTVER: 2.4.0+), it is found to find the highest homologous gene, and then the BLASTX on the NCBI website has been conserved. The domain and ORF conducted online analysis, eventually selected and designed, and the 15 candida...

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Abstract

The invention discloses a cryptomeria fortunei fluorescent quantitative internal reference gene under abiotic stress and a special primer and application thereof, and belongs to the technical field of plant molecular biology. The abiotic stress comprises drought stress, heat damage stress, low temperature stress and salt stress. 15 candidate internal reference genes are screened and designed, the stability of the candidate genes is evaluated through five algorithms (delta-CT, geNorm, NormFinder, BestKeeper and RefFinder), the fluorescent quantitative internal reference genes suitable for the cryptomeria fortunei under abiotic stress are obtained, real-time fluorescent quantitative PCR primers of the reference genes are designed. The primer has the advantages of strong specificity and high amplification efficiency, can greatly improve the detection efficiency when the cryptomeria fortunei gene is quantitatively detected by real-time fluorescence, and improves the credibility of the detection result.

Description

Technical field[0001]The present invention belongs to the field of plant molecular biology, and specifically, the fluorescence quantitative inner ginsence and its special primers and applications, including drought stress, thermal damage, low temperature stress and salt stress.Background technique[0002]Cryptomeria Fortunei Hooibrenk, also known as long leafquaro, a paraffler for Shanko Liu Pread. Nonbiobiostatic factors such as drought, heat removal, low temperature, salt damage, etc., can cause a series of physiological metabolic reactions in plants. However, the study of genes under non-biological stress in the will now have not been reported. Differences in gene expression in non-biological stress in Tulu, involves the use of real-time fluorescence quantitative techniques analysis and verify gene expression levels, requiring stable and reliable interference, so that steady expression of cocci biodias is screened. The internal parametric gene is a key factor in real-time fluoresce...

Claims

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Application Information

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IPC IPC(8): C12Q1/6851C12Q1/6895C12N15/11
CPCC12Q1/6851C12Q1/6895C12Q2600/166C12Q2531/113C12Q2563/107C12Q2545/101
Inventor 徐进崔洁冰张莹婷胡海亮
Owner NANJING FORESTRY UNIV
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