Fluorescent quantitative reference genes of different tissue of cryptomeria fortunei as well as special primer and application of fluorescent quantitative reference genes

A fluorescence quantitative, internal reference gene technology, applied in the field of plant molecular biology, to achieve the effects of high amplification efficiency, improved detection efficiency, and improved reliability

Pending Publication Date: 2021-06-01
NANJING FORESTRY UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

In the process of molecular biology research on Cedar, it is inevitable to involve the study of Cedar gene expression, but there are basically no reports on the internal reference genes used to analyze and verify the expression level of Cedar gene by qRT-PCR, and the stable

Method used

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  • Fluorescent quantitative reference genes of different tissue of cryptomeria fortunei as well as special primer and application of fluorescent quantitative reference genes
  • Fluorescent quantitative reference genes of different tissue of cryptomeria fortunei as well as special primer and application of fluorescent quantitative reference genes
  • Fluorescent quantitative reference genes of different tissue of cryptomeria fortunei as well as special primer and application of fluorescent quantitative reference genes

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Experimental program
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Effect test

Embodiment 1

[0030] The screening of the most stably expressed internal reference gene in different tissues of Cryptomeria comprises the following steps:

[0031] 1. Sampling of test materials: Collect cedars that grow well in the Baima base of Nanjing Forestry University, and collect cedar roots, stems, leaves and fruits in July 2020, store them in dry ice and bring them back to the laboratory, and place them in a -80°C refrigerator save.

[0032] 2. Extraction of total RNA from different tissues: The total RNA from different tissues of Cedar cedar was extracted by using the general-purpose total RNA extraction kit of Biotech Biotechnology Company according to the instructions.

[0033] 3. Synthesis of the first strand of cDNA:

[0034] The cDNA reverse transcription kit ( III RT SuperMix for qPCR (+gDNA wiper) R323-01) reverses the total RNA to eDNA as a template for fluorescent quantitative PCR.

[0035] 1) Prepare the following mixture in an RNase-free centrifuge tube: 4x gDNA wipe...

Embodiment 2

[0063] Three internal reference genes UBI, CYP, and SDH1 with comprehensive ranking stability and unstable internal reference gene 18S rRNA were used for verification.

[0064] In order to verify the stability of the internal reference genes screened, the expression patterns of CESA, CHS and PYL in different tissues of Cryptomeria were analyzed.

[0065] The specific primers for the CESA gene are:

[0066] CESA-F: 5'-CCAACGGTCAAGTGGTGTCT-3';

[0067] CESA-R: 5'-GGTTAGGGTCAGCATAGGGA-3'.

[0068] The specific primers for the CHS gene are:

[0069] CHS-F: 5'-ATGCCAAATGTCGCCAAAGT-3';

[0070] CHS-R: 5'-GTCGTTTCTCAAGAGCGTGCC-3'.

[0071] The specific primers for the PYL gene are:

[0072] PYL-F: 5'-CACAGGCTACGGGATTACCA-3':

[0073] PYL-R: 5'-GCAAATTACACCGCACAACG-3'.

[0074] In the expression of CESA, CHS and PYL ( Figure 4 , Figure 5 and Figure 6 ), when the unstable internal reference gene 18S rRNA was used as the internal reference gene, it was highly expressed in s...

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Abstract

The invention discloses fluorescent quantitative reference genes of different tissue of Cryptomeria fortunei as well as special primers and application thereof, and belongs to the technical field of plant molecular biology. The fluorescent quantitative reference gene is a UBI gene, a CYP gene and an SDH1 gene, and the nucleotide sequences of the UBI gene, the CYP gene and the SDH1 gene are respectively shown as SEQ ID NO.1, SEQ ID NO.2 and SEQ ID NO.3. Twelve candidate reference gene sequences are screened through cryptomeria fortunei transcriptome data, the stability of the candidate reference genes is evaluated by using three reference gene stability evaluation software, and the optimal genes UBI, CYP and SDH1 for real-time fluorescent quantitative PCR of different tissue of cryptomeria fortunei are obtained. And a real-time fluorescent quantitative PCR primer of the reference gene is designed, and the primer is high in specificity and amplification efficiency, so that the detection efficiency of detecting the cryptomeria fortunei gene in a real-time fluorescent quantitative manner can be greatly improved, and the credibility of a detection result is improved.

Description

technical field [0001] The invention belongs to the technical field of plant molecular biology, and in particular relates to fluorescent quantitative internal reference genes of different tissues of cedar cedar, special primers and applications thereof. Background technique [0002] Real-time fluorescent quantitative PCR (qRT-PCR) has become the main method for studying gene expression due to its high sensitivity, strong reproducibility, strong specificity, rapidity and high efficiency. However, qRT-PCR will be affected by RNA purity, quantity and reverse transcription efficiency, so it is necessary to introduce internal reference genes as standards to accurately quantify gene expression. [0003] An internal reference gene refers to a gene that can be stably expressed in different plant tissues, different growth stages and under different conditions under ideal conditions, and is not affected by the external environment. A large number of reports have identified suitable i...

Claims

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Application Information

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