Screening method for reference genes in different tissues of cunninghamia lanceolata and application of screened genes as reference genes

A technology of internal reference genes and screening methods, which is applied in the field of fluorescent quantitative PCR detection, and can solve the problems of screening internal reference genes, etc.

Active Publication Date: 2018-05-29
FUJIAN AGRI & FORESTRY UNIV
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  • Application Information

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Problems solved by technology

[0004] In recent years, there have been many reports on the screening of plant internal reference genes. At the same time, more and more scholars have studied the molecular regulation mechanism of Chinese fir. The molecular regulation mechanism largely depends on the expression of the target gene, and the expression of the target gene It is usual

Method used

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  • Screening method for reference genes in different tissues of cunninghamia lanceolata and application of screened genes as reference genes
  • Screening method for reference genes in different tissues of cunninghamia lanceolata and application of screened genes as reference genes
  • Screening method for reference genes in different tissues of cunninghamia lanceolata and application of screened genes as reference genes

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Experimental program
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Effect test

Embodiment 1

[0078] The roots, stems and leaves of Chinese fir seedlings were selected as the research objects of this experiment. The Hoagland nutrient solution was used for sand culture, the pH of the nutrient solution was 5.8, and the formula of the nutrient solution was shown in Table 1. After the cultivation, the roots, stems and leaves of Chinese fir were collected and quickly frozen in liquid nitrogen and stored at -80°C for later use.

[0079] Table 1 Hoagland complete nutrient solution

[0080]

[0081]

[0082] 1.2 Total RNA extraction and cDNA synthesis

[0083] Based on the RNAprep Pure PlantKit kit from TIANGEN, total RNA was extracted from roots, stems, and leaves of Chinese fir seedlings. The integrity of total RNA was detected by 1.0% agarose gel electrophoresis and stored in a -80°C ultra-low temperature refrigerator.

[0084] The GoScriptTM Reverse Transcription System kit from Promega Company was selected, and a cDNA strand of Chinese fir roots, stems, and leaves ...

Embodiment 2

[0113] Detection of target gene expression

[0114] 18S rRNA was used as an internal reference gene to detect the target gene, which is the transcription factor ClPHR1 of fir phosphorus transporter. Real-time fluorescent quantitative PCR was used to detect the phosphorus supply (1.0mmol / L KH 2 PO 4 ) for 3 days, the expression of ClPHR1 gene in the roots, stems and leaves of No. 32 phosphorus-efficient Chinese fir family. and leaves, with lower expression levels in stems.

[0115] Table 5 Expression analysis of ClPHR1 gene in different tissues of Chinese fir No. 32

[0116] tissue site

Embodiment 3

[0118] 18S rRNA was used as an internal reference gene to detect the expression of Chinese fir cellulose synthase genes ClCesA1 and ClCesA2 in the roots, stems and leaves of No. 32 Chinese fir family. The expression level of ClCesA1 and ClCesA2 is much higher than that in stems and leaves, and the expression level in leaves is lower. The results show that ClCesA1 and ClCesA2 genes are mainly involved in regulating the synthesis of cellulose in Chinese fir stems and roots, which is consistent with the basic functions of these two genes.

[0119] Table 6 Expression analysis of ClCesA1 and ClCesA2 genes in different tissues of Chinese fir No. 32

[0120] tissue site

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Abstract

The invention provides a screening method for reference genes in different tissues of cunninghamia lanceolata and application of the reference genes and belongs to the field of fluorescent quantitation PCR (Polymerase Chain Reaction) detection. The screening method comprises the following steps: taking a glyceraldehyde-3-phosphate dehydrogenase gene, a transcriptional elongation factor, 18S rRNA,28S rRNA and a ubiquitin gene as candidate reference genes, and using the candidate reference genes as template design primers; extracting total RNA of roots, stems and leaf tissues of cunninghamia lanceolata seedlings and synthesizing cDNA with the total RNA as a template; carrying out qPCR amplification with the cDNA as the template to obtain a Ct value and relative expression Q; carrying out stability evaluation on the candidate reference genes by adopting a plurality of software according to the relative expression Q value and Ct value; sorting the stability of the candidate reference genes from strong to weak, and selecting candidate genes ranking the top two as the reference genes of the cunninghamia lanceolata. The 18S rRNA and/or ubiquitin gene obtained by screening is applied as the reference gene in different tissues of the cunninghamia lanceolata.

Description

technical field [0001] The invention belongs to the technical field of fluorescent quantitative PCR detection, and in particular relates to a screening method for internal reference genes of Chinese fir in different tissues and the application of screening genes as internal reference genes. Background technique [0002] Chinese fir (Cunninghamia lanceolata) is one of the most important coniferous fast-growing timber species in China. It has the characteristics of fast growth, high yield per unit area, and good material quality. Chinese fir has straight wood texture, uniform structure, strong corrosion resistance, and good insect resistance. It is widely used in making furniture, building bridges, ships, etc. At the same time, as an evergreen coniferous tree species, fir has a straight and round trunk, a conical crown, light-loving, good germination and renewal, and rapid growth. It is often used as an excellent tree species promoted by the garden department, and has good lan...

Claims

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Application Information

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IPC IPC(8): C12Q1/6895C12Q1/686
CPCC12Q1/686C12Q1/6895C12Q2600/166C12Q2563/159
Inventor 李明张颖胡霞吴鹏飞帅鹏邹显花马祥庆
Owner FUJIAN AGRI & FORESTRY UNIV
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