Reference gene suitable for gene expression analysis of different tissues of cigar and application thereof
An internal reference gene and gene expression technology, applied in the field of molecular biology, can solve the problems of unreported internal reference gene screening and unstable expression in cigar tobacco
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Embodiment 1
[0033] Example 1 Screening of Candidate Internal Reference Genes
[0034] 1. Screening of cigar tobacco candidate internal reference genes
[0035] From the transcriptome sequencing data (uploaded to China Nucleic Acid Database https: / / bigd.big.ac.cn / gsa. Retrieval number: CRA003020), 10 gene sequences with high and consistent expression in roots, stems and leaves were selected as candidate internal reference genes (Table 1). Primer design was performed using Primer Premier 5.0 (Table 2).
[0036] Table 1 Candidate internal reference gene information
[0037] gene name gene description Seq ID RPL8 60S ribosomal protein L8 transcript_19714 RPL14A 60S ribosomal protein L14-1 transcript_22219 RPS16 40S ribosomal protein S16 transcript_22287 UBC27 Ubiquitin-conjugating enzyme E2 27 transcript_20180 UBC28 Ubiquitin-conjugating enzyme E2 28 transcript_20220 UBC35 Ubiquitin-conjugating enzyme E2 35 transcript_21000...
Embodiment 2
[0052] Example 2 Candidate Internal Reference Gene Primer Design and qRT-PCR
[0053] Through the method of three-generation sequencing combined with transcriptome sequencing, the sequence of the gene was obtained, and then specific qPCR primers were designed. The primer sequences of candidate internal reference genes are shown in Table 3. The cDNA of 10 samples (full flowering stage (root, stem, leaf, calyx, corolla, stigma, ovary, stamen, pistil) and seeds) was mixed in equal amounts as a template, and diluted according to a 5-fold gradient, that is, the concentration of each template 1, 10 respectively -1 、10 -2 、10 -3 、10 -4 , a total of 5 gradients were carried out for fluorescent quantitative PCR, and a standard curve was drawn (such as figure 2 shown).
[0054] Use Roche Light Cycler 96 real-time fluorescence quantitative PCR instrument to carry out RT-qPCR amplification reaction, add the following reagents in sequence to the 20 μL reaction system: 10 μL 2×SYBR I ...
Embodiment 3
[0060] Example 3 Candidate Internal Reference Gene Stability Analysis
[0061] In order to screen the best internal reference gene, the stability of each candidate internal reference gene was analyzed and evaluated by three softwares: geNorm, NormFinder and Bestkeeper. Among them, geNorm software uses C t The value is the original data, and the expression stability of the candidate internal reference gene is judged by calculating the expression stability value (M) in different samples. The larger the M value, the lower the gene stability. On the contrary, the smaller the M value, the higher the gene stability. high. Usually, when the M value is less than or equal to 1.5, it is considered that the expression of the gene is stable and suitable for use as an internal reference gene. Among the 10 candidate genes, RPL14A and UBC27 were the most stable, and PSAP was the least stable.
[0062] The NormFinder software is similar to geNorm and also uses C t The value is the origina...
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