An internal reference gene suitable for gene expression analysis in different tissues of cigars and its application
An internal reference gene and gene technology, applied in the field of molecular biology, can solve the problems of unstable expression and unreported screening of internal reference genes in cigars, and achieve the effect of improving accuracy
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Embodiment 1
[0033] Example 1 Screening of candidate internal reference genes
[0034] 1. Screening of candidate reference genes for cigars
[0035] From the transcriptome sequencing data (uploaded to the China Nucleic Acid Database https: / / bigd.big.ac.cn / gsa. Accession 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 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 EIF2 Eula...
Embodiment 2
[0052] Example 2 Candidate internal reference gene primer design and qRT-PCR
[0053] The sequence of the gene was obtained by three-generation sequencing combined with transcriptome sequencing, and then specific qPCR primers were designed. The candidate internal reference gene primer sequences are shown in Table 3. Mix equal amounts of cDNA from 10 samples (full bloom (root, stem, leaf, calyx, corolla, stigma, ovary, stamen, pistil) and seeds) as templates, and dilute them according to a 5-fold gradient, that is, the concentration of each template 1 and 10 respectively -1 , 10 -2 , 10 -3 , 10 -4 , a total of 5 gradients were used to perform fluorescence 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 20 μL reaction system: 10 μL 2×SYBR I Master, 0.5 μL upstream and downstre...
Embodiment 3
[0060] Example 3 Stability analysis of candidate internal reference genes
[0061] In order to screen the best reference gene, the stability of each candidate reference gene was analyzed and evaluated by three softwares: geNorm, NormFinder and Bestkeeper. Among them, geNorm software starts with C t The value is the original data, and the expression stability (M) of the candidate internal reference gene in different samples is calculated to determine its expression stability. The larger the M value, the lower the gene stability. On the contrary, the smaller the M value, the higher the gene stability. high. Generally, when the M value is less than or equal to 1.5, the gene expression is considered to be 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 most unstable.
[0062] NormFinder software is similar to geNorm, but also uses C t The value is the original data, and the express...
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