Dandelion sterol synthase, gene encoding dandelion sterol synthase and its preparation and application
A dandelion sterol and gene technology, applied in the fields of application, genetic engineering, plant gene improvement, etc., can solve problems that have not been reported
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Embodiment 1
[0039] Example 1. Acquisition of the full-length cDNA sequence of the dandelion sterol synthase gene in the root, stem, leaf and milk transcriptome data and metabolite integration analysis
[0040] 1. According to the integrated analysis of the transcriptome data of the roots, stems, leaves and milk of Eucalyptus japonica (NCBI accession number: root, SRR6282416; stem, SRR6282415; leaf, SRR6282414; milk, SRP073643), after removing redundant and repetitive sequences, use Trinity software (Grabherr et al., 2011) reintegrated the transcriptome data and screened a total of 7 oxidative squalene cyclase (OSC) genes. The abundance of the transcripts of the seven OSC genes in each tissue sample was calculated and normalized using the FPKM method, as figure 1 As shown in A.
[0041] 2. At the same time, GC-MS was used to detect the content of dandelion sterol in four different tissues, such as figure 1 Shown in B. The specific method is as follows:
[0042]Take 100 mg of fresh root...
Embodiment 2
[0044] Embodiment 2, the acquisition of the full-length cDNA sequence of dandelion sterol synthase gene
[0045] 1. Extract total RNA from different tissues of Eucalyptus japonica, such as figure 2 shown. The specific method is as follows:
[0046] Weigh about 100mg of Eucalyptus japonica roots, stems, and leaves for rapid grinding in liquid nitrogen, and use the EASYspin plus Plant RNA Rapid Extraction Kit (Adelaide) to extract total RNA from the ground sample powder.
[0047] 2. Reverse transcription of RNA into cDNA. The specific method is as follows:
[0048] Add the following components to an RNase-free PCR tube: RNA: 8 μl (about 1.5 μg), DNase I: 1 μl, 10×DNaseI buffer: 1 μl, RNase inhibitor: 0.5 μl. Mix the above substances and incubate at 37°C for 30min. Then add 1 μl of 100mM EDTA, and incubate at 65°C for 10min to terminate the reaction. Add 1 μl Oligo(dT)18 and 1 μl dNTPMIX (10 mM) to the PCR tube of the above mixture, pre-denature at 65°C for 5 min, and then...
Embodiment 3
[0054] Embodiment 3, the application of dandelion sterol synthase gene
[0055] The dandelion sterol synthase gene with the correct sequencing result in Example 2 was cloned into the yeast expression vector pESC-Ura to form the yeast expression plasmid pESC-Ura-EtOSC6 containing the dandelion sterol synthase gene. Transfer the recombinant plasmid pESC-Ura-EtOSC6 into Saccharomyces cerevisiae, use the empty vector of yeast cells (pESC-Ura) as the control group experiment, pick multiple transgenic yeast single colonies in 5ml containing 20μg / ml ergosterol, 13μg / ml chlorine Heme and 0.5% Tween 80 in the SD-Ura deficient medium, cultured at 250 rpm at 30°C for 24 hours, took 1ml of glycerol to preserve the strains, and obtained pESC-Ura-EtOSC6 as the vector, and Saccharomyces cerevisiae as the vector. The dandelion sterol-producing engineering bacteria constructed by the host bacteria. The remaining bacterial liquid was collected by centrifugation at 5000 rpm, washed once with do...
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