Diosgenin synthesis related protein as well as coding gene and application thereof
A technology of diosgenin and related proteins is applied in the directions of application, genetic engineering, plant genetic improvement, etc., can solve problems such as acid pollution, and achieve the effect of good application prospect.
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Embodiment 1
[0050] Example 1 Content analysis of diosgenin in leaves and tuber tissues of Dioscorea shield
[0051] Wild Dioscorea scutellaria was collected in Zhuxi County, Hubei Province, China, and its tubers were transplanted into the soil for plant propagation. The leaves and tubers of 5-month seedling age and the tubers of 8-month seedling age were collected (representative plants such as figure 1 Shown in A and B) for the extraction of diosgenin and diosgenin, the extraction method is as follows:
[0052] Take about 1 g of fresh plant tissue and grind the sample into powder in liquid nitrogen. The sample powder is ultrasonically extracted in 1 ml of methanol at 30°C for 20 min (3 biological replicates for each sample), and the methanol extract is evaporated to dryness in vacuum. , Adding sulfuric acid with a final concentration of 1.5M, acidolysis at 100°C for 4 hours, the residue after acidolysis was extracted with n-hexane, washed with water until neutral, the n-hexane extract wa...
Embodiment 2
[0055] Example 2 Obtaining the full-length cDNA sequence of the diosgenin synthesis gene
[0056] 1, get the total RNA ( figure 2 ), the specific method is as follows:
[0057] About 100 mg of Dioscorea scutellaria plant tubers were weighed and quickly ground in liquid nitrogen, and the ground sample powder was extracted with the EASYspin plus plant RNA rapid extraction kit (Adelaide) to extract total RNA.
[0058] 2. Reverse transcribe RNA into cDNA, the specific method is as follows:
[0059] Add the following components to an RNase-free PCR tube: 4 μl RNA (about 1.0 μg), 1 μl Oligo(dT)18, and 1 μl ldNTP MIX (10 mM). The above mixture was pre-denatured at 65°C for 5 minutes, and then immediately placed on ice, followed by adding 4 μl of 5× reverse transcriptase buffer, 0.5 μl of RNase inhibitor and 1 μl of reverse transcriptase, mixed well and incubated at 42°C for 1 hour to synthesize First strand cDNA. The synthesized cDNA was stored in a -80°C refrigerator.
[0060]...
Embodiment 3
[0071] Example 3 Application of diosgenin synthesis gene
[0072] The CYP90B71 and CYP90G6 genes were cloned into the same yeast expression vector pESC-URA to obtain the recombinant vector pESC-URA-CYP90B71-CYP90G6, and the CYP94N8 gene was cloned into the yeast expression plasmid pESC-Leu2d-CPR to obtain the recombinant vector pESC-Leu2d-CPR- CYP94N8. The recombinant plasmids pESC-URA-CYP90B71-CYP90G6 and pESC-Leu2d-CPR-CYP94N8 were simultaneously transferred into the cholesterol-producing Saccharomyces cerevisiae RH6829 strain (this strain has been described in the literature Souza, C.M. et al. A stable yeast strain efficiently producing cholesterol instead ofergosterol is functional for tryptophan uptake, but not weak organic acid resistance. Metab.Eng.13, 555-569 (2011), which can be provided by Zhang Yansheng's research group of Shanghai University), the control is the co-expression of empty vector pESC-URA and pESC-Leu2d-CPR For transformation, the transgenic yeast was ...
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