Method for constructing monascus strain capable of achieving high yield of acid protease
A technology of acid protease and monascus, which is applied in the direction of microorganism-based methods, botany equipment and methods, biochemical equipment and methods, etc., can solve the problems of non-directional stability and large working range of natural breeding methods, and achieve Strong stability, good market application prospects, and the effect of genetically stable transformants
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Embodiment 1
[0030] Embodiment 1: Cloning of Monascus acid protease gene
[0031] 1. Monascus RNA extraction and cDNA double-strand synthesis:
[0032] Scrape off the Monascus hyphae cultured on malt agar medium for 12 days with a sterile spoon, grind with liquid nitrogen and extract its RNA with OMEGA’s fungal RNA extraction kit; take 500ng of the extracted Monascus RNA and use reverse transcription reagents The cassette synthesizes the first strand and the second strand, and the synthesized cDNA double strands are stored at -20°C.
[0033] 2. Primer design and gene amplification:
[0034] According to the Monascus purple acid protease gene published in GenBank Asp (AB090877.1) sequence (SEQIDNO.1) and features of the multiple cloning site of plasmid pBC-Hygro, designed with software PrimerPremier5.0 Asp Gene primers. The primer sequences are:
[0035] Asp -ORF-F:5'-TCCCCCGGGATGGTCGTCTTTCAGCAAGATCAC-3'
[0036] Asp -ORF-R: 5'-TCCCCCGGGTTATGCCTGAGGGGCAAATCCGA-3'
[0037] Using the...
Embodiment 2
[0038] Example 2 Acid protease gene Monascus expression vector pBC-Hygro- Asp The build:
[0039] The sequence of Example 1 is correct Asp Fragment and vector pBC-Hygro were used separately Sma Carry out enzyme digestion with I restriction endonuclease, use gel extraction kit to recover and purify the digested Asp and linear pBC-Hygro vector, then use T4DNA ligase to Asp The fragment is cloned into the pBC-Hygro vector; the recombinant expression vector pBC-Hygro- Asp The construction map is as figure 2 shown.
[0040] Use pBC-Hygro empty vector and recombinant expression vector pBC-Hygro- Asp Electrophoresis together, the results show that the recombined plasmid is about 1200bp longer than the empty plasmid vector, as shown in image 3 as shown, image 3 Middle M: DL23130DNAMarker, 1: vector pBC-Hygro- Asp , 2: pBC-Hygro vector; primers for recombinant plasmid Asp -ORF-F and Asp -ORF-R was amplified by PCR to obtain a specific band of about 1200bp, such as Fi...
Embodiment 3
[0041] Example 3 Agrobacterium-mediated homologous transformation of Monascus
[0042] 1. Mediated transformation:
[0043] The carrier pBC-Hygro- Asp The recombinant plasmid was transformed into Agrobacterium tumefaciens. Pick a single colony of recombinant Agrobacterium from the YEB plate (containing 34 μg / mL chloramphenicol, 50 μg / mL rifampicin) and inoculate it in YEB liquid medium (containing 34 μg / mL chloramphenicol, 50 μg / mL rifampicin), Shake the bacteria at 28°C and 200r / min in the dark for 2 days.
[0044] 2. PCR verification:
[0045] Take the bacterial liquid cultivated in step 1 for PCR verification to determine whether the recombinant vector has been transferred into Agrobacterium. Add the inducer of Agrobacterium tumefaciens to the recombinant Agrobacterium bacterium cultured in step 1, and shake the bacteria at 200r / min for 4h; the inducer is 200μmol / mL acetosyringone solution; -80 (v / v) of 0.85% saline (m / v) eluted Monascus spores from solid culture.
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