Construction method and application of monascus purpureus YJX-8 cDNA library

[0017] Example 1 Construction of Monascus purpureus YJX-8 cDNA library

[0018] 1. Monascus purpureus YJX-8 culture

[0019] Monascus purpureus YJX-8 was inoculated into PDA slant medium, and cultured at 30°C for 72h, so that the mycelium covered the medium. The spores were washed with sterile water, and the spore suspension was incubated at 30°C for 5 days at 180 rpm.

[0020] 2. Monascus purpureus YJX-8 total RNA extraction

[0021] The hyphae were rinsed with DEPC water, dried with filter paper, quickly placed in a pre-cooled mortar, and fully ground into powder by adding liquid nitrogen.

[0022] 3. First-strand synthesis of Monascus purpureus YJX-8 cDNA

[0023] Using Monascus purpureus YJX-8 total RNA as template, according to Gold yeast two-hybrid system (Clontech company) instructions to synthesize the first strand of cDNA.

[0024] Add 2μL of RNA, 2μL of Oligo-dT Primer, ddH to the centrifuge tube 2 O 1 μL, the reaction conditions are 72 °C, 2 min; ice bath for...

[0036] Example 2 Screening of Monascus purpureus YJX-8 ester synthase LIP05 interacting protein

[0037] 1. Construction of pGBKT7-LIP05 bait vector

[0038] The primers were designed with the LIP05 sequence after removing the signal peptide as the template. The primer sequences are as follows:

[0039] LIP05-F: 5'-ATGGAGGCC GAATTC CTCCCCCCTAACACCC-3';

[0040] LIP05-R: 5’-GATCCCCGG GAATTC TCACGATGAAGCAGC-3'.

[0041] The plasmid containing the LIP05 gene was used as a template for PCR amplification to obtain the target gene fragment, and the bait vector pGBKT7 was digested with EcoR I to obtain the target vector fragment. After the fragment was recovered and purified by gel, it was ligated and transformed, and the recombinant vector pGBKT7-LIP05 was successfully obtained. .

[0042] 2. Decoy vector pGBKT7-LIP05 toxicity and transcriptional autoactivation activity detection

[0043] Take 1 μL of pGBKT7-LIP05 and pGBKT7 plasmids to transform Y2HGold yeast strains, respe...