A kind of filamentous fungal promoter and its application
A filamentous fungus, promoter technology, applied in fungi, using vectors to introduce foreign genetic material, DNA/RNA fragments, etc.
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Embodiment 1
[0043] Obtaining Aspergillus terreus citrate synthase (CS) promoter sequence
[0044] Primers Pcs-F1 (5'-CAAGCTCGAGTGGTTGGTAGTGTGGATTATG-3') and Pcs-R (5'-CACGCCATGGTTCGAATCCAACAAGAGTC-3') were designed according to the information published in the Aspergillus terreus NIH2624 genome database, and were purchased from Aspergillus terreus strain NRRL 1960 (ATCC 10020, CICC40205, The genome from China Industrial Microorganism Culture Collection Management Center) was used as a template, and pfu DNA polymerase (Fermentas, Catalog No.: EP0501) was used for PCR to amplify Pcs1, and the length of Pcs1 was detected by 1.0% agarose gel electrophoresis to be about 1895 bp The band of the target product was cloned into the pMD18-T vector (Takara, Catalog No.: D101A) by TA, and the plasmid pJL-1 was obtained after sequencing. Sequencing confirmed that the DNA sequence of the promoter Pcs1 of Aspergillus terreus citrate synthase is shown in SEQ ID NO.1.
[0045] SEQ ID NO.1:
[0046] tggt...
Embodiment 2
[0083] Construction of promoter activity analysis vector
[0084] Using PgpdAT-F1 (5'-GACCTCGAGATATGGCCTACGAACGAATTGC-3') and PgpdAT-R2 (5'-CCCGGGTCTAGATGTGATGATTGATGAGTTGTTGG-3') as a primer pair, PCR was performed using the genome of Aspergillus terreus NRRL1960 as a template, and the PCR product was gelled by 1% agarose The band with a size of about 2000 bp detected by gel electrophoresis was the target fragment, and the target fragment was recovered by tapping the gel and cloned into the pMD18-T-simple vector (Takara, Catalog No.: D103A) by TA to obtain the plasmid pJL-2.
[0085] Using mtpMD18-F (5'-GGATAACGCAGGAAAGAAGATGTGAGCAAAAGGC CAG-3') and mtpMD18-R (5'-CTGGCCTTTTGCTCACATCTTCTTTCCTGCGTTAT CC-3') as primers and pJL-2 as template, pJL-2 was subjected to site-directed mutation to remove pMD18-T- The PciI restriction site on the simple to obtain the plasmid pJL-3. The specific process is as follows: PCR amplification under the action of pfu DNA polymerase (pre-denatura...
Embodiment 3
[0089] Construction of Aspergillus terreus citrate synthase promoter activity analysis vector
[0090] Plasmid pJL-5 was digested with XhoI (Fermentas, Catalog No.: ER0691) and PciI, and a gth fragment with a size of 7.4 kb was recovered.
[0091] Using Pcs-F1 (5'-CAAGCTCGAGTGGTTGGTAGTGTGGATTATG-3') and Pcs-R (5'-CACGCCATGGTTCGAATCCAACAAGAGTC-3') as primers and plasmid pJL-1 as a template, PCR was performed to amplify Pcs1, and the PCR product was passed through 1% agarose gel The size detected by electrophoresis is about 1.9kb. After the PCR product is double-digested with XhoI (Fermentas, Catalog No.: FD0694) and NcoI (Fermentas, Catalog No.: FD0573), a band with a size of about 1.9kb is recovered, namely SEQ ID NO .1, connect with the above-mentioned recovered gth fragment, pick positive clones for verification after transformation, obtain plasmid pJL-6 after sequencing verification is correct (see figure 2 ).
[0092] Using Pcs-F2 (5'-CAAGCTCGAGCGAATCTGGCAACGTCCTCCG-3')...
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