Cloning and expression of beta-1, 4-endo-mannanase (An Man5A) gene
A mannanase and gene technology, applied in the field of bioengineering, can solve problems that have not been reported in research, and achieve the effects of large industrial production and application potential, economic value, high catalytic activity and thermal stability.
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Embodiment 1A
[0038] Cloning of embodiment 1An man5A 3' end mRNA sequence
[0039] The first strand of cDNA was synthesized by reverse transcription with Oligo dT-Adaptor Primer; the first round of PCR was performed with M13 Primer M4 and ManF1 as primers (94°C for 2min; 30 cycles, 94°C for 30s, 53°C for 30s, 72 ℃90s; 72℃10min); the second round of PCR was performed with the first round PCR product as template and M13Primer M4 and ManF2 as primers (94℃2min; 30 cycles, 94℃30s, 53℃30s, 72℃90s; 72 ℃ 10min). The two rounds of PCR products were analyzed by 1% agarose gel electrophoresis, the target band was recovered by slicing the gel and ligated with pUCm-T (pUCm-T-Anman5A3′), transformed into JM109, and sent to Shanghai Sangon for sequencing.
Embodiment 2A
[0040] Cloning of Example 2An man5A 5' end mRNA sequence
[0041] The first round of PCR was performed with 5′RACE Outer Primer (TaKaRa Company) and ManR1 as primers (94°C 3min; 30 cycles, 94°C 30s, 55°C 30s, 72°C 1min; 72°C 10min); with 5′RACE Inner Primer and ManR2 were used as primers for the second round of PCR (94°C for 3min; 30 cycles, 94°C for 30s, 53°C for 30s, 72°C for 1min; 72°C for 10min). The two rounds of PCR products were analyzed by 1% agarose gel electrophoresis, the target band was recovered and ligated with pUCm-T (pUCm-T-Anman5A5′), transformed into JM109, and then sent to Shanghai Sangon for sequencing.
Embodiment 3A
[0042] Cloning of Example 3 An man5A 5' end promoter sequence
[0043] Using the processed A.niger LW-1 genomic DNA as a template, the first round of PCR uses LD and ManR1 as primers, and the reaction conditions are: 94°C for 4min; 30 cycles, 94°C for 30s, 53°C for 30s, and 72°C for 30s; 10 min at 72°C; the second round of PCR was performed using LD and ManR2 as primers (4 min at 94°C; 30 cycles, 30 s at 94°C, 30 s at 55°C, 30 s at 72°C; 10 min at 72°C). The two rounds of PCR products were analyzed by 2% agarose gel electrophoresis, the target band was recovered by slicing the gel and ligated with pUCm-T (pUCm-T-Anman5AP), transformed into JM109, and sent to Shanghai Sangon for sequencing.
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