Fungal nitrilase and gene sequence thereof
A technology of nitrilase and fungal nitrile, applied in the directions of hydrolase, genetic engineering, plant gene improvement, etc., can solve the problem that the fungal nitrilase has not been paid enough attention, the coding gene of nitrilase has not been elucidated in large quantities, and it is impossible to obtain large-scale nitrilase. Scale enzyme products and other issues
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Embodiment 1
[0036] This example illustrates Gibberella ( Gibberella intermedia ) Extraction of CA3-1 total RNA.
[0037] Gibberella ( Gibberella intermedia ) CA3-1 strain in enzyme production medium (glucose 10 g / L, yeast extract 7.5 g / L, NaCl 1.16 g / L, KH 2 PO 4 2.72 g / L, FeSO 4 0.03 g / L, caprolactam 3.39 g / L) for 2 days at 30°C. Collect the bacteria by vacuum filtration and wash with sterile water 2-3 times, put the wet bacteria in a glass homogenizer, add 1 mL Trizol reagent, homogenize at low temperature for 2 min; transfer the homogenate to a 1.5 mL centrifuge tube , add 0.2 mL of chloroform, shake vigorously for 30 s, place at room temperature for 3 min, and centrifuge at 12,000 rpm at 4 °C for 10 min; transfer the upper aqueous phase to a clean centrifuge tube, add 1 / 2 times absolute ethanol (v / v), Mix well; transfer the mixture to the adsorption column with a pipette, let stand at room temperature for 2 minutes, centrifuge at 12,000 rpm for 3 minutes, discard the waste liqu...
Embodiment 2
[0039] This example illustrates the process of obtaining cDNA sequence by reverse transcription reaction.
[0040] Using total RNA as a template, reverse transcription was performed using AMV reverse transcriptase to synthesize the first strand of cDNA. The reverse transcription reaction was carried out according to the following conditions: incubation at 42-60°C for 15-30 minutes, inactivation of AMV reverse transcriptase at 99°C for 5 minutes, and storage at 5°C for 5 minutes.
[0041] In the reverse transcription tube add Ex Taq ? HS enzyme and related reagents synthesize the second strand of cDNA, and further amplify the cDNA sequence by PCR. The nucleotide sequence am (ATGTCCAAGWCYCTCAARGT) is used as the upstream primer, and the M13 Primer M4 universal primer is used as the downstream sequence. The PCR reaction was carried out in a 50 μL system, and the reaction conditions were as follows: pre-denaturation at 94°C for 2 min and cycle; denaturation at 94°C for 30 s, ann...
Embodiment 3
[0043] This example illustrates the method for cloning the gene encoding fungal nitrilase.
[0044] Gibberella ( Gibberella intermedia ) The cDNA sequence of CA3-1 is used as a template, and the following nucleotide sequences are used as primers to amplify the coding gene of fungal nitrilase by PCR.
[0045] Primer P1: 5'-CCGGAATTCATGTCCAAGACTTCTCAAAGTCG-3'
[0046] Primer P2: 5'-CCCAAGCTTTCACAGGTCGTTGGCAAAG-3'
[0047] The PCR reaction was carried out in a 50 μL system, and the reaction conditions were pre-denaturation at 94°C for 2 min and cycling; denaturation at 94°C for 30 s, annealing at 61°C for 30 s, and extension at 72°C for 1 min, a total of 30 cycles; final extension at 72°C 10 min. The PCR products were recovered by slicing gel after agarose gel electrophoresis, connected with pMD-19 T vector and transformed into Escherichia coli E. coli JM109, positive transformants were screened on LB plates containing ampicillin resistance (100 mg / L). The positive transfor...
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