1,3-specific lipase, its coding gene sequence and its use
A lipase, specific technology, applied in genetic engineering, plant genetic improvement, application, etc., can solve the problems of the research on the lipase gene of Acremonium compactum that have not been seen
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Embodiment 1
[0133] Example 1. Cloning of Acremonium compacta lipase gene
[0134] The 1,3-specific lipase gene lipAS was cloned from Acremonium compactus strain CGMCC4420 by molecular biology methods, and the specific steps were as follows:
[0135] 1. Obtaining the Conserved Sequence of the Lipase Gene
[0136] The PCR primers Blip-1 and Blip-2 for amplifying the conserved sequence of the lipase gene were designed and synthesized, and their sequences are as follows:
[0137] Blip-1 (SEQ ID NO:3): 5'-GTS(C / G)GTS(C / G)CTCGCCTTCCGCGG-3'
[0138] Blip-2 (SEQ ID NO:4): 5'-GCCGCACCAGR(A / G)CTR(A / G)TGR(A / G)CC-3'
[0139] For S(C / G) and R(A / G) in the sequence, the content in brackets refers to the possible situation of degenerate bases outside the brackets.
[0140] The SV Total RNA Isolation System Kit (SV Total RNA Isolation System Kit, Cat#Z3100) from Promega Company was used to extract the total RNA of Acremonium compactus, and the first strand cDNA synthesis kit (First Strand cDNA Synt...
Embodiment 2
[0152] Example 2. Recombinant expression of Acremonium compacta lipase gene in Aspergillus niger
[0153] 1. Cloning of gene lipAS
[0154] According to the full-length gene sequence of lipAS, the amplification primers qh-lip1 and qh-lip2 were designed and synthesized, and their sequences are as follows:
[0155] Upstream qh-lip1 (SEQ ID NO:9):
[0156] 5'-AAA ctgcag ATGCGGCATCCGCAGTCTCT-3'
[0157] Downstream qh-lip2 (SEQ ID NO: 10):
[0158] 5'-CC aagctt TTAATGGTGGTGATGATGGTGTAACTCATCGCCATTCACG-3'
[0159] Wherein, PstI and HindIII restriction sites (shown underlined) were respectively introduced into the primers qh-lip1 and qh-lip2. Downstream qh-lip2 introduces a 6×His-tag tag (ATGGTGGTGATGATGGTG, shown in bold).
[0160] Total RNA was extracted from Acremonium compacta and reverse transcribed. The cDNA obtained by reverse transcription was used as a template, and the primers qh-lip1 and qh-lip2 were used as a primer pair for PCR amplification. The specific c...
Embodiment 3
[0183] Embodiment 3. Enzymatic property analysis of recombinant lipase
[0184] 1. Effect of reaction temperature on pure enzyme activity of Acremonium compactus recombinant lipase
[0185] At 20°C, 30°C, 40°C, 50°C, and 60°C, the activity of the pure enzyme lipase was measured according to the pNPP method described in Example 2, and the highest enzyme activity was taken as the relative enzyme activity, and the activity of the pure enzyme at other temperatures was calculated. Relative vitality.
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