Scylla paramamosain resistance factor SpALF6 single nucleotide mutant as well as preparation method and application thereof
A single nucleotide mutation, Scylla sinensis technology, applied in the directions of botanical equipment and methods, biochemical equipment and methods, applications, etc., can solve the problem of little anti-Candida albicans activity and the like
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Embodiment 1
[0068] Example 1: Acquisition of the full-length nucleic acid sequence and amino acid sequence of the disease resistance factor SpALF6 of Scylla pseudomae
[0069] Total RNA was extracted from the blood cell samples of Scylla pseudocarpus, and after testing to meet the requirements, the cDNA sequences of a large number of genes were obtained through transcriptome sequencing and data analysis. Using the online software blastx to analyze one of the full-length 756bp sequences, it was found that the sequence was 78% homologous to Portunus trituberculatus PtALF6 in the Genbank database, indicating that the sequence was a new member of the ALF family of Scylla cryptae, named SpALF6, its nucleotide sequence and predicted amino acid sequence are shown in SEQ ID NO: 1 and SEQ ID NO: 2, respectively.
[0070] Among them, the full-length cDNA of SpALF6 (SEQ ID NO: 1) contains 681 bp, wherein the 79th to 426th nucleotides are the open reading frame (SEQ ID NO: 5) encoding the amino acid ...
Embodiment 2
[0072] Example 2: Acquisition of SpALF6 single nucleotide mutant SpALF6-M sequence
[0073] The DNA sequence of the single nucleotide mutant SpALF6-M was obtained by fusion PCR technology. The primers SpALF6EF:5'-TACTCAGAATTCTTCGTGAAGGAACTACTCT-3'(SEQ ID NO:6) and SpALF6MR:5'-TACTCGGTAGTTACAGACACGGTC-3'(SEQ ID NO:8) were paired, and the mutation site was obtained by PCR using the SpALF6 nucleic acid sequence as a template (Containing) the upstream nucleic acid fragment; SpALF6MF:5'-TCCTTGACCGTGTCTGTAACTAC-3'(SEQ ID NO:9) and primer SpALF6ER:5'-TACTCACTCGAGCTATTCTAAGAAGAGTCG-3'(SEQ ID NO:7) are paired, using the SpALF6 nucleic acid sequence as a template, Obtain nucleic acid fragments downstream of the mutation site (including) by PCR; take 0.5 μL of the above two PCR products, mix them as subsequent PCR templates, and use SpALF6EF and SpALF6ER as paired primers to obtain the corresponding SpALF6-M mature peptide region by PCR. nucleic acid sequence. Finally, compared with th...
Embodiment 3
[0074] Embodiment 3: Construction of SpALF6 and SpALF6-M prokaryotic expression vector
[0075] The DNA sequence corresponding to the SpALF6 mature peptide was amplified with the upstream primer SpALF6EF and the downstream primer SpALF6ER. The PCR reaction conditions were: pre-denaturation at 95°C for 3 min, denaturation at 95°C for 30 s, annealing at 53°C for 45 s, extension at 72°C for 45 s, a total of 35 cycles, and a final extension at 72°C for 5 min. PCR products were analyzed by 1.5% agarose gel electrophoresis and positive fragments were recovered.
[0076] The above-mentioned purified and recovered nucleic acid fragments and the expression vector pET30a were respectively digested with endonucleases EcoRI and XhoI, and after digesting at 37°C for 3 hours, 1 μL of the digested products were subjected to 1% agarose gel electrophoresis to detect the digestion effect. The electrophoresis results of the purified and recovered nucleic acid fragments and the expression vector...
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