Molecular cloning and characterization of a lysozyme cDNA from the mole cricket Gryllotalpa orientalis (Orthoptera: Gryllotalpidae)
Molecular cloning and characterization of a lysozyme cDNA from the mole cricket Gryllotalpa orientalis (Orthoptera: Gryllotalpidae)
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DOI：
10.1007/s11033-014-3446-5
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发表时间：
2014-09-01
影响因子：
2.8
通讯作者：
Cho, Saeyoull
中科院分区：
文献类型：
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作者：
Kwon, Hyojung;Bang, Kyeongrin;Cho, Saeyoull
克隆了东方绿僵菌溶菌酶全长基因，并进行了序列测定。该溶菌酶蛋白氨基酸序列全长143个氨基酸，分子量为15.84 kDa，等电点为4.74。序列分析、比对和系统发育分析结果表明，东方棉溶菌酶属于C(鸡)型溶菌酶蛋白家族。侧耳溶菌酶的蛋白质序列与果蝇溶菌酶有很高的同源性(51.7%)，但与黑腹果蝇溶菌酶不同的是，侧耳溶菌酶是免疫诱导型的，在多种组织中都有表达。东方落叶松溶菌酶基因在感染后8h表达最高，随后随感染时间延长而下降。我们还利用pET表达系统在体外表达了侧耳乳杆菌溶菌酶蛋白。与阴性对照相比，过量表达的侧耳溶菌酶对革兰氏阴性杆菌大肠杆菌和革兰氏阳性菌枯草芽孢杆菌均表现出抗菌活性，其最小抑菌浓度分别为30.3和7.55AµM。这些结果表明，侧耳溶菌酶可能比其他溶菌酶对多种微生物具有更强的抗菌活性。
A full-length lysozyme cDNA from Gryllotalpa orientalis was cloned and sequenced. The deduced amino acid sequence of the lysozyme protein was 143 amino acids in length, with a calculated molecular mass of 15.84 kDa and an isoelectric point of 4.74. Sequence motifs, together with alignment and phylogenetic results, confirmed that G. orientalis lysozyme belongs to the C (chicken)-type lysozyme family of proteins. The protein sequence of lysozyme from G. orientalis showed high identity to that of Drosophila melanogaster (51.7 %); however, in contrast to D. melanogaster lysozyme, G. orientalis lysozyme was immune inducible and expressed in a wide range of tissues. Expression of G. orientalis lysozyme mRNA was highest at 8 h post-infection and subsequently decreased with time after bacterial infection. We also expressed G. orientalis lysozyme protein in vitro using the pET expression system. Compared with the negative control, over-expressed G. orientalis lysozyme showed antimicrobial activity against Gram-negative bacteria Escherichia coli and Gram-positive bacteria Bacillus subtilis by radial diffusion assay, with minimal inhibitory concentration values of 30.3 and 7.55 A mu M, respectively. These results indicate that G. orientalis lysozyme may have stronger antimicrobial activity than other lysozymes against a broad range of microorganisms.