Phage display cDNA cloning and expression analysis of hydrophobins from the entomopathogenic fungus Beauveria (Cordyceps) bassiana
Phage display cDNA cloning and expression analysis of hydrophobins from the entomopathogenic fungus Beauveria (Cordyceps) bassiana
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DOI：
10.1099/mic.0.2007/008532-0
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发表时间：
2007-10-01
期刊：
影响因子：
2.8
通讯作者：
Keyhani, Nemat O.
中科院分区：
文献类型：
--
作者：
Cho, Eun-Min;Kirkland, Brett H.;Keyhani, Nemat O.
疏水素是一种小的两亲性蛋白质，在真菌的广泛生长和发育过程中发挥作用。它们参与气生结构的形成，真菌细胞与表面的附着，并在响应表面线索和病程的信号传递中发挥作用。球孢白僵菌是一种重要的昆虫病原真菌，可用作节肢动物生防剂。为了验证利用噬菌体展示技术克隆疏水素编码基因的可行性，我们用N，N‘-二乙酰壳二糖、岩藻糖、乳糖、麦芽糖和蜂蜜二糖偶联的琼脂糖珠进行了生物扫描实验。经过五轮迭代生物扫描后，使用二糖或乳糖偶联的琼脂糖珠选择性地富集了两个球孢杆菌(1类)疏水蛋白对应的cDNA。表达分析表明，Hyd1基因在所有供试样品中均有表达，包括气生分生孢子、体外培养的胚芽孢子、沉水分生孢子以及在甲壳素和昆虫角质层上形成孢子的细胞，其中Hyd1基因在生长菌丝体中的表达最高。相反，Hyd2基因在任何单细胞类型(气生分生孢子、胚芽孢子和沉水分生孢子)中都不表达，但在生长中的菌丝体和细胞在甲壳素和昆虫角质层上产孢时表达。MS指纹图谱在沸腾的小岛屿发展中国家中发现了类似10 kDa的蛋白质-气生分生孢子不溶于三氟醋酸的提取物，鉴定出球孢杆菌小棒层的主要成分是hy2基因产物。这些结果揭示了分离的疏水蛋白的差异调控，表明噬菌体展示技术是一种克隆疏水蛋白基因的新方法。
Hydrophobins are small amphipathic proteins that function in a broad range of growth and developmental processes in fungi. They are involved in the formation of aerial structures, the attachment of fungal cells to surfaces, and act in signalling in response to surface cues and pathogenesis. Beauveria bassiana is an important entomopathogenic fungus used as an arthropod biological control agent. To examine the feasibility of using phage display technology to clone cDNAs encoding hydrophobins, biopanning experiments were performed using a variety of affinity resins, including N,N'-diacetylchitobiose-, fucose-, lactose-, maltose- and melibiose-coupled agarose beads. After five rounds of iterative biopanning, cDNAs corresponding to two B. bassiana (class 1) hydrophobins were selectively enriched using melibiose- or lactose-coupled agarose beads. Expression analysis revealed that the hyd1 gene was expressed in all samples tested, including aerial conidia, in vitro blastospores, submerged conidia, and cells sporulating on chitin and insect cuticle, with hyd1 expression peaking in growing mycelia. In contrast, the hyd2 gene was not appreciably expressed in any of the single-cell types (aerial conidia, blastospores and submerged conidia), but was constitutively expressed in growing mycelia and when cells were sporulating on chitin and insect cuticle. MS fingerprinting of an similar to 10 kDa protein found in boiling SIDS-insoluble, trifluoroacetic acid-soluble extracts from aerial conidia identified the major component of the B. bassiana rodlet layer to be the hyd2 gene product. These results reveal the differential regulation of the isolated hydrophobins and indicate that phage display represents a novel approach to cDNA cloning of hydrophobins.