灰葡萄孢鞘脂代谢通路基因Bcsphl1克隆及功能分析

Cloning and sequence expression analysis of sphingolipid metabolic pathway gene Bcsphl1 in necrotrophic fungus Botrytis cinerea

为明确灰葡萄孢Botrytis cinerea鞘脂代谢通路基因Bcsphl1的生物信息学特征及其在菌核发育中的潜在功能,采用CTAB法和Trizol法提取灰葡萄孢菌丝、菌核的gDNA和总RNA,利用PCR技术克隆其Bcsphl1基因,运用生物信息学软件对其进行鉴定和分析,并利用半定量反转录-聚合酶链反应(semi-quantitative reverse transcription and polymerase chain reaction,SqRT-PCR)技术测定其在灰葡萄孢菌核不同形成阶段的表达情况。结果表明:Bcsphl1基因序列全长1833 bp,编码360个氨基酸;Bcsphl1基因编码蛋白的分子质量为40.94 kD,理论等电点为7.73,分子式为C_(1889)H_(2868)N_(492)O_(507)S_(11),不稳定系数为31.19,脂肪指数为100.47,亲水性平均指数为0.160,属于结构稳定的疏水性蛋白。该基因编码的蛋白质无信号肽,定位于细胞质膜,含有3个跨膜结构域和1个脂肪酸羟化酶保守结构域。氨基酸序列同源性比对发现Bcsphl1氨基酸序列与灰葡萄孢T4菌株鞘氨醇羟化酶类蛋白的一致性达98.23%。Bcsphl1基因在灰葡萄孢菌核形成初期(6 d)、中期(9 d)和后期(12 d)均有较高水平表达,其中在中期表达水平最高,表明其可能参与灰葡萄孢菌核形成的调控。

To clarify bioinformatics characteristics and evaluate the potential functions of sphingolipid metabolic pathway gene Bcsphl1 in sclerotial development,CTAB and Trizol methods were used to extract the gDNA and total RNA from sclerotia of necrotrophic fungus Botrytis cinerea,the Bcsphl1 gene was cloned using PCR and sequenced,bioinformatic analyses were conducted on the sequence data,and the expression of Bcsphl1 in different sclerotial developmental periods was quantified using semiquantitative reverse transcription and polymerase chain reaction(SqRT-PCR).The results were as follows:the length of the Bcsphl1 gene was 1833 bp in total;and the gene encodes a protein of 360 amino acids,with a molecular weight of 40.94 kD,a theoretical isoelectric point pI of 7.73,a molecular formula of C_(1889)H_(2868)N_(492)O_(507)S_(11),an instability index of 31.19,an aliphatic index of 100.47,and an average hydrophilicity of 0.160;the protein,which contains three transmembrane domains and one fatty acid hydroxylase conservative domain,has no signal peptide and was located in the plasma membrane of cells.The amino acid sequence and structures of the protein encoded by Bcsphl1 have high similarities with sphingosine hydroxylase proteins in Botrytis cinerea T4 strain(98.23%).Furthermore,Bcsphl1 was expressed at the early(6 d),middle(9 d)and late stages(12 d)of sclerotial development of Botrytis cinerea,and the highest expression level was found at the middle stage,indicating that Bcsphl1 may be participated in regulating sclerotial development.