甘蔗镰孢菌FsANT基因的克隆及表达模式分析

甘蔗是我国主要的糖料作物，是生产蔗糖最主要的原料，甘蔗生产过程中受到的病虫害威胁给蔗糖产业造成了严重的损失。甘蔗镰孢菌（Fusarium sacchari）引起的梢腐病是一种真菌性病害，严重影响甘蔗作物生产。镰孢菌致病基因的研究对于梢腐病的防治具有重要意义。腺苷酸转移酶（adenine nucleotide translocase, ANT）介导线粒体与细胞质基质之间ADP/ATP的交换，在真核细胞能量代谢中发挥重要作用，与细胞的生长、发育和凋亡密切相关。本研究克隆获得了长936 bp具有完整开放阅读框（open reading frame, ORF）的甘蔗镰孢菌ANT基因序列，命名为FsANT，其编码311个氨基酸，并且与小麦条锈菌病原菌ANT蛋白和小麦白粉病病原菌ANT序列相似性较高。蛋白软件分析显示，该基因编码的蛋白为稳定的疏水蛋白，含有5个跨膜结构，一个ADP/ATP transporter结构域，有3个同源重复的MCF基序，与粒体转运蛋白家族（mitochondrial carrier family，MCF）的基本结构特点相吻合。软件预测分析发现FsANT基因可能定位在线粒体。qRT-PCR结果显示，FsANT基因在菌丝生长时期的表达相对稳定无显著差异；在与甘蔗互作过程的表达模式表现为：接种后12 h该基因开始上调表达，24 h表达量显著升高，72 h达到表达高峰。推测FsANT基因的表达不仅与甘蔗镰孢菌细胞的生长相关，同时可能参与F. sacchari与甘蔗的互作过程，且主要在侵染后期发挥功能。研究病原菌生长保守基因为甘蔗抗梢腐病育种提供新思路。

Cloning of an FsANT Gene in Fusarium sacchari and Analysis on Its Expression Pattern During Infection Process

Sugarcane is the main sugar crop in China and the most important raw material for sugar production. The threat of diseases and insect pests in sugarcane production has caused serious losses to the sugar industry. Tip rot caused by Fusarium sacchari is a fungal disease that seriously affects sugarcane crop production. It is of great significance to study the pathogenic genes of F. sacchari. Adenine nucleotide translocase (ANT) mediates the exchange of ADP/ATP between mitochondria and cytoplasmic matrix, which plays an important role in eukaryotic cell energy metabolism and is closely related to cell growth, development and apoptosis. In this study, a 936 bp ANT gene sequence of F. sacchari with a complete open reading frame (ORF) was cloned, tentatively named FsANT, encoding 311 amino acids. FsANT protein of F. sacchari was found to cluster with ANT protein of other pathogens, indicating that FsANT may had the similar function. Softwares analysis showed that the protein encoded by this gene was a stable hydrophobic protein, containing five transmembrane structures. In addition, ANT has three homologous and repeated MCF motifs in one ADP/ATP transporter domain, which conforms to the basic structural characteristics of Mitochondrial carrier family (MCF). The software predicted that the gene might target mitochondria. qRT-PCR results showed that FsANT gene expression was relatively stable during mycelia growth period without significant difference. In the process of interaction with sugarcane, the expression pattern of this gene began to be up-regulated 12 h after inoculation, and increased significantly 24 h after inoculation, and reached its peak at 72 h. It is speculated that the expression of FsANT gene was not only related to the growth of F. sacchari cells, but also maight participate in the interaction between F. sacchari and sugarcane, and mainly played a role in the later stage of infection. The study of pathogenic growth conserved genes would provide a new idea for sugarcane breeding against pokkah boeng disease.