茯砖茶“金花菌”vcx基因克隆及表达分析

Cloning and Expression Analysis of vcx Gene of Aspergillus cristatus in Fuzhuan Brick Tea

茯砖茶是一种紧压黑茶,冠突曲霉是茯砖茶发花阶段的主要微生物,其有性发育产生闭囊壳又名"金花菌",其数量和质量常用来判断茯砖茶的品质好坏。冠突曲霉可以用Na Cl来控制其产有性孢子及无性孢子,为了研究这一特殊产孢机理,及钙信号和液泡在这产孢过程中的作用。通过克隆冠突曲霉钙信号途径中的液泡上钙氢交换子vcx基因,并对这一基因进行结构和表达分析。结果表示:冠突曲霉中的vcx基因编码区1 326 bp,不含内含子,预测编码441个氨基酸,不含信号肽,是一个疏水蛋白,含有11个跨膜结构,符合钙氢交换蛋白的基本结构。通过对其在冠突曲霉的有性、无性、菌丝各生长阶段的表达量进行测定,结果显示:vcx基因对其发育产生正调控,可能参与了冠突曲霉的无性产孢及盐压力响应过程。实验结果不仅为该蛋白的后续研究提供了条件,而且为钙信号调控丝状真菌在盐压力应答下的产孢机制奠定基础。

Aspergillus cristatus is the dominant fungus involved in the fermentation of Chinese Fuzhuan brick tea.It can develop asexual or sexual spores which are produced under different osmotic pressure, with Na Cl as osmotic pressure regulators. It produces the sexual morph when grown in a hypotonic medium（0.5 mol/L Na Cl）, whereas the asexual morph is formed under high concentration of salts（3 mol/L Na Cl）. In order to explore whether the Ca2＋signaling pathway responds to the changes in the salt stress in A. cristatus, vcx gene of A. cristatus was cloned and characterized. The full length of vcx gene c DNA of A. cristatus is 1 326 bp, including a 1 326 bp open reading frame encoding 441 amino acids. The calculated molecular mass of Vcx protein is 47.439 9 k D, which have 11 TH domain.The expression levels of vcx gene ware up-regulated in the asexual stage. The study results provide the basis for further exploring the relationship between the salt stress response and spore germination mechanism in fungi.