马尔尼菲青霉在d1hA基因的克隆及基因功能的初步研究

【作者】 赵允谦；

【导师】 任双喜； 赵国屏；

【作者基本信息】 复旦大学 ， 微生物， 2008， 硕士

【摘要】 马尔尼菲青霉菌是一类两相条件致病真菌,在25℃培养呈现腐生菌丝态,无致病性,37℃早现酵母态,有致病性。尽管它具有非常重要的医学研究价值以及独特的温度调控的两相转换机制,但对马尔尼菲青霉的研究由于基因组信息和有效操作方法的缺乏而受到局限。在我们的研究中,一个3936bp的新基因dlhA被成功克隆到。dlhA和在皮生芽孢菌中发现的起全局调控作用的基因drk1具有高度保守性。我们构建了RNA干扰表达盒,表达盒内具有目的基因编码序列430bp的反向重复序列并被gus基因隔开。整个干扰载体借助根癌农杆菌体系成功转化马尔尼菲青霉,并在一个可控的木糖诱导启动子xylP调控RNA干扰。研究发现dlhA基因沉默的转化子形态学发生了明显的变化,而且丧失了产孢能力。实验结果表明dlhA在不同温度下调节不同的信号途径,对于研究的极性生长,可能的两相转换机制以及作为药物靶点的筛选都具有重要的意义。更多还原

【Abstract】 Penicillium marneffei is a thermally dimorphic opportunistic human pathogen with a saprophytic filamentous hyphal form at 25°C and a pathogenic unicellular yeast form at 37°C. Despite its medical importance and its unusual thermal dimorphic character, a large part of P.marneffei still remains unknown and relative molecular researches have been limited due to the lack of genomic information and effective genetic tools. We cloned a 3936 gene dlhA highly conserved to the histidine kinase gene drk1, which has the globe regulation in morphogenesis, cell wall composition, sporulation and expression of virulence factors in Blastomyces dermatitidis. One RNAi expression cassette was designed with inverted repeats of coding sequence of the targeted gene separated by a segment of gus~ gene. Constructs were transferred by Agrobacterium -mediated transformation and under the control of promoter xylP, RNAi can be regulated in a controllable and reversible manner. dlhA-silenced transformants exhibited severe morphological defects and incapability of conidiogenesis compared with their parent strains. These evidences imply that dlhA plays a role regulating the expression of downstream effectors, which are necessary exploring the propable mechanisms of correct polarized growth, dimorphic switch, and further research for drug design.更多还原