Physiological Mechanism Of Transcription Factors Asg1p And Hal9p In Acid Stress Response Of Candida Glabrata

This dissertation chose the mutant strain C. glabrata ATCC 55 as a model system to identify the function of transcription factor Cg Asg1 p and Cg Hal9 p under acidic conditions. The regulation mechanism of Cg Asg1 p and Cg Hal9 p and the effect of gene Cg ASG1 or Cg HAL9 deletion on cell growth, intracellular environment, intracellular localization and transcription level in response to acid stress were studied in detail with the theory and method of molecular biotechnology and microbial physiology. The main results were described as follows.1. The mutant strain Cgasg1D and the revertant strain Cgasg1D/Cg ASG1 were constructed and it was found that changes were exhibited as follows: the growth of Cgasg1D in the medium at p H 5.2 were 15% slower than that of wild-type strain while no growth was detected in the medium at p H 2.0; the mutant strain Cgasg1D exhibited a 90% reduction in growth viability after incubation in the medium at p H 2.0 for 12 h; incubating the mutant strain Cgasg1D in the medium at p H 2.0 led to a 10% reduction in H+-ATPase activity, an 13-fold increase in the m RNA level of Cg PMA1, a reduction from 6.0 to 5.2 in p Hin and an 2-fold increase in ROS content; when extracellular p H decreased to 2.0, Cg Asg1 p was transferred from cytoplasm to nucleus in response to acid stress.2. The mutant strain Cghal9D and the revertant strain Cghal9D/Cg HAL9 were constructed and it was found that changes were exhibited as follows: growth of Cghal9D in the medium at p H 5.2 were 13% slower than that of wild-type strain, while no growth was detected in the medium at p H 2.0; the mutant strain Cghal9D exhibited a 90% reduction in growth viability after incubation in the medium at p H 2.0 for 12 h; after incubated in p H 2.0, the mutant strain Cghal9D exhibited a constant level of H+-ATPase activity, an increase from 60% Cg PMA1 m RNA level of wt to the same level of wt, a reduction from 6.0 to 5.3 in p Hin and an 2-fold increase in ROS content; transcription factor Cg Hal9 p was constitutively localized in the cytoplasm in the medium at p H 5.2 and p H 2.0.3. The mutant strain Cgasg1Dhal9D was constructed and it was found that Cgasg1Dhal9D showed the same growth as wt in the medium at p H 5.2 and p H 2.0. The m RNA level of gene Cg HAL9 and Cg ASG1 was analyzed by q RT-PCR and it was found that of gene Cg ASG1 and Cg HAL9, either gene deletion could not affect the m RNA level and the intracellular localization of the other one in the medium at p H 5.2 or p H 2.0; Cg Asg1 p and Cg Hal9 p are required for tolerance of acid stress via the regulation of multiple pathways, such as the MAPK signaling pathway, plasma membrane or cell wall organization, trehalose accumulation, and the RIM101 signaling pathway; changes of Cgasg1D and Cghal9D in several biological process, such as oxidation-reduction process, transcriptional regulation and transmembrane transport, could cancel each other out, resulting in the same growth phenotype as wt of the mutant strain Cgasg1Dhal9D. The RNAseq data was verified to be accurate by q RT-PCR.