生物学杂志

生物学杂志 ›› 2020, Vol. 37 ›› Issue (3): 7-.doi: 10.3969/j.issn.2095-1736.2020.03.007

• 研究报告 • 上一篇    下一篇

巴斯德毕赤酵母MAPK/HOG信号通路的分子互作研究

  

  1. 1. 江南大学 粮食发酵工艺与技术国家工程实验室， 无锡 214122； 2. 江南大学 工业生物技术教育部重点实验室， 无锡 214122；
    3. 江南大学 糖化学与生物技术教育部重点实验室， 无锡 214122
  • 出版日期:2020-06-18 发布日期:2020-06-10
  • 通讯作者: 白仲虎，教授，博士生导师，主要从事发酵工程和生物医药过程工程等研究，E-mail：baizhonghu@jiangnan.edu.cn；杨艳坤，副教授，硕士生导师，主要从事微生物学、分子生物学、发酵工程研究，E-mail：yangyankun@jiangnan.edu.cn
  • 作者简介:王荣斌，硕士，主要从事发酵工程、分子生物学研究，E-mail：wangrb100@gmail.com
  • 基金资助:
    国家自然科学基金（31570034）；江苏省第十五批“六大人才高峰”（SWYY-180）；教育部工业生物技术重点实验室开放课题（KLIB-KF201802）

The interactions of MAPK/HOG signal pathway factors in Pichia pastoris

  1. 1. National Engineering Laboratory for Cereal Fermentation Technology; 2. The Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology; 3. The Key Laboratory of Carbohydrate Chemistry and Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi 214122, China
  • Online:2020-06-18 Published:2020-06-10

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摘要： MAPK/HOG信号通路在酵母适应外界渗透压变化中起着非常关键的作用。酵母中对该通路的研究大都基于酿酒酵母，而在巴斯德毕赤酵母中的研究非常少。应用CRISPR/Cas9系统敲除了PBS2、HOG1、HOT1和HOT2基因，并检测了4种敲除菌株和对照菌株在高渗和正常条件下生长情况和相关基因的转录水平，发现HOG1、HOT1和HOT2的缺失均会影响细胞抵抗高渗的能力。PBS2和HOG1在高渗和正常条件下转录水平不变，同时PBS2和HOG1可在转录水平调控MSN2和HOT1，但不调控HOT2；MSN2和HOT1在pbs2Δ、hog1Δ仍可响应高渗高表达，表明存在其他转录因子参与对其的调控。确认了MAPK/HOG信号通路中的下游相关基因GT1、DOG2、DAK1、HXT1、HSP12和CTT1，其中Hog1对HSP12和CTT1的表达起到了重要作用，Hot2参与对HXT1的调控，而Hot1不参与对这些基因的调控。同时，可能存在其他转录因子参与调控下游基因。最后，建立了毕赤酵母MAPK/HOG信号通路的初步模型，为之后的深入研究奠定了基础。

关键词: 巴斯德毕赤酵母, MAPK/HOG, 信号通路, 高渗, 调控

Abstract: MAPK/HOG signal pathway of yeast plays an important role in adapting the change of hyperosmotic stress outside. The current studies on this pathway, however, are practically based on Saccharomyces cerevisiae instead of Pichia pastoris, which limits the application of this excellent heterologous protein expression system. In this study, CRISPR/Cas9 system was applied to knock out the key transcription factor genes PBS2, HOG1, HOT1 and HOT2 in P. pastoris. Then, these mutant strains and GS115 strain were treated both in pre-stress and hyperosmotic condition. The results revealed that HOG1, HOT1 and HOT2 played important roles in protecting cells from hyperosmotic stress. The transcriptional levels of PBS2 and HOG1 were constitutive and they up-regulated MSN2 and HOT1, but no significant changes were found in the transcription level of HOT2. However, MSN2 and HOT1 were still induced by hyperosmotic stress indicating that more transcription factors were involved in the regulation network. Several genes in the MAPK/HOG pathway, including GT1, DOG2, DAK1, HXT1, HSP12 and CTT1, were confirmed. Hog1 was a key transcription factor for up-regulating HSP12 and CTT1. The expression of HXT1 was regulated by Hot2. However, Hot1 was not involved in the regulation network. Finally, a novel MAPK/HOG pathway model in P. pastoris was established.

Key words: Pichia pastoris, MAPK/HOG, signal pathway, hyperosmotic stress, regulation

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