HOG通路激酶SakA在灰盖鬼伞菌柄伸长过程中生理功能的研究

【作者】 赵静；

【导师】 刘中华；

【作者基本信息】 南京师范大学 ， 微生物， 2021， 硕士

【摘要】 灰盖鬼伞是用于研究担子菌子实体发育机制的模式真菌。在光照、低温、营养匮乏等外界不良环境条件的刺激下,担子菌的生长阶段会由营养菌丝生长阶段转入子实体生长发育阶段。HOG通路（高渗透压甘油信号通路）是真核生物响应外界不良环境信号刺激的重要通路。在子囊菌类的真菌中,HOG pathway能够响应高渗压力、氧化胁迫等环境信号,参与无性孢子的形成及应激反应的生理过程。但是目前,HOG pathway在担子菌子实体生长发育中的功能和机制还尚不清楚。本研究提取灰盖鬼伞AmutBmut菌株子实体菌柄不同部位的蛋白,利用Western Blot技术检测HOG pathway中关键激酶SakA在菌柄不同部位的磷酸化水平情况。结果显示,激酶SakA在菌柄顶部快速伸长区的的磷酸化水平高于菌柄中部慢速伸长区和基部不伸长区。说明,HOG pathway中的关键激酶SakA参与菌柄的伸长生长过程。进一步的检测分析显示,菌柄顶部快速伸长区的组织渗透压较低、内源性活性氧（ROS）水平较高,表明在菌柄顶部,激酶SakA可能是通过响应渗透胁迫或氧化应激胁迫信号被激活。本研究构建了SakA干扰（pCC-SakAi）载体,利用PEG-Ca Cl₂介导的遗传转化,将SakA干扰质粒与营养缺陷型标签质粒pEASY-zero-pab1共同转入到灰盖鬼伞AmutBmut菌株中,成功获得SakA沉默（SakAi）菌株。实时荧光定量PCR检测结果表明,SakAi菌株中SakA的表达量下降了76.8%。分析空载对照（Mock）菌株和SakAi菌株的菌株生长情况后,发现SakAi菌株的菌丝径向生长速度明显减慢,并且SakAi菌株的子实体高度也显著低于Mock菌株;通过检测与菌柄伸长相关基因表达量后,我们发现细胞壁扩张蛋白ChiE1在SakAi菌株子实体菌柄顶部的表达量显著降低。结果表明,SakA基因沉默可能导致了细胞壁扩张蛋白ChiE1的表达量降低,从而造成了灰盖鬼伞子实体菌柄的伸长速率减慢。此外,本研究还构建了SakA组成型磷酸化突变蛋白（T170E+Y172D）的重组表达质粒。分析Mock菌株和SakA组成型磷酸化突变蛋白的重组表达(SakA^T170E+Y172D)菌株的生长情况,我们发现SakA^T170E+Y172D菌株的菌丝径向生长速度明显变快,子实体菌柄的高度也显著高于Mock菌株,并且在SakA^T170E+Y172D菌株中ChiE1的表达量也显著升高。以上结果表明,HOG pathway中的关键激酶SakA可能是通过调控细胞壁扩张蛋白几丁质酶ChiE1的表达量参与灰盖鬼伞子实体的菌柄伸长过程。本研究证明了在灰盖鬼伞子实体发育过程中,HOG通路的关键激酶SakA可能是通过调控细胞壁扩张蛋白ChiE1的表达参与调控子实体菌柄的伸长生长。更多还原

【Abstract】 Coprinopsis cinerea is a model fungus used to study the developmental mechanism of basidiomycete fruiting bodies.Under the stimulus of adverse external environmental conditions such as light,low temperature,lack of nutrients and other external environmental conditions,the growth stage of basidiomycetes will be transferred from the growth stage of vegetative hyphal to the growth and development stage of fruiting bodies.The HOG pathway is important for eukaryotes to respond to adverse environmental signal stimuli from the outside world.In fungi of ascomycetes,HOG pathway responds to environmental signals such as hyperosmotic pressure and oxidative stress,and participates in the physiological process of asexual spore formation and stress resistance.But at present,the function and mechanism of HOG pathway in the development of macrofungi fruit bodies are still unclear.In this study,the protein in different regions of the fruiting body stipes in Coprinopsis cinerea AmutBmut strain was extracted,and the phosphorylation level of the key kinase SakA in the different regions of the stipes was detected by Western Blot.The results showed that the phosphorylation level of the kinase SakA in the faster elongation stipe regions in the apical was higher than the slower elongation stipe regions in the median and the non-elongation stipe regions in the basal.It shows that the key kinase SakA in the HOG pathway is involved in the elongation and growth of the stipe.Further detection and analysis showed that the tissue osmotic pressure in the fast elongation region of the apical was lower and the endogenous reactive oxygen species（ROS）level was higher,it indicates that the key kinase SakA in the HOG pathway in the apical of the stipes may be activated by responding to osmotic stress or oxidative stress signals.In this study,a SakA interference（pCC-SakAi）vector was constructed.Using a PEG-Ca Cl₂ mediated genetic transformation system,the SakAi strain was obtained.The real-time quantitative PCR detecting system results showed that the expression of SakA in SakAi strains decreased by 76.8%.After analyzing the growth of the fruiting bodies in Mock strains and SakAi strains,we found that the radial growth rate of the hyphae in SakAi strains were significantly slowed down,and the length of the fruiting bodies were also significantly lower than that in Mock strains;After detecting the expression of genes related to stipe elongation,we found that the expression of the cell wall expansion protein chitinase ChiE1 in the apical of the fruiting bodies in SakAi strains were significantly reduced.The results showed that SakA gene silencing may lead to a decrease in the expression of the cell wall expansion protein ChiE1,which resulted in a slowdown in the stipe elongation rate of Coprinopsis cinerea fruiting bodies.In addition,our study also constructed a recombinant expression plasmid of SakA constitutively phosphorylated mutant protein（T170E+Y172D）.Analyzing the growth of Mock strains and recombinant expression strains of SakA constitutively phosphorylated mutant protein(SakA^T170E+Y172D),we found that the radial growth rate of the hyphae in SakA^T170E+Y172D strains were significantly faster,and the length of stipes were also significantly higher than that in Mock strains,and the expression of ChiE1 in SakA^T170E+Y172D strains also increased significantly.The above results indicate that the key kinase SakA in the HOG pathway may be involved in the stipe elongation process of Coprinopsis cinerea fruiting bodies by regulating the expression of the cell wall expansion protein chitinase ChiE1.This study proves that during the development of fruiting bodies in Coprinopsis cinerea,the key kinase SakA of the HOG pathway may be involved in regulating the elongation and growth of the fruiting body stipes by regulating the expression of the cell wall expansion protein ChiE1.更多还原