[Objective] The objective of this paper is to study the relationship between STK1 and appressorium development,clarify the regulation of STK1 on the glycogen and lipid accumulation,and to make a foundation for elucidation of the molecular mechanism of appressorium development of Setosphaeria turcica.[Method] The appressoria were induced with hypha tips of wild type (WT) isolate and STK1 knock-out mutant (△ STK1) on the hydrophobic surface of glass slide by the method or "hypha separation with slide insertion" and incubation in a petri-dish moist chamber at 22℃ and under alternative changes of 14 h lightness and 10 h darkness,to observe the course of appressrium development under the microscope at intervals of 12 h.The glycogen in mycelia and appressoria of WT and △ STK1 on the slides were stained 48 h with I2/KI before and after 48 h of appressorium induction,and the changes of glycogen metabolism during appressorium development were microscopically observed.The lipid in mycelia and appressoria of WT and △ STK1 on the slides were placed in a-70℃ ultra-low temperature freezer refrigeration for 30 min before stained for 24 h with Oil Red O,and the changes of lipid metabolism during appressorium development were microscopically observed.The expression of key genes in glycogen and lipid synthesis during appressorium development was detected by real-time PCR.[Result] The appressoria from hypha tips could be induced on the hydrophobic surface of glass slide for both WT isolate and △ STK1 mutant.However,the appressoria of △ STK1 were differentiated into several different abnormal types,which were obviously different from WT isolate.The appressorium of WT was single cell within 48 h of appressorium induction and only a few multicellular appressoria were found after 48 h of induction.Nevertheless,the twisted appressoria were found after only 24 h of appressorium induction of △STK1,and some other abnormal appressorium forms,such as double-bifurcation,multi-bifurcation and "O" type,were found after 48 h of induction.After the glycogen and lipid staining in hypha and appressoria of WT and △STK1,it was found that the accumulation of glycogen and lipid was equally distributed in the mycelia and appressoria of WT isolate,but there was almost no accumulation of glycogen and lipid in the appressoria of △STK1.The glycogen accumulation was significantly reduced in the mycelium of △STK1 and the lipid was mainly distributed in the septum parts of hyphal cells,which were significantly different from WT.The gene expression of glycogen synthase (GS) and diacylglycerol acyltransferase (DGAT) increased by 6.6％ and 40.3％,respectively,after the appressoria of WT isolate were induced after 48 hours.However,the GS gene expression decreased by 9.0％ and the DGAT gene expression increased by only 24.5％ in the △ STK1 mutant.[Conclusion]The loss of STK1 function resulted in abnormal appressorium development,decreases of glycogen accumulation,uneven distribution of lipid,and a significant reduction in the expression of key genes of glycogen and lipid synthesis,indicating the accumulation of glycogen and lipid is closely related to the appressoriurn development of S.turcica.%[目的]通过研究STK1与附着胞发育的关系,明确附着胞发育过程中STK1对糖原和脂肪合成的调控作用,为阐明玉米大斑病菌(Setosphaeria turcica)附着胞发育的分子机制打下基础.[方法]以玻璃平板为疏水基质表面,通过“插片分离菌丝”法使菌丝附着于载玻平板表面,然后将附着有菌丝的玻璃平板置于保湿培养皿中22℃、14h光照和10h黑暗交替培养,诱导野生型菌株(WT)和STK1基因敲除突变体(△STK1)的菌丝形成附着胞,每隔12 h显微观测附着胞的形态和发育过程;分别将附着有未经诱导的WT和△STK1菌丝的玻璃平板和经过48 h附着胞诱导的玻璃平板浸没在I2/KI染色液中静置染色48 h,显微观察附着胞发育过程中糖原的变化;分别将附着有未经诱导的WT和△STK1菌丝的玻璃平板和经过48 h附着胞诱导的玻璃平板置于-70℃的超低温冰箱中冷冻处理30 min,然后将玻璃平板置于0i 1-red 0染色液中静置染色24 h,显微观察附着胞发育过程中脂肪的代谢变化;利用real-time PCR技术检测附着胞发育过程中糖原和脂肪合成关键酶基因的表达情况.[结果]玉米大斑病菌WT菌株和△ STK1菌株利用菌丝尖端在玻璃平板的疏水表面均能够产生附着胞,但△ STK1菌株的附着胞发育与WT菌株显著不同,WT菌株48 h内为单胞附着胞,诱导48 h后少数附着胞形成了多细胞附着胞,而△ STK1菌株在诱导24 h后即出现了扭曲附着胞的异形附着胞形态,48 h后还出现了双权、多杈和0型等多种异常的附着胞类型;WT菌株和△STK1菌株的菌丝和附着胞进行糖原和脂肪的染色后,发现WT菌株的菌丝和附着胞都有均匀分布的糖原和脂肪,而△ STK1菌株的附着胞内几乎没有糖原和脂肪的积累,与WT菌丝的结果不同,在△ STK1菌株的菌丝内糖原沉积减少,脂肪主要分布于菌隔部位;附着胞诱导48h后,WY菌株糖原合酶(glycogen synthase,GS)和二酰甘油酰基转移酶(diacylglycerol 0-acyltransferase,DGAT)基因的表达量比诱导前分别增加了6.6％和40.3％,而△STK1菌株GS基因的表达量则下降了9.0％,DGAT基因的表达量仅上升了24.5％.[结论]STK1的功能缺失使玉米大斑病菌的附着胞发育形态异常,糖原积累下降,脂肪分布不均,糖原和脂肪合成的关键酶基因表达量均显著下降,表明糖原和脂肪代谢与玉米大斑病菌的附着胞发育密切相关.
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