光周期蛋白Frq和自噬蛋白在稻瘟菌及禾谷镰刀菌中的功能分析

发布时间：2019-04-10 10:30

【摘要】：生物钟虽然是生物体内部的生理进程,但是与外界环境也是紧密联系的,能够使生物体更好地适应外界环境条件的变化。frq基因是粗糙脉孢菌生物钟的中心作用元件之一,为了明确生物钟基因对稻瘟菌的重要性,我们对frq同源基因MoFRQ的功能进行了分析。结果表明,在持续黑暗的条件下MoFRQ的表达不具有周期性。但是与黑暗条件相比,光照处理可以提高MoFRQ的表达水平。另外,光照不仅可以促进MoFRQ的表达,而且会使MoFrq-GFP定位在细胞核中。光照条件下,Mofrq突变体气生菌丝的生长受到抑制。与野生型菌株Guy11相比,Mofrq突变体分生孢子的产生数量明显降低。酵母双杂交发现,MoFrq和Htf1可以直接发生互作,从而共同影响分生孢子的形成。Mofrq突变体附着胞的形成和穿透能力出现下降,从而使其致病力明显降低。Mofrq突变体的糖原和脂肪粒从分生孢子向附着胞的移动出现延迟,从而可能会对附着胞的膨压产生影响。与野生型菌株相比,Mofrq突变体对外界活性氧压力敏感性增强,但是对渗透压的敏感性变化不大。另外,Mofrq突变体中的PKA活性水平明显降低。以上结果说明,MoFRQ缺失后会对气生菌丝的生长、分生孢子的产生、附着胞的穿透、活性氧压力的敏感性以及信号通路产生影响。当敲除禾谷镰刀菌的FgFRQ基因后,不会对菌丝生长和致病力产生影响。在Mofrq突变体中表达FgFRQ后,不能使菌落生长和致病力方面的缺陷得以回复,但是用粗糙脉孢菌的FRQ却可以回复菌落生长和致病力方面的缺陷。除Fg Frq外,Frq和MoFrq都具有非最优密码子使用的偏好性,从而会对FRQ的功能产生影响。对于植物病原真菌来说,自噬过程能够使细胞内的物质运输到液泡中进行降解,从而实现细胞内合成与降解的自我平衡,而且自噬对于病原真菌实现对寄主植物的侵染同样十分重要。为了明确自噬对禾谷镰刀菌的重要作用,我们分别对所预测得到的26个自噬相关基因(ATGs)进行了敲除。在禾谷镰刀菌不同生长发育阶段中,大多数ATG基因都在菌丝或分生孢子阶段有着相对较高的表达量。与野生型菌株PH-1相比,15个ATG基因缺失突变体气生菌丝的生长明显受到抑制,其中有12个突变体的生长速率出现下降。有10个突变体的分生孢子产生数量也明显降低。在有性生殖方面,有13个突变体无法产生子囊壳。在能够形成正常子囊壳的突变体中,有7个突变体子囊孢子的形成与释放也受到影响。当接种小麦穗后,有17个突变体的致病力都明显降低。在DON的生物合成方面,有7个突变体的DON合成发生变化。综上所述,ATG基因对于禾谷镰刀菌的生长速率、气生菌丝的生长、分生孢子的产生、有性生殖和致病力等多个方面的生物学过程都具有重要作用。
[Abstract]:Although the biological clock is the internal physiological process of the organism, it is also closely related to the external environment, which can make the organism better adapt to the changes of the external environmental conditions. The frq gene is one of the central acting elements of the biological clock of Paeosporium rubricosporium. In order to clarify the importance of biological clock gene to blast fungus, we analyzed the function of frq homologous gene MoFRQ. The results show that the expression of MoFRQ is not periodic under the condition of continuous darkness. However, compared with the dark condition, light treatment can improve the expression level of MoFRQ. In addition, light can not only promote the expression of MoFRQ, but also localize MoFrq-GFP in the nucleus. Under light condition, the growth of aerated hyphae of Mofrq mutant was inhibited. Compared with wild-type strain Guy11, the number of conidia produced by Mofrq mutant was significantly lower than that of wild-type strain. Yeast two-hybrid showed that MoFrq and Htf1 could interact directly, which together affected the formation of conidia and decreased the ability of appressorium formation and penetration in Mofrq mutant. The migration of glycogen and fat grains from conidia to appressorium of Mofrq mutant was delayed, which might affect the swelling pressure of appressorium. Compared with wild-type strains, the sensitivity of Mofrq mutants to external reactive oxygen species (Ros) pressure was increased, but the sensitivity to osmotic pressure changed little. In addition, the activity level of PKA in Mofrq mutant decreased significantly. These results suggest that MoFRQ deletion may affect the growth of aerated hyphae, conidia production, appressorium penetration, sensitivity of reactive oxygen species pressure and signaling pathway. When the FgFRQ gene of Fusarium graminearum was knocked out, the mycelium growth and pathogenicity were not affected. When FgFRQ was expressed in Mofrq mutants, the defects in colony growth and pathogenicity could not be recovered, but the defects in colony growth and pathogenicity could be recovered by using FRQ. With the exception of Fg Frq, both Frq and MoFrq have the preference of non-optimal codon usage, which will affect the function of FRQ. For plant pathogenic fungi, autophagy processes can transport intracellular substances to vacuoles for degradation, thus achieving the self-balance of intracellular synthesis and degradation. Autophagy is also important for pathogenic fungi to infect host plants. In order to clarify the important role of autophagy on Fusarium graminearum, 26 autophagy-related genes (ATGs) were knockout respectively. In different stages of growth and development of Fusarium graminearum, most of the ATG genes were expressed in hyphae or conidia. Compared with wild type strain PH-1, the growth of aerated hyphae of 15 ATG gene deletion mutants was significantly inhibited, and the growth rate of 12 mutants was decreased. The number of conidia produced by 10 mutants also decreased significantly. In sexual reproduction, 13 mutants were unable to produce ascomystic shells. Among the mutants capable of forming normal ascomystic shells, the formation and release of ascospores of seven mutants were also affected. When the wheat panicle was inoculated, the pathogenicity of 17 mutants was significantly decreased. In the biosynthesis of DON, the DON synthesis of 7 mutants changed. In conclusion, ATG gene plays an important role in the biological processes of Fusarium graminearum, such as the growth rate of Fusarium graminearum, the growth of aerated hyphae, the production of conidia, the sexual reproduction and pathogenicity of Fusarium graminearum.
【学位授予单位】：西北农林科技大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：S435.111.4;S435.121.45

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