柑橘褐斑病菌SNF1和FTR1基因生物学功能初步研究

发布时间：2018-06-16 19:06

本文选题：柑橘褐斑病菌 + 原生质体　；参考：《西南大学》2017年硕士论文

【摘要】：由交链格孢菌(Alternaria alternata)引起的柑橘褐斑病主要为害感病品种的叶片、枝梢、花瓣和果实,严重时造成叶片和果实大量脱落,导致柑橘减产。研究发现柑橘褐斑病菌产生的ACT毒素及对活性氧的解毒机制是其侵染寄主致病所必需的,但当前对于毒素合成机制及解毒活性氧的分子机理仍缺乏深入的系统研究,同时对病原菌基础代谢相关研究开展尚不充分。蔗糖非发酵蛋白激酶SNF1在多种植物病原真菌中被证实参与对生长发育及致病性的调控,且转录组测序结果显示H2O2可诱导柑橘褐斑病菌Aa SNF1基因的表达。高亲和铁离子通透酶FTR1参与真菌胞内铁代谢,研究人员推测柑橘褐斑病菌可能存在一套由还原性铁离子摄取系统参与的对活性氧的解毒。因此,为进一步了解柑橘褐斑病菌致病机理,本研究聚焦柑橘褐斑病菌SNF1和FTR1基因,初步分析其在生长致病过程中发挥的作用。本试验优化柑橘褐斑病菌原生质体制备条件,初步完善PEG介导的原生质体遗传转化体系。同时通过基因敲除及回补,初步分析SNF1和FTR1在柑橘褐斑病菌生长发育及致病过程中的作用,得到如下结果:1.柑橘褐斑病菌在PDB中150 rpm培养36 h,以0.7 M Na Cl为稳渗剂,1%Kitalase于30℃酶解2.5 h时所释放原生质体数量最多,达4.36×108个/m L,可满足转化需要。以柑橘褐斑病菌不同基因转化该方法制备的原生质体,在抗性平板上均能收获足量的转化子,表明该优化方法达到预期效果。2.应用Split-marker方法构建Aa SNF1、Aa FTR1基因敲除盒,利用PEG介导原生质体遗传转化,经抗生素筛选、PCR及Souhern杂交验证得到Aa SNF1和Aa FTR1基因敲除菌株。利用酵母高效同源重组原理构建基因回补载体,遗传转化后得到回补菌株CP-S-2和CP-F-2。3.野生型与突变体表型分析结果显示:Aa SNF1基因参与调控柑橘褐斑病菌的菌丝生长、产孢、碳源利用、细胞壁功能及致病性。具体结果如下:(1)ΔAa Snf1菌株气生菌丝不发达,菌落颜色变深,在不同培养基上生长速度下降,PDA培养基上菌落直径减小19%。(2)与野生型相比,ΔAa Snf1突变体产孢量下降70%,分生孢子颜色变浅,直径变小。PDB培养基中,突变体孢子萌发延滞明显,野生型在接种2 h时萌发率为30%左右,而突变体仅有5%的孢子正常萌发。直至8 h时,野生型已有90%以上的孢子萌发,而ΔAa Snf1突变体萌发率仅50%左右。野生型在接种12 h时孢子基本全部萌发,变体菌株萌发率上升至80%。(3)Aa SNF1的缺失导致菌株致病力显著下降,突变体无病斑或仅形成小斑。在多聚半乳糖醛酸、蔗糖、酒精为单一碳源的培养上,ΔAa Snf1突变体出现生长缺陷,菌落直径比野生型分别下降35%、37%和45%。(4)相比于野生型,ΔAa Snf1突变体在含细胞壁胁迫因子的培养基上表现出更强的耐受性,并且随胁迫因子浓度的升高表现更为显著。但ΔAa Snf1突变体在含不同稳渗剂及H2O2的培养基上与野生型生长无明显差异。回补菌株CP-S-2生长致病恢复至野生型水平。4.Aa FTR1在低铁环境下被诱导表达,相对表达量为对照组的32倍。Aa FTR1的缺失导致柑橘褐斑病菌胞内铁含量显著下降,且低铁环境下降更明显。但突变体表型分析结果显示Aa FTR1基因的缺失并不影响菌株的生长、产孢、孢子萌发、渗透压胁迫、氧化胁迫及致病性。虽然突变体致病力与野生型无差异,但RT-q PCR分析表明,Aa FTR1基因在侵染柑橘致病的过程中表达水平呈现明显的先上升后下降趋势,在侵染初期(接种48 h)达到峰值,为对照组的7倍。
[Abstract]:The citrus brown spots caused by Alternaria alternata are mainly the leaves, shoots, petals and fruits of the susceptible varieties, which seriously cause the loss of leaves and fruits, resulting in the reduction of citrus production. The study found that the ACT toxin produced by the citrus brown spot pathogen and the detoxification mechanism of the active oxygen are essential for the infection of the host. However, there is still a lack of systematic research on the mechanism of toxin synthesis and the molecular mechanism of detoxification of reactive oxygen species. At the same time, the research on basic metabolism related to pathogenic bacteria is not sufficient. The non fermented protein kinase SNF1 of sucrose is proved to be involved in the regulation of growth and pathogenicity in a variety of plant pathogenic fungi, and the sequencing results of the transcriptional group are significant. H2O2 can induce the expression of Aa SNF1 gene of Citrus brown spot pathogen. The high affinity iron ion permeable enzyme FTR1 participates in the intracellular iron metabolism of fungi. The researchers speculate that the citrus brown spot pathogen may have a set of detoxification of reactive oxygen species involved in the reductive iron uptake system. Therefore, this study is a further study of the pathogenesis of Citrus brown spot pathogen. Focusing on the SNF1 and FTR1 genes of Citrus brown spot pathogen, this paper preliminarily analyzes its role in the process of growth pathogenicity. This experiment optimizes the preparation conditions of the protoplast of Citrus brown spot pathogen and preliminarily perfected the genetic transformation system of protoplast mediated by PEG. At the same time, the growth and development of Citrus brown spot pathogen by SNF1 and FTR1 were analyzed by gene knockout and supplementation. The following results were obtained as follows: 1. the citrus brown spot pathogen was cultured in PDB with 150 rpm, 36 h, 0.7 M Na Cl as a stabilizing agent, and the number of protoplasts released by 1%Kitalase at 2.5 h at 30 degrees centigrade, reaching 4.36 * 108 /m L, which could meet the transformation needs. On the resistant plate, a full amount of transformants can be harvested on the resistant plate, indicating that the optimized method has achieved the desired effect by using the Split-marker method to construct Aa SNF1, Aa FTR1 gene knockbox, PEG mediated genetic transformation of protoplast, screening by antibiotics, PCR and Souhern hybridization to obtain Aa SNF1 and Aa polymerase gene knockout strains. CP-S-2 and CP-F-2.3. wild type and mutant body surface type analysis showed that Aa SNF1 gene was involved in the regulation of mycelium growth, sporulation, carbon source utilization, cell wall function and pathogenicity of Citrus brown spot pathogen. The specific results are as follows: (1) Qi Aa Snf1 strain gas mycelium In the underdeveloped, the colony color became deeper and the growth rate decreased on the different medium. The colony diameter of the PDA medium decreased by 19%. (2). Compared with the wild type, the sporulation of the delta Aa Snf1 mutant decreased by 70%, the conidium color became shallow, and the diameter of the mutant was smaller in the.PDB medium. The germination rate of the mutant was obvious, and the germination rate of the wild type was about 30% when inoculated 2 h. And only 5% of the spore germinated normally. Up to 8 h, more than 90% of the spore germinated in the wild type, and the germination rate of the delta Aa Snf1 mutant was only 50%. The spores basically germinated when the wild type was inoculated 12 h. The germination rate of the mutant strain rose to 80%. (3) Aa SNF1 and the pathogenicity of the mutant was significantly decreased, the mutant had no disease spots or only the mutant. Formation of small spots. In the cultivation of polygalactose acid, sucrose and alcohol as a single carbon source, the delta Aa Snf1 mutant appeared growth defects, the colony diameter decreased by 35% than the wild type, 37% and 45%. (4) compared to the wild type, and the delta Aa Snf1 mutant showed stronger tolerance on the culture medium containing cell wall stress factor, and with the stress factor. There was no significant difference in the concentration of Aa Snf1, but there was no significant difference between the mutant and the wild type in the medium containing the different osmotic agents and H2O2. The recovery strain CP-S-2 was induced to the wild type and.4.Aa FTR1 was induced in the low iron environment, and the relative expression was 32 times the absence of.Aa FTR1. The intracellular iron content of the pathogen was significantly decreased, and the low iron environment decreased more obviously. But the mutant phenotypic analysis showed that the deletion of Aa FTR1 gene did not affect the growth, sporulation, spore germination, osmotic stress, oxidative stress and pathogenicity. Although the mutant pathogenicity was not different from the wild type, the RT-q PCR analysis showed that the Aa FTR1 gene was found. In the process of Citrus infection, the level of expression increased obviously and then decreased, and reached the peak at the initial stage of infection (48 h), which was 7 times that of the control group.
【学位授予单位】：西南大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S436.66

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