本文選題：柑橘褐斑病菌 + 原生質(zhì)體�。� 參考：《西南大學(xué)》2017年碩士論文

【摘要】：由交鏈格孢菌(Alternaria alternata)引起的柑橘褐斑病主要為害感病品種的葉片、枝梢、花瓣和果實(shí),嚴(yán)重時(shí)造成葉片和果實(shí)大量脫落,導(dǎo)致柑橘減產(chǎn)。研究發(fā)現(xiàn)柑橘褐斑病菌產(chǎn)生的ACT毒素及對(duì)活性氧的解毒機(jī)制是其侵染寄主致病所必需的,但當(dāng)前對(duì)于毒素合成機(jī)制及解毒活性氧的分子機(jī)理仍缺乏深入的系統(tǒng)研究,同時(shí)對(duì)病原菌基礎(chǔ)代謝相關(guān)研究開展尚不充分。蔗糖非發(fā)酵蛋白激酶SNF1在多種植物病原真菌中被證實(shí)參與對(duì)生長(zhǎng)發(fā)育及致病性的調(diào)控,且轉(zhuǎn)錄組測(cè)序結(jié)果顯示H2O2可誘導(dǎo)柑橘褐斑病菌Aa SNF1基因的表達(dá)。高親和鐵離子通透酶FTR1參與真菌胞內(nèi)鐵代謝,研究人員推測(cè)柑橘褐斑病菌可能存在一套由還原性鐵離子攝取系統(tǒng)參與的對(duì)活性氧的解毒。因此,為進(jìn)一步了解柑橘褐斑病菌致病機(jī)理,本研究聚焦柑橘褐斑病菌SNF1和FTR1基因,初步分析其在生長(zhǎng)致病過程中發(fā)揮的作用。本試驗(yàn)優(yōu)化柑橘褐斑病菌原生質(zhì)體制備條件,初步完善PEG介導(dǎo)的原生質(zhì)體遺傳轉(zhuǎn)化體系。同時(shí)通過基因敲除及回補(bǔ),初步分析SNF1和FTR1在柑橘褐斑病菌生長(zhǎng)發(fā)育及致病過程中的作用,得到如下結(jié)果:1.柑橘褐斑病菌在PDB中150 rpm培養(yǎng)36 h,以0.7 M Na Cl為穩(wěn)滲劑,1%Kitalase于30℃酶解2.5 h時(shí)所釋放原生質(zhì)體數(shù)量最多,達(dá)4.36×108個(gè)/m L,可滿足轉(zhuǎn)化需要。以柑橘褐斑病菌不同基因轉(zhuǎn)化該方法制備的原生質(zhì)體,在抗性平板上均能收獲足量的轉(zhuǎn)化子,表明該優(yōu)化方法達(dá)到預(yù)期效果。2.應(yīng)用Split-marker方法構(gòu)建Aa SNF1、Aa FTR1基因敲除盒,利用PEG介導(dǎo)原生質(zhì)體遺傳轉(zhuǎn)化,經(jīng)抗生素篩選、PCR及Souhern雜交驗(yàn)證得到Aa SNF1和Aa FTR1基因敲除菌株。利用酵母高效同源重組原理構(gòu)建基因回補(bǔ)載體,遺傳轉(zhuǎn)化后得到回補(bǔ)菌株CP-S-2和CP-F-2。3.野生型與突變體表型分析結(jié)果顯示:Aa SNF1基因參與調(diào)控柑橘褐斑病菌的菌絲生長(zhǎng)、產(chǎn)孢、碳源利用、細(xì)胞壁功能及致病性。具體結(jié)果如下:(1)ΔAa Snf1菌株氣生菌絲不發(fā)達(dá),菌落顏色變深,在不同培養(yǎng)基上生長(zhǎng)速度下降,PDA培養(yǎng)基上菌落直徑減小19%。(2)與野生型相比,ΔAa Snf1突變體產(chǎn)孢量下降70%,分生孢子顏色變淺,直徑變小。PDB培養(yǎng)基中,突變體孢子萌發(fā)延滯明顯,野生型在接種2 h時(shí)萌發(fā)率為30%左右,而突變體僅有5%的孢子正常萌發(fā)。直至8 h時(shí),野生型已有90%以上的孢子萌發(fā),而ΔAa Snf1突變體萌發(fā)率僅50%左右。野生型在接種12 h時(shí)孢子基本全部萌發(fā),變體菌株萌發(fā)率上升至80%。(3)Aa SNF1的缺失導(dǎo)致菌株致病力顯著下降,突變體無病斑或僅形成小斑。在多聚半乳糖醛酸、蔗糖、酒精為單一碳源的培養(yǎng)上,ΔAa Snf1突變體出現(xiàn)生長(zhǎng)缺陷,菌落直徑比野生型分別下降35%、37%和45%。(4)相比于野生型,ΔAa Snf1突變體在含細(xì)胞壁脅迫因子的培養(yǎng)基上表現(xiàn)出更強(qiáng)的耐受性,并且隨脅迫因子濃度的升高表現(xiàn)更為顯著。但ΔAa Snf1突變體在含不同穩(wěn)滲劑及H2O2的培養(yǎng)基上與野生型生長(zhǎng)無明顯差異。回補(bǔ)菌株CP-S-2生長(zhǎng)致病恢復(fù)至野生型水平。4.Aa FTR1在低鐵環(huán)境下被誘導(dǎo)表達(dá),相對(duì)表達(dá)量為對(duì)照組的32倍。Aa FTR1的缺失導(dǎo)致柑橘褐斑病菌胞內(nèi)鐵含量顯著下降,且低鐵環(huán)境下降更明顯。但突變體表型分析結(jié)果顯示Aa FTR1基因的缺失并不影響菌株的生長(zhǎng)、產(chǎn)孢、孢子萌發(fā)、滲透壓脅迫、氧化脅迫及致病性。雖然突變體致病力與野生型無差異,但RT-q PCR分析表明,Aa FTR1基因在侵染柑橘致病的過程中表達(dá)水平呈現(xiàn)明顯的先上升后下降趨勢(shì),在侵染初期(接種48 h)達(dá)到峰值,為對(duì)照組的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.
【學(xué)位授予單位】：西南大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：S436.66

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