本文關鍵詞：柑橘褐斑病菌比較基因組和轉錄組分析及柑橘綠霉病菌產(chǎn)孢中心調控途徑和高滲甘油途徑的功能基因研究　出處：《浙江大學》2016年博士論文　論文類型：學位論文

  更多相關文章： 柑橘褐斑病菌 基因組測序 非必需染色體 線粒體 轉錄組 氧化脅迫 柑橘綠霉病菌 產(chǎn)孢中心調控途徑 PdbrlA PdabaA PdwetA 高滲甘油途徑 Pdos2 甾醇合成

【摘要】：一、柑橘褐斑病菌比較基因組和轉錄組分析柑橘褐斑病(Alternaria brown spot, ABS)是部分重要橘,以及這些橘和柚或橘和橙雜交柑橘上的重要病害,引起落葉、落果、枯梢,帶病果實無法鮮銷,常給感病的柑橘品種生產(chǎn)帶來巨大困難。柑橘褐斑病的病原為交鏈格孢菌橘致病型(A. alternata pathotype tangerine,也稱A. alternata pv. citri),已有的研究發(fā)現(xiàn)該病原菌能夠產(chǎn)生寄主選擇性ACT毒素,而該毒素是病菌致病所必需的；同時還發(fā)現(xiàn)活性氧解毒系統(tǒng)在病菌致病中也有重要的作用。然而,目前對柑橘褐斑病菌毒素的合成和調控過程以及抗氧化調控網(wǎng)絡仍缺乏深入的認識,對該病菌的基礎代謝過程,如生長和繁殖,對環(huán)境的適應以及次生代謝物產(chǎn)生等方面的研究也很少。深入研究柑橘褐斑病菌的生長、發(fā)育、繁殖、適應、次生代謝等基礎生物學以及致病機理可為探索全新病害防治途徑,改善現(xiàn)有防治策略提供新的思路,而一個完整的基因組信息是開展相關研究的基礎。因此,我們對一個來自浙江甌柑的柑橘褐斑病菌菌株Z7進行了全基因組測序,并與已知其他鏈格孢菌的基因組序列進行了比較,同時還分析了H202處理后的轉錄組變化,得到以下結果：1.比較基因組學研究揭示了柑橘褐斑病菌橘致病型的特異基因柑橘褐斑病菌Z7菌株基因組包含161個contigs,總長34.41Mb,平均G+C含量為51.0%。Z7基因組共編碼12062個基因和115個tRNA,平均基因長度1726bp,序列重復率為0.55%。通過不同致病型交鏈格孢菌株直系同源基因的分類與比較,得到10個橘致病型特有的基因。這些基因成簇聚集在基因組上,后續(xù)分析認為它們是合成ACT毒素的關鍵組分,決定了柑橘致病型的分化�；蚪M水平上構建的系統(tǒng)進化樹與Lawrence等提出的鏈格孢屬下劃分4個組的觀點相吻合,為鏈格孢屬真菌新分類系統(tǒng)提供了強有力的支撐。交鏈格孢菌橘致病型基因組中發(fā)現(xiàn)了18個次生代謝基因簇,其中柑橘�；韵嚓P的ACT毒素基因簇長91.2kb,包括25個基因,大部分基因在基因組中都有2-3個拷貝,推測在進化過程中這些基因發(fā)生了復制,也暗示Z7具有較強的產(chǎn)生ACT毒素的能力。柑橘褐斑病菌含有較多的細胞壁降解酶,這與它們死體營養(yǎng)的生活方式相一致。2.比較基因組揭示柑橘褐斑病菌非必需染色體起源于鏈格孢菌的祖先我們用比較基因組的方法,獲得了Z7總長1.88Mb的非必需染色體(Conditionally Dispensable Chromosome, CDC)序列。CDC和EC基因組的組成結構具有明顯的不同,CDC的平均G+C含量是47.7%,序列重復率為1.23%；Z7CDC編碼525個蛋白,不含有tRNA。利用GO數(shù)據(jù)庫分析發(fā)現(xiàn)CDC上的基因主要富集在‘細胞代謝過程’、‘初級代謝過程’、‘氧化還原反應’和‘大分子代謝過程’等生物學過程。蛋白家族分析表明CDC上含有13個碳水化合物酶,29個分泌蛋白,3個激酶,21個轉錄因子,25個轉運蛋白和13個細胞色素P450單加氧酶。密碼子適應性指數(shù)(CAI)分析表明EC和CDC基因的密碼子偏好性明顯不同,暗示CDC的進化歷史可能與EC不同。通過Ka/Ks分析發(fā)現(xiàn),CDC上的基因都受到強烈的純化選擇。直系同源聚類發(fā)現(xiàn)接近77%的CDC基因的同源基因至少在5個其他鏈格孢菌種中出現(xiàn),通過比較由24個鏈格孢菌屬蛋白和29個其他真菌屬的蛋白組成的兩個數(shù)據(jù)庫,超過95%的CDC基因都與鏈格孢菌數(shù)據(jù)庫具有較高的相似性,表明CDC上絕大多數(shù)基因來自于鏈格孢屬真菌的祖先。3.線粒體比較基因組揭示柑橘褐斑病菌與其他種屬線粒體的保守基因在排布上顯示巨大差異柑橘褐斑病菌線粒體基因組大小50,625bp,平均A+T含量為70.8%,含有13個標準的蛋白基因,2個核糖體大小亞基和31個tRNA,線粒體基因組編碼效率為63.7%,整體表現(xiàn)出偏好使用富含A/T的密碼子。腔菌目(Pleosporales)三個菌株線粒體基因組的基因較為保守,但基因的排布則表現(xiàn)出巨大的差異,暗示它們進化歷史不同。用線粒體蛋白構建的不同物種系統(tǒng)進化樹與核基因組構建的進化樹-致,說明雖然線粒體基因相對基因組基因進化速度較快,但總體上與物種進化趨勢吻合。不同真菌線粒體內基因組內含子及基因間區(qū)存在多樣性,是造成線粒體大小差異的重要原因。4.轉錄組分析揭示谷胱甘肽系統(tǒng)、過氧化物酶和轉運蛋白等基因家族在H202脅迫適應中發(fā)揮重要作用通過對柑橘褐斑病菌在H202處理30min后其轉錄組表達變化分析,發(fā)現(xiàn)1108個上調表達基因和498個下調表達基因。差異表達的基因主要富集在細胞代謝、氧化還原和細胞轉運等生物過程。谷胱甘肽系統(tǒng)、硫氧還蛋白、過氧化氫酶、半胱氨酸過氧化物氧化還原酶等可能是該病菌清除H202的主要成員。此外,我們還發(fā)現(xiàn)轉運蛋白、激酶家族、轉錄因子、細胞色素P450、泛素和熱激蛋白等在柑橘褐斑病菌H202脅迫適應中發(fā)揮重要作用。在非必需染色體上也有29個基因受到H202的明顯誘導,其中包括ACT毒素合成的關鍵基因聚酮合成酶CDCn|11750。二、柑橘綠霉病菌產(chǎn)孢中心調控途徑和高滲甘油途徑的功能基因研究柑橘綠霉病(Penicillium digitatum)是柑橘貯藏、運輸和銷售環(huán)節(jié)中的主要問題,在我國每年因綠霉病導致柑橘損失高達數(shù)百萬噸。迄今,已有3個柑橘綠霉病菌菌株的全基因組公布,但對該病菌的生長發(fā)育、適應和致病等的分子機制了解甚少。形成大量的分生孢子是構成病害流行的基礎,而抵抗適應高滲透勢等逆境脅迫條件是病菌賴以寄生高糖柑橘果實的必要基礎。為了解柑橘綠霉病菌產(chǎn)孢和對滲透勢的適應機制,本文研究了該病菌的brlA、abA和wetA 3個產(chǎn)孢中心調控基因和雙組份組氨酸激酶基因os2的生物學功能,取得如下結果：1. PdbrlA、PdabA和PdwetA調控柑橘綠霉病菌產(chǎn)孢的不同階段病菌繁殖產(chǎn)生的大量無性孢子是綠霉病菌病賴以傳播擴散的必要條件。brlA、 abaA和wetA是調節(jié)分生孢子形成的重要元件。柑橘綠霉病菌中PdbrlA敲除突變體完全喪失了形成分生孢子梗的能力；PdabaA敲除突變體雖能形成分生孢子梗,但所形成的分生孢子�；�,不具有產(chǎn)孢能力；PdwetA的缺失突變體能夠形成正常的分生孢子梗,也可以產(chǎn)孢,但分生孢子的細胞壁明顯疏松,加厚,分生孢子色素不沉積,分生孢子萌發(fā)延遲,病菌對滲透脅迫,去垢劑和熱激反應的耐受性明顯下降,但對H202的耐受性卻變強。qRT-PCR結果表明綠霉菌中存在PdbrlA→PdabA→PdwetA級聯(lián)調控模式,而且這種模式可能存在負反饋調節(jié)機制�；虮磉_譜分析結果表明：與野生型相比,缺失突變體中的401個基因下調表達,144個基因上調表達。Go富集表明下調表達的基因功能主要富集在細胞膜完整性,跨膜轉運和碳水化合物活性等方面,上調的基因主要參與細胞膜轉運的過程。KEGG分析預測PdbrlA與淀粉和蔗糖代謝途徑有關。根據(jù)煙曲霉和構巢曲霉中產(chǎn)孢相關基因的報道,我們在綠霉菌中找到了39個與產(chǎn)孢相關的同源基因。其中,12個基因(abr1、alb1、arp1、arp2、ayg1、aspf4、rodA、rodB、ppoC、axl2、 abaA和wetA)在PdbrlA中表達明顯下調,2個基因(flbA和flbB)表達明顯上升,12個表達下調的基因啟動子區(qū)都具有brlA和abaA的轉錄結合位點。2.柑橘綠霉病菌通過Pdos2正調控甘油含量、負調控甾醇含量適應高鹽環(huán)境高滲甘油途徑在真核生物體內廣泛存在,對機體適應環(huán)境變化具有極其重要的作用。Os2是該途徑的一個重要激酶,柑橘綠霉病菌△PdoS2突變體對NaCl滲透壓和細胞壁干擾制劑剛果紅和十二烷基苯磺酸鈉的敏感性顯著上升,對氟咯菌腈和異菌脲兩種殺菌劑的抗性也有部分提高,表明Pdos2參與了高滲透脅迫適應、細胞壁完整性和藥劑抗性等生命過程。然而突變體對由H202引起的氧化壓的敏感性并沒有明顯改變�！鱌dos2突變體在接種4天后引起的病斑大小相比野生型減少了約25%,表明Pdos2對維持柑橘綠霉病菌的致病性起到部分作用。0.7M NaCl處理后,野生型菌絲體內甘油的含量明顯提高而麥角甾醇的含量卻顯著降低�！鱌doS2突變體菌絲內的甘油含量僅有輕微地上升,而麥角甾醇的含量保持與野生型相同的狀態(tài),表明Pdos2參與調節(jié)的滲透適應是與它對甘油合成的正調控和麥角甾醇合成的負調控作用有關。Pdos2基因能夠直接或者間接負調控甾醇合成途徑關鍵基因Pderg11C、Pderg1、Pderg3A/B和Pderg25等的表達。
[Abstract]:A citrus acicola comparative genome and transcriptome analysis of citrus leaf spot (Alternaria brown, spot, ABS) is an important part of orange, orange and grapefruit or orange and orange and citrus hybrid an important disease caused by defoliation, dieback, fruit abscission, fruit can not be sold fresh sick, often susceptible to citrus production bring great difficulties. Citrus brown spot pathogen Alternaria tenuis pathotype (A. alternata pathotype orange tangerine, also known as A. alternata pv. citri), it has been found that the pathogenic bacteria can produce host selectivity of ACT toxin, and the toxin is required for pathogenicity; it was also found that the active oxygen detoxification system also has an important role in pathogenicity. However, the toxin synthesis and citrus acicola regulation and antioxidant regulatory networks is the lack of in-depth understanding of the basic metabolism of the pathogen, such as raw Long and reproduction, and the adaptability of secondary metabolites on the environment are few. The further study of citrus acicola growth, development, reproduction, adaptation, secondary metabolic biology and pathogenic mechanism for exploring new ways to improve the disease prevention and treatment, provide new ideas of prevention strategies, and a complete the genomic information is the basis of related research. Therefore, we performed whole genome sequencing of a Zhejiang mandarin orange citrus acicola strain Z7, and the other known species of Alternaria genomic sequences were compared, and also analyzed the transcriptome changes after H202 treatment, the results are as following: 1. comparison genomics research reveals acicola pathotypes of citrus orange citrus acicola specific gene Z7 strain genome contains 161 contigs, length 34.41Mb, the average content of G+C is 51.0%.Z7 Because group encoding 12062 genes and 115 tRNA genes, the average length of the 1726bp sequence, 0.55%. repetition rate by different pathotype of Alternaria alternata strains of orthologous gene classification and comparison, get the unique 10 orange pathotype genes. These genes are clustered in genome and subsequent analysis and they are considered as the key group synthesis of ACT toxin, determines the differentiation of pathogenicity. Citrus genomic division on the level of phylogenetic tree and Lawrence of Alternaria under 4 groups of views coincide, provides strong support for Alternaria fungi. A new classification system found 18 gene clusters to secondary metabolism Alternaria tangerine pathotype genome, including citrus specific ACT toxin gene cluster length 91.2kb, including 25 genes, most of the genes are 2-3 copies in the genome, which showed that these radicals in the evolutionary process Because the copy also suggests that Z7 has a strong ability to produce ACT toxin. Cell wall degrading enzymes of citrus acicola contain more, with their necrotrophic lifestyle consistent.2. citrus genome reveals acicola nonessential chromosome originated from Alternaria ancestor we use comparative genomics method. The Z7 of 1.88Mb (Conditionally Dispensable Chromosome non essential chromosome, CDC) structure and EC sequence of.CDC genome is obviously different, the average content of G+C is 47.7% CDC, repetition rate of 1.23% Z7CDC encoding sequence; 525 proteins, not containing tRNA. using the GO database analysis found on the CDC gene is mainly concentrated in the "cell metabolism", "primary metabolic process", "redox reaction" and "high molecular metabolism" and other biological processes. Analysis showed that the CDC protein family Containing 13 carbohydrate enzymes, 29 secreted proteins, 3 kinase, 21 transcription factors, 25 transporters and 13 cytochrome P450 monooxygenase. Codon adaptation index (CAI) analysis showed that the codon preference of EC and CDC genes were significantly different, suggesting that the evolutionary history and may CDC EC. The Ka/Ks analysis showed that the CDC genes are subject to strong purifying selection. Orthologous cluster found homologous gene of CDC gene close to 77% in at least 5 other Alternaria species in two databases by comparing 24 Alternaria genus proteins and 29 other fungal genera the protein, more than 95% of the CDC gene with Alternaria database showed high similarity, the vast majority of mitochondrial CDC gene from Alternaria fungi ancestor.3. comparative genomic reveal citrus acicola with other species of mitochondria The conserved genes revealed great differences of citrus acicola mitochondrial genome size 50625bp in the arrangement, the average A+T content was 70.8%, protein gene contains 13 standard, 2 ribosomal subunits and 31 tRNA mitochondrial genome encoding efficiency is 63.7%, the overall performance of a preference for the use of A/T enriched codon. Loculoascomycetes (. Pleosporales) three strains of mitochondrial genes are more conservative, but the gene arrangement showed great differences, suggesting that they are different. The evolutionary history constructed by mitochondrial protein systems of different phylogenetic trees and nuclear genome constructed phylogenetic tree - induced mitochondrial genes, although relative genome evolution speed, but on the whole and the evolutionary trend. There is diversity of different fungal mitochondrial genome intron and intergenic region, is the important reason causing mitochondrial size differences. Transcriptome analysis revealed 4. glutathione peroxidase system and transporter gene family in H202 stress play an important role in the adaptation of citrus acicola in H202 group the expression of 30min after the change analysis, found 1108 up-regulated genes and 498 down regulated genes. Differentially expressed genes mainly enriched in cell metabolism, redox and cellular transport and other biological processes. The glutathione system, thioredoxin, catalase, cysteine peroxiredoxin may be the major members of the bacteria removal of H202. In addition, we also found that the transporter protein kinases, transcription factor, cytochrome P450, ubiquitin and heat shock protein play an important role in stress adaptation in citrus leaf spot of H202. In the non essential chromosome 29 gene was also induced by H202, including ACT toxin synthesis The key gene of polyketide synthase CDCn|11750. two digitatum spore center regulatory pathways and hypertonic glycerol pathways to study functional gene of Citrus green mold (Penicillium digitatum) is the main problem of Citrus storage, transportation and sale, in our country every year because of Citrus green mold caused the loss of up to millions of tons. So far, there 3 strains of Penicillium digitatum genome published, but for the bacteria growth, to understand the molecular mechanism and pathogenic yet. The formation of a large number of conidia are constitute the basis of disease epidemic, and resistance to osmotic potential stress condition is a necessary basis for citrus fruit sugar parasitic bacteria to understand digitatum spore and adaptive mechanism of osmotic potential, was studied in this paper. BrlA, abA and wetA 3 sporulation center regulatory genes and two-component histidine induced The biological function of OS2 gene, the results were as follows: 1. PdbrlA, a large number of asexual spores in different stages of bacteria PdabA and PdwetA regulation digitatum spore breeding is a necessary condition for green mildew disease spreading.BrlA, abaA and wetA are important components of regulating spore formation. Citrus green mold bacteria in PdbrlA knockout mutants completely lost the ability to form conidiophores; PdabaA knockout mutant can form conidiophores, but the formation of conidiophore deformity, does not have the ability to produce spores; deletion mutant PdwetA can form conidiophores are normal, but also can sporulation the cell wall of conidia was loose, thickened, spore pigment deposition, conidia germination delay, resistant to osmotic stress tolerance, detergent and heat shock response decreased significantly, but the tolerance of H202 is The results show that there is strong.QRT-PCR PdbrlA - PdabA - PdwetA cascade control model of green mold, and this pattern may exist negative feedback regulation mechanism. Gene expression spectrum analysis results showed that: compared with the wild type, expression of 401 gene deletion mutants, 144 genes up-regulated expression of.Go gene showed that enrichment downregulated the expression of the main enrichment function in the cell membrane integrity, transmembrane transport and carbohydrate activity, genes involved in cell membrane transport process.KEGG analysis of PdbrlA and starch and sucrose metabolism prediction. According to Aspergillus fumigatus and Aspergillus nidulans sporulation related genes reported, we found 39 and sporulation related homeobox gene in green mold. Among them, 12 genes (abr1, alb1, ARP1, Arp2, ayg1, aspf4, rodA, rodB, ppoC, axl2, abaA and wetA) expression was markedly reduced in PdbrlA, 2 genes (flbA And flbB) was significantly increased in 12, down regulated expression of transcription gene promoter with brlA and abaA binding sites.2. digitatum positive regulation of glycerol content by Pdos2, the negative regulation of sterol content adaptation to high salt environment of hypertonic glycerol pathways in eukaryotic organisms widely exist, has the function of.Os2 is very important an important way to the kinase to the body to adapt to environmental changes, digitatum PdoS2 sensitivity of NaCl mutant on osmotic pressure and cell wall interference preparation of Congo red and twelve sodium increased significantly, resistant to fludioxonil and iprodione two fungicides have increased, indicating that Pdos2 participates in the high adaptation to osmotic stress, cell wall integrity and drug resistance and so on. However the sensitivity of mutant to oxidation caused by H202 pressure did not change significantly. The Pdos2 mutant inoculation in 4 days After the lesion size was reduced by about 25% compared to the wild type, showed that the pathogenicity of Pdos2 to maintain digitatum play a part of.0.7M after NaCl treatment, content of wild type in the mycelium of glycerol increased obviously and ergosterol content was significantly decreased. The content of glycerol PdoS2 mutants in silk only slightly increased. The ergosterol content remains the same as the wild type state that Pdos2 participate in the regulation of the permeability and it is fit for glycerol on.Pdos2 gene negative regulation is regulation of synthesis and synthesis of ergosterol can directly or indirectly negative regulation of sterol biosynthesis pathway genes Pderg11C, Pderg1, expression of Pderg3A/B and Pderg25.

【學位授予單位】：浙江大學
【學位級別】：博士
【學位授予年份】：2016
【分類號】：S436.66
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本文編號：1355891