申克孢子丝菌酵母相和菌丝相cDNA消减文库的构建及差异表达基因的生物信息学分析
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摘要
申克孢子丝菌(Sporothrix Schenckii, SSchenckii)是孢子丝菌病(Sporotrichosis)的病原菌,该菌遍布世界各地,人类可因皮肤轻微外伤后接触被该菌污染的物质而感染。由申克孢子丝菌引起的孢子丝菌病是最常见的深部真菌感染之一,在全世界均有发生。临床表现为皮肤、皮下组织及其附近淋巴系统的慢性感染,皮损多局限于暴露部位,形成沿淋巴走行分布的特征性结节,可引起皮肤化脓、溃烂及渗出,少数病人可发生多系统播散。
申克孢子丝菌为一种双相型真菌,在室温25℃表现为菌丝相,体内37℃为酵母相,这种酵母相与菌丝相之间的转换称为菌相转换。申克孢子丝菌酵母相可在感染者体内组织增殖,形成小的芽生孢子而致病。
申克孢子丝菌的双相性表明该菌不同菌相存在差异表达基因,在不同的环境条件下,通过菌体内信号转导的调控,表现为酵母相或菌丝相。目前申克孢子丝菌双相转换的相关研究仅涉及少数几个基因,双相转换的分子机制尚未明了,研究申克孢子丝菌双相转换机制对揭示其发病机理有重要意义。
本研究应用抑制性消减杂交( Suppression Subtractive Hybridization,SSH)技术,构建高特异性的申克孢子丝菌菌丝相(Mycelium,M)和酵母相(Yeast,Y)的正反cDNA消减文库,并对其差异表达的基因进行生物信息学分析,以探讨差异表达基因与酵母相与菌丝相双相转换的相关性。
本研究选择申克孢子丝菌标准株CMCC(F)D1a作为研究对象,以体外诱导形成的纯的菌丝相(M)与酵母相(Y)细胞作为实验材料,分别提取M与Y细胞的总RNA,逆转录并扩增M与Y细胞的cDNA,纯化扩增的cDNA,经RsaI酶切处理,将酶切后的M与Y细胞的cDNA分别作为测试子(Tester)和驱动子(driver),将M与Y细胞的cDNA分别分为两份,每份连接不同的接头,即:接头1(adaptor1)和接头2(adaptor2)。之后进行两轮消减杂交及两轮PCR扩增,纯化第二轮PCR产物,以紫外分光光度计检测cDNA纯度与浓度。连接载体pGEM-T并转化感受态细胞TOPO10,蓝白斑筛选阳性克隆,采用T7引物,PCR扩增插入片段,挑取插入片断大于250bp、带型单一的克隆进行大规模测序。判别测序的有效性,去除非单一克隆的数据,并对所有数据进行去载、拼接、聚类和Blastn分析及整理。
结果:M+Y文库(即在M相高表达,Y相低表达或不表达)获得751条表达序列标签(Expressed Sequence Tags,ESTs),平均长度为690.98bp,ESTs经拼接后获得101条unigenes;Y+M文库(即在Y相高表达,M相低表达或不表达)获得875条ESTs,平均长度为575.9bp,拼接获得249条unigenes。Blastn分析发现两个消减文库中都出现多个重复的基因序列,去除重复序列后,M+Y文库共获得45条unigenes,Y+M文库获得91条unigenes。申克孢子丝菌酵母相菌丝相的转换伴随着不同菌相细胞差异基因的高表达,这些高表达的差异基因可分为四类:(1)结构基因类,如18S、25sRNA基因,AJ496242.1,FJ882050.1,AF343676.1等,某些结构基因在申克孢子丝菌酵母相菌丝相状态下的独特表达,可能是菌相转换的结果;(2)代谢酶类,如gb|CP001656.1|,ref|NM_001006571.1|,DQ298384.1等,参与碳水化合物、氨基酸、脂质和辅酶运输和代谢;(3)细胞表面分子类,如gi|30721613|,gi|45383668,gb|GQ379464.1|等,这些分子参与细胞内的信号转导,推测其在菌相转换中起介导细胞基因表达的调控或细胞行为的改变;(4)功能不明的细胞分子,如gb|DQ510714.1|,gb|EU923089.1|,gb|DQ298391.1|等。它们在申克孢子丝菌双相转换机制中的作用也有待进一步验证。
申克孢子丝菌菌丝相和酵母相cDNA消减文库的构建及生物信息学分析为进一步筛选鉴定菌相转换相关差异基因奠定了基础,有助于阐明申克孢子丝菌双相转换的分子机制。
Sporothrix schenckii is the etiological agent of sporotrichosis, a subcutaneous mycosis, with an increasing worldwide incidence. It enters through small cuts and abrasions in the skin to cause the infection. Symptoms of sporotrichosis include localized nodular leisions, bumps and chronic ulcers at the point of entry and also along lymph nodes and vessels. However, it can disseminate in immunocompromised patients .
S.schenckii is a pathogenic fungus that undergoes a dimorphic transition from yeast to mycelium in response to environment conditions such as temperature. It appears as mycelium form at 25°C and yeast form at 37°C. Sporotrichosis is caused by the multiplication of yeast form colonies.
By some approaches, several genes have been identified that are expressed differently in transition from yeast to mycelium. Signal transduction modulated by some elements such as mitogen-activated protein kinases, cAMP and pH-responsive modules, appear to regulate this transition. However, the mechanism of dimorphic transition is still unclear. Therefore, study on the mechanism of dimorphic transition is important to reveal the pathogenesis of S.schenckii.
To screen the other differentially expressed genes about dimorphic transition of S.Schenckii, cDNA subtractive library between yeast and mycelium phase was constructed by suppression subtractive hybridization(SSH)and bioinformatics analysis was performed to profile the relationship between those differently expressed genes and dimorphic transition.
In this study, CMCC(F)D1a, a S. schenckii standard strain, was induced to form mycelium(M)and yeas(Y)cells. Total RNA isolated from M and Y cells was reverse transciptased and amplified respectively, the average length of cDNA after purification and RsaI enzyme digestion is 500bp. cDNA of M and Y cells after RsaI enzyme restriction was regarded as tester and driver respectively, and then were divided into two fragments to ligate adaptor1 and adaptor2. The PCR products were ligated with pGEM-T plasmid vectors, transformed into E.coli TOP10. The positive recombinant clones were picked up through the blue-white screening system, and confirmed by PCR method with P7 primer. Finally, the differentially expressed cDNA fragments were sequenced and compared with EST database in Genbank.
Results: 751 expressed sequence tags(ESTs)were obtained in M+Y library(that means ESTs were over expressed in M-phase, low expressed or no expressed in Y-phase), the average length is 690.98bp. Meanwhile, 875 ESTs were obtained in Y+M library, the average length is575.9bp. After splicing of ESTs, 101 unigenes were obtained in M+Y library and 249 unigenes in Y+M library. However, several repeated sequences were found by Blastn analysis in both two subtractive libraries. Finally, 45 unigenes in M+Y library and 91 unigenes in Y+M library were obtained after the removal of repetitive sequences.
During the construction of cDNA subtractive libraries, the ?distribution of differently expressed genes varied with dimorphic transition. The over expressed genes with diversity and complexity were divided into four types.(1)Structural genes, such as 18S, 25sRNA gene_AJ496242.1,_FJ882050.1,_AF343676.1, some expressed only in yeast or mycelium phase, were the consequence of phase transition of Sporothrix schenckii.(2)Metabolic enzymes,such as gb|CP001656.1, _ref|NM_001006571.1, _DQ298384.1, which participated in carbohydrate, amino acid, lipid and coenzyme transport and metabolism(.3)Molecule on cell surface, such as gi|30721613|,_gi|45383668,_gb|GQ379464.1|, which participated in signaling transduction of cell, could modulate the expression of genes or morphogenesis of cells(.4)Molecule with indistinct function, such as gb|DQ510714.1| _gb|EU923089.1| _gb|DQ298391.1|.
Construction of cDNA subtractive libraries between yeast and mycelium phase of S.schenckii and bioinformatics analysis could play a role in the understanding of the phase transition mechanisms.
申克孢子丝菌为一种双相型真菌,在室温25℃表现为菌丝相,体内37℃为酵母相,这种酵母相与菌丝相之间的转换称为菌相转换。申克孢子丝菌酵母相可在感染者体内组织增殖,形成小的芽生孢子而致病。
申克孢子丝菌的双相性表明该菌不同菌相存在差异表达基因,在不同的环境条件下,通过菌体内信号转导的调控,表现为酵母相或菌丝相。目前申克孢子丝菌双相转换的相关研究仅涉及少数几个基因,双相转换的分子机制尚未明了,研究申克孢子丝菌双相转换机制对揭示其发病机理有重要意义。
本研究应用抑制性消减杂交( Suppression Subtractive Hybridization,SSH)技术,构建高特异性的申克孢子丝菌菌丝相(Mycelium,M)和酵母相(Yeast,Y)的正反cDNA消减文库,并对其差异表达的基因进行生物信息学分析,以探讨差异表达基因与酵母相与菌丝相双相转换的相关性。
本研究选择申克孢子丝菌标准株CMCC(F)D1a作为研究对象,以体外诱导形成的纯的菌丝相(M)与酵母相(Y)细胞作为实验材料,分别提取M与Y细胞的总RNA,逆转录并扩增M与Y细胞的cDNA,纯化扩增的cDNA,经RsaI酶切处理,将酶切后的M与Y细胞的cDNA分别作为测试子(Tester)和驱动子(driver),将M与Y细胞的cDNA分别分为两份,每份连接不同的接头,即:接头1(adaptor1)和接头2(adaptor2)。之后进行两轮消减杂交及两轮PCR扩增,纯化第二轮PCR产物,以紫外分光光度计检测cDNA纯度与浓度。连接载体pGEM-T并转化感受态细胞TOPO10,蓝白斑筛选阳性克隆,采用T7引物,PCR扩增插入片段,挑取插入片断大于250bp、带型单一的克隆进行大规模测序。判别测序的有效性,去除非单一克隆的数据,并对所有数据进行去载、拼接、聚类和Blastn分析及整理。
结果:M+Y文库(即在M相高表达,Y相低表达或不表达)获得751条表达序列标签(Expressed Sequence Tags,ESTs),平均长度为690.98bp,ESTs经拼接后获得101条unigenes;Y+M文库(即在Y相高表达,M相低表达或不表达)获得875条ESTs,平均长度为575.9bp,拼接获得249条unigenes。Blastn分析发现两个消减文库中都出现多个重复的基因序列,去除重复序列后,M+Y文库共获得45条unigenes,Y+M文库获得91条unigenes。申克孢子丝菌酵母相菌丝相的转换伴随着不同菌相细胞差异基因的高表达,这些高表达的差异基因可分为四类:(1)结构基因类,如18S、25sRNA基因,AJ496242.1,FJ882050.1,AF343676.1等,某些结构基因在申克孢子丝菌酵母相菌丝相状态下的独特表达,可能是菌相转换的结果;(2)代谢酶类,如gb|CP001656.1|,ref|NM_001006571.1|,DQ298384.1等,参与碳水化合物、氨基酸、脂质和辅酶运输和代谢;(3)细胞表面分子类,如gi|30721613|,gi|45383668,gb|GQ379464.1|等,这些分子参与细胞内的信号转导,推测其在菌相转换中起介导细胞基因表达的调控或细胞行为的改变;(4)功能不明的细胞分子,如gb|DQ510714.1|,gb|EU923089.1|,gb|DQ298391.1|等。它们在申克孢子丝菌双相转换机制中的作用也有待进一步验证。
申克孢子丝菌菌丝相和酵母相cDNA消减文库的构建及生物信息学分析为进一步筛选鉴定菌相转换相关差异基因奠定了基础,有助于阐明申克孢子丝菌双相转换的分子机制。
Sporothrix schenckii is the etiological agent of sporotrichosis, a subcutaneous mycosis, with an increasing worldwide incidence. It enters through small cuts and abrasions in the skin to cause the infection. Symptoms of sporotrichosis include localized nodular leisions, bumps and chronic ulcers at the point of entry and also along lymph nodes and vessels. However, it can disseminate in immunocompromised patients .
S.schenckii is a pathogenic fungus that undergoes a dimorphic transition from yeast to mycelium in response to environment conditions such as temperature. It appears as mycelium form at 25°C and yeast form at 37°C. Sporotrichosis is caused by the multiplication of yeast form colonies.
By some approaches, several genes have been identified that are expressed differently in transition from yeast to mycelium. Signal transduction modulated by some elements such as mitogen-activated protein kinases, cAMP and pH-responsive modules, appear to regulate this transition. However, the mechanism of dimorphic transition is still unclear. Therefore, study on the mechanism of dimorphic transition is important to reveal the pathogenesis of S.schenckii.
To screen the other differentially expressed genes about dimorphic transition of S.Schenckii, cDNA subtractive library between yeast and mycelium phase was constructed by suppression subtractive hybridization(SSH)and bioinformatics analysis was performed to profile the relationship between those differently expressed genes and dimorphic transition.
In this study, CMCC(F)D1a, a S. schenckii standard strain, was induced to form mycelium(M)and yeas(Y)cells. Total RNA isolated from M and Y cells was reverse transciptased and amplified respectively, the average length of cDNA after purification and RsaI enzyme digestion is 500bp. cDNA of M and Y cells after RsaI enzyme restriction was regarded as tester and driver respectively, and then were divided into two fragments to ligate adaptor1 and adaptor2. The PCR products were ligated with pGEM-T plasmid vectors, transformed into E.coli TOP10. The positive recombinant clones were picked up through the blue-white screening system, and confirmed by PCR method with P7 primer. Finally, the differentially expressed cDNA fragments were sequenced and compared with EST database in Genbank.
Results: 751 expressed sequence tags(ESTs)were obtained in M+Y library(that means ESTs were over expressed in M-phase, low expressed or no expressed in Y-phase), the average length is 690.98bp. Meanwhile, 875 ESTs were obtained in Y+M library, the average length is575.9bp. After splicing of ESTs, 101 unigenes were obtained in M+Y library and 249 unigenes in Y+M library. However, several repeated sequences were found by Blastn analysis in both two subtractive libraries. Finally, 45 unigenes in M+Y library and 91 unigenes in Y+M library were obtained after the removal of repetitive sequences.
During the construction of cDNA subtractive libraries, the ?distribution of differently expressed genes varied with dimorphic transition. The over expressed genes with diversity and complexity were divided into four types.(1)Structural genes, such as 18S, 25sRNA gene_AJ496242.1,_FJ882050.1,_AF343676.1, some expressed only in yeast or mycelium phase, were the consequence of phase transition of Sporothrix schenckii.(2)Metabolic enzymes,such as gb|CP001656.1, _ref|NM_001006571.1, _DQ298384.1, which participated in carbohydrate, amino acid, lipid and coenzyme transport and metabolism(.3)Molecule on cell surface, such as gi|30721613|,_gi|45383668,_gb|GQ379464.1|, which participated in signaling transduction of cell, could modulate the expression of genes or morphogenesis of cells(.4)Molecule with indistinct function, such as gb|DQ510714.1| _gb|EU923089.1| _gb|DQ298391.1|.
Construction of cDNA subtractive libraries between yeast and mycelium phase of S.schenckii and bioinformatics analysis could play a role in the understanding of the phase transition mechanisms.
引文
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[37] Ranganathan S, Menon R, Gasser RB. Advanced in silico analysis of expressed sequence tag (EST) data for parasitic nematodes of major socio-economic importance--fundamental insights toward biotechnological outcomes[J]. Biotechnol Adv. 2009; 27(4): 439-48.
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[1] Valentin-Berrios S, Gonzalez-Mendez RR, et al. Functional, genetic and bioinformatic characterization of a calcium/calmodulin kinase gene in Sporothrix schenckii[J]. BMC Microbiol. 2007 Nov 29;7:107.
[2] Aquino-Pi?ero EE, Rodríguez del Valle N. Different protein kinase C isoforms are present in the yeast and mycelium forms of Sporothrix schenckii[J]. Mycopathologia. 1997; 138(3):109-15.
[3] Nakao S, Inoue D. Involvement of protein kinase C in IL-1beta-induced expression of cyclooxygenase-2 in human gingival fibroblasts[J]. J Oral Sci. 2009 Sep;51(3): 417-23.
[4] Aquino-Pi?ero E, Rodríguez-del Valle N. Characterization of a protein kinase C gene in Sporothrix schenckii and its expression during the yeast-to-mycelium transition[J]. Med Mycol. 2002 Apr;40(2):185-99.
[5] de Jesús-Berríos M, Rodríguez-del Valle N. Expression of a Pho85 cyclin– dependent kinase is repressed during the dimorphic transition in Sporothrix schenckii[J]. Fungal Genet Biol. 2002 Oct; 37(1):39-48.
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[18] Means AR, Dedman JR. Calmodulin--an intracellular calcium receptor[J]. Nature. 1980;285:73–77. doi: 10.1038/285073a0.
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[39] Gusev Y. Computational methods for analysis of cellular functions and pathways collectively targeted by differentially expressed microRNA. Methods[J],2008 Jan; 44(1): 61-72.
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[1] Valentin-Berrios S, Gonzalez-Mendez RR, et al. Functional, genetic and bioinformatic characterization of a calcium/calmodulin kinase gene in Sporothrix schenckii[J]. BMC Microbiol. 2007 Nov 29;7:107.
[2] Aquino-Pi?ero EE, Rodríguez del Valle N. Different protein kinase C isoforms are present in the yeast and mycelium forms of Sporothrix schenckii[J]. Mycopathologia. 1997; 138(3):109-15.
[3] Nakao S, Inoue D. Involvement of protein kinase C in IL-1beta-induced expression of cyclooxygenase-2 in human gingival fibroblasts[J]. J Oral Sci. 2009 Sep;51(3): 417-23.
[4] Aquino-Pi?ero E, Rodríguez-del Valle N. Characterization of a protein kinase C gene in Sporothrix schenckii and its expression during the yeast-to-mycelium transition[J]. Med Mycol. 2002 Apr;40(2):185-99.
[5] de Jesús-Berríos M, Rodríguez-del Valle N. Expression of a Pho85 cyclin– dependent kinase is repressed during the dimorphic transition in Sporothrix schenckii[J]. Fungal Genet Biol. 2002 Oct; 37(1):39-48.
[6] Huang D, Friesen H, Andrews B. Pho85, a multifunctional cyclin-dependent protein kinase in budding yeast[J]. Mol Microbiol. 2007 Oct;66(2):303-14.
[7] Nishizawa M, Suzuki K, Fujino M, et al. The Pho85 kinase, a member of the yeast cyclin-dependent kinase (Cdk) family, has a regulation mechanism different from Cdks functioning throughout the cell cycle[J]. Genes Cells. 1999 Nov; 4(11): 627-42.
[8] Bootman MD, Collins TJ, Peppiatt CM, et al. Calcium signalling--an overview[J]. Semin Cell Dev Biol. 2001;12:3–10. doi: 10.1006/scdb.2000. 2011.
[9] Bootman MD, Lipp P, Berridge MJ. The organisation and functions of local Ca(2+) signals[J]. J Cell Sci. 2001;114:2213–2222.
[10] Berridge MJ, Bootman MD, Roderick HL. Calcium signalling: dynamics, homeostasis and remodelling[J]. Nat Rev Mol Cell Biol. 2003;4:517–529.
[11] Berridge MJ. Calcium signal transduction and cellular control mechanisms[J]. Biochim Biophys Acta. 2004;1742:3–7.
[12] Berridge MJ. The versatility and complexity of calcium signalling[J]. Novartis Found Symp. 2001;239:52–64; discussion 64-7, 150-9.
[13] Chin D, Means AR. Calmodulin: a prototypical calcium sensor[J]. Trends Cell Biol. 2000;10:322–328.
[14]. Hook SS, Means AR. Ca(2+)/CaM-dependent kinases: from activation to function. Annu Rev Pharmacol Toxicol[J]. 2001;41:471–505.
[15]. Nairn AC, Picciotto MR. Calcium/calmodulin-dependent protein kinases[J]. Semin Cancer Biol. 1994;5:295–303.
[16]Hudmon A, Schulman H. Structure-function of the multifunctional Ca2+ / calmodulin-dependent protein kinase II[J]. Biochem J. 2002;364:593–611.
[17] Means AR. Regulatory cascades involving calmodulin-dependent protein kinases[J]. Mol Endocrinol. 2000;14:4–13. doi: 10.1210/me.14.1.4.
[18] Means AR, Dedman JR. Calmodulin--an intracellular calcium receptor[J]. Nature. 1980;285:73–77. doi: 10.1038/285073a0.
[19] Tombes RM, Faison MO, Turbeville JM. Organization and evolution of multifunctional Ca(2+)/CaM-dependent protein kinase genes[J]. Gene. 2003 Dec 11;322:17-31.
[20] Liz Valle-Aviles, Shirley Valentin-Berrios, Ricardo R Gonzalez-Mendez, et al. Functional, genetic and bioinformatic characterization of a calcium/calmodulin kinase gene in Sporothrix schenckii[J]. BMC Microbiol. 2007(7): 107-117.
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