灰葡萄孢钙调磷酸酶基因β亚基及其启动子的克隆与序列分析
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摘要
植物病原真菌的生长、发育和致病性均受到真菌的细胞外信号转导途径调控。如:Ca~(2+)途径、cAMP途径和MAPK途径。CNB基因通过去磷酸化作用可对其他信号转导通路进行调节,本论文克隆了CNB基因及其5'上游调控序列,并对其进行了生物信息学分析、拷贝数的验证,构建了CNB基因正反义表达载体,为进一步明确CNB基因与灰葡萄孢病菌的致病性的关系奠定基础。
本试验根据CNB基因核苷酸序列的保守区域设计简并引物,以番茄灰霉病菌BC1菌株的cDNA为模板,通过PCR技术获得CNB基因的部分片段,通过NCBI的同源性分析,在灰葡萄孢EST表达序列标签库中发现两条与该片段同源性达到100%的EST标签,通过DNAMAN软件分析,拼接获得了CNB基因525 bp的cDNA开放阅读框(ORF)。然后根据CNB基因cDNA全长序列设计特异性引物进行PCR扩增,以番茄灰霉病菌BC1基因组DNA为模板,扩增获得了该基因810 bp的DNA全长序列。利用DNAMAN软件对DNA序列和cDNA序列进行对比分析,发现CNB基因cDNA开放阅读框编码174个氨基酸,DNA序列含有3个大小分别为40 bp、120bp和84 bp的内含子,在内含子的边缘都显示出典型的内含子边缘特征序列“GT...AG”。
利用NCBI对CNB蛋白进行分析,结果表明CNB蛋白含有4个EF手型Ca~(2+)结合区域,相对分子量为19.749 8 KD,等电点为4.28,其中含有32个带负电荷的氨基酸,21个带正电荷的氨基酸,不含有半胱氨酰、组氨酸和色氨酸,在进行蛋白纯化时应该选择弱阳离子介质进行离子交换色谱。利用Genome walking技术获得了CNB基因5'上游转录调控序列,通过Softberry软件进行同源性分析,结果表明该序列中具有1个重要的核转录因子NF-Kb结合位点,1个激活蛋白质AP-2结合位点,以及Sp1、E2F、BKLF转录因子结合位点各1个,该启动子具有如此多的转录因子位点,而这些作用元件大多与细胞增殖有关,这否与灰霉的强致病性有关,还需要进一步的研究证明的。
以CNB基因的DNA序列为探针进行Southern杂交试验,结果表明CNB基因在番茄灰霉病菌的基因组中以单拷贝形式存在,为下一步进行CNB基因的敲除或缺失试验,验证CNB基因在灰葡萄孢病菌的致病性中的作用提供了有利的证据。
Signal transduction cascades regulating fungal growth, morphology, and pathogenicity are remarkably conserved between distantly related fungi, such as Ca~(2+) cascades, the cAMP-dependent protein kinase and MAP kinases. In order to identify the relationship between Calcineurinβsubunit(CNB)and the pathogenicity of Botrytis cinerea of tomato, CNB were cloned and the functional analysis were done by bioinformatics analysis, CNB copies' confirmation and constructed the sence and antisence CNB gene expression induction.
Homologous cDNA fragments of CNB were obtained by polymerase chain reaction (PCR) amplification from degenerated primer sets designed on the basis of the conserved amino acid regions of CNB from others fungi. Using NCBI blastn, the sequences of CNB showed 100% similarity to two ESTs of B. cinerea. The 525 bp completed cDNA sequence of CNB in B. cinerea was obtained with DNAMAN. The 810 bp completed DNA sequence of CNB in B. cinerea was obtained with primer sets designed on the basis of cDNA sequence of CNB. There is one open reading frame composed of 174 aa and three deduced introns of 40 bp, 120 bp and 84 bp. All introns were accordance with GT-AG rules.
Analyzing CNB protein sequences by NCBI, conclusion showed it contained molecular weight of 19.7498 KD and PI of 4.28 and four EF-hand calcium-binding domains, containing 32 aa with negative electric charge and 21 aa with positive electric charge, no containing Cys, His, Trp. The promoter sequence of CNB was amplified using the Genomic walking method. Promoter sequence analysis showed that the promoter sequence contained a nucleus transcription factor NF-Kb union position spot, an activation protein AP-2 union position spot, and other regulatory elements, such as TF II -I, AP-2, BKLF, Sp1. We proofed furtherly if the regulatory elements relating cell multiplication were critical for B. cinerea pathogenicity.
The result of Southern blotting showed that CNB was single copy in the genome of B.cinerea, which provided evidence for us to identify furtherly the relationship between CNB and the pathogenicity of B. cinerea.
本试验根据CNB基因核苷酸序列的保守区域设计简并引物,以番茄灰霉病菌BC1菌株的cDNA为模板,通过PCR技术获得CNB基因的部分片段,通过NCBI的同源性分析,在灰葡萄孢EST表达序列标签库中发现两条与该片段同源性达到100%的EST标签,通过DNAMAN软件分析,拼接获得了CNB基因525 bp的cDNA开放阅读框(ORF)。然后根据CNB基因cDNA全长序列设计特异性引物进行PCR扩增,以番茄灰霉病菌BC1基因组DNA为模板,扩增获得了该基因810 bp的DNA全长序列。利用DNAMAN软件对DNA序列和cDNA序列进行对比分析,发现CNB基因cDNA开放阅读框编码174个氨基酸,DNA序列含有3个大小分别为40 bp、120bp和84 bp的内含子,在内含子的边缘都显示出典型的内含子边缘特征序列“GT...AG”。
利用NCBI对CNB蛋白进行分析,结果表明CNB蛋白含有4个EF手型Ca~(2+)结合区域,相对分子量为19.749 8 KD,等电点为4.28,其中含有32个带负电荷的氨基酸,21个带正电荷的氨基酸,不含有半胱氨酰、组氨酸和色氨酸,在进行蛋白纯化时应该选择弱阳离子介质进行离子交换色谱。利用Genome walking技术获得了CNB基因5'上游转录调控序列,通过Softberry软件进行同源性分析,结果表明该序列中具有1个重要的核转录因子NF-Kb结合位点,1个激活蛋白质AP-2结合位点,以及Sp1、E2F、BKLF转录因子结合位点各1个,该启动子具有如此多的转录因子位点,而这些作用元件大多与细胞增殖有关,这否与灰霉的强致病性有关,还需要进一步的研究证明的。
以CNB基因的DNA序列为探针进行Southern杂交试验,结果表明CNB基因在番茄灰霉病菌的基因组中以单拷贝形式存在,为下一步进行CNB基因的敲除或缺失试验,验证CNB基因在灰葡萄孢病菌的致病性中的作用提供了有利的证据。
Signal transduction cascades regulating fungal growth, morphology, and pathogenicity are remarkably conserved between distantly related fungi, such as Ca~(2+) cascades, the cAMP-dependent protein kinase and MAP kinases. In order to identify the relationship between Calcineurinβsubunit(CNB)and the pathogenicity of Botrytis cinerea of tomato, CNB were cloned and the functional analysis were done by bioinformatics analysis, CNB copies' confirmation and constructed the sence and antisence CNB gene expression induction.
Homologous cDNA fragments of CNB were obtained by polymerase chain reaction (PCR) amplification from degenerated primer sets designed on the basis of the conserved amino acid regions of CNB from others fungi. Using NCBI blastn, the sequences of CNB showed 100% similarity to two ESTs of B. cinerea. The 525 bp completed cDNA sequence of CNB in B. cinerea was obtained with DNAMAN. The 810 bp completed DNA sequence of CNB in B. cinerea was obtained with primer sets designed on the basis of cDNA sequence of CNB. There is one open reading frame composed of 174 aa and three deduced introns of 40 bp, 120 bp and 84 bp. All introns were accordance with GT-AG rules.
Analyzing CNB protein sequences by NCBI, conclusion showed it contained molecular weight of 19.7498 KD and PI of 4.28 and four EF-hand calcium-binding domains, containing 32 aa with negative electric charge and 21 aa with positive electric charge, no containing Cys, His, Trp. The promoter sequence of CNB was amplified using the Genomic walking method. Promoter sequence analysis showed that the promoter sequence contained a nucleus transcription factor NF-Kb union position spot, an activation protein AP-2 union position spot, and other regulatory elements, such as TF II -I, AP-2, BKLF, Sp1. We proofed furtherly if the regulatory elements relating cell multiplication were critical for B. cinerea pathogenicity.
The result of Southern blotting showed that CNB was single copy in the genome of B.cinerea, which provided evidence for us to identify furtherly the relationship between CNB and the pathogenicity of B. cinerea.
引文
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