梨果黑斑病菌钙调磷酸酶基因生物信息学分析及其对侵染结构分化的调控作用

Bioinformatics Analysis of Calcineurin Gene and Its Regulatory Role in Infection Structure Differentiation of Alternaria alternata

为揭示梨果黑斑病菌Alternaria alternata CaN基因的调控作用,采用RT-PCR技术克隆A.alternata CaN基因,利用在线工具对其基因及其编码蛋白进行生物信息学分析;同时分析其在A.alternata侵染结构分化过程中的表达特性;并进一步以钙调磷酸酶专一性抑制剂环孢菌素A(CsA)处理研究其对A.alternata侵染结构分化和致病性的影响。结果A.alternata克隆得到CNA和CNB基因,分别命名为AaCNA(GenBank NW_017306222)和AaCNB(GenBank NW_017306193),生物信息学分析表明AaCNA和AaCNB均无跨膜结构,且具有多个磷酸化位点,主要定位于细胞核上;AaCNA包括一个N端催化结构域、CNB结合结构域、钙调素CaM结合结构域和自抑制蛋白结构域,AaCNB具有4个EF-hand Ca 2+结合位点。qRT-PCR分析表明,在疏水及果蜡诱导A.alternata侵染结构分化过程中AaCNA和AaCNB基因表达量均显著上调,且果蜡诱导作用更明显。药理学表明,环孢菌素A处理显著抑制疏水性和蜡质诱导的A.alternata孢子萌发和附着胞形成,同时还可抑制损伤接种A.alternata早酥梨黑斑病的扩展。综上结果表明AaCNA和AaCNB参与A.alternata侵染结构形成的调控。

Bioinformatics of CaN genes and their encoded protein were analyzed by using online tools,and RT-PCR was used to identify the regulatory role of CaN gene in Alternaria alternata based on cloning CaN of Alternaria alternata,the expression characteristics of CaN during infection structure differentiation of A.alternata were also analyzed.As a specific inhibitor of calcineurin,cyclosporine A(CsA)was used to examine the regulatory role of AaCaN in infection structure differentiation and pathogenicity of A.alternata.CNA and CNB of A.alternata were cloned and assigned as AaCNA(GenBank NW_017306222)and AaCNB(GenBank NW_017306193),respectively.Bioinformatics analysis showed that AaCNA and AaCNB had no transmembrane structure and both had multiple phosphorylation sites,which were located on the nucleus.AaCNA includes an N-terminal catalytic calmodulin binding,calcineurin B subunit binding,and autoinhibitory domains.AaCNB comprises four EF-hand calcium binding domains.qRT-PCR analysis showed that gene expression levels of AaCNA and AaCNB were significantly upregulated during infection structure differentiation of A.alternata on hydrophobic and pear wax extract coated surface,and the fruit wax extract coated surface had a greater stimulation impact.Pharmacological experiment indicated that CsA treatment significantly inhibited spore germination and appressorium formation rate of A.alternata on hydrophobic and pear wax extract coated surface,and also inhibited the development of pear black spot.These findings suggested that AaCNA and AaCNB would be involved in the regulation of A.alternata infection structure formation.