梨果黑斑病菌互隔交链孢(Alternaria alternata) hnr基因的克隆及其对侵染结构分化的调控

Cloning of hnr and characterization of its regulatory role in the infection structure differentiation of Alternaria alternata

摘要:

【背景】羟基萘还原酶(hydroxynaphthalene reductase，HNR)是1,8-间苯二酚(1,8-dihydroxynaphthalene，DHN)黑色素合成途径中起关键作用的酶，研究表明HNR不仅参与真菌黑色素合成，而且对其生长发育及致病性也具有一定的调控作用，但HNR对真菌病原物侵染结构分化的调控研究鲜见报道。【目的】在对梨果黑斑病菌互隔交链孢(Alternaria alternata) HNR的基因进行克隆与生物信息学分析的基础上，通过药理学方法初步探讨HNR对A.alternata生长及侵染结构分化的调控作用，为进一步揭示HNR在A.alternata侵染结构分化形成过程中的分子机制提供理论依据。【方法】对梨果黑斑病菌A.alternata的2个hnr基因进行了克隆；通过gene structure display server、open reading frame (ORF) Finder及conserved domain search等数据库及相关软件，对hnr基因及蛋白进行生物信息学分析，并利用HNR特异性抑制剂三环唑处理分析其对A.alternata生长发育、黑色素合成和侵染结构形成的影响，同时采用实时荧光定量PCR (RT-qPCR)技术分析了hnr基因在A.alternata不同侵染结构分化时期的表达特性。【结果】从梨果黑斑病菌A.alternata克隆得到2个羟基萘还原酶基因hnr的编码区全长，分别命名为Aa4hnr和Aa3hnr，其中Aa4hnr基因全长为1 266 bp，编码了268个氨基酸，无内含子，有9个ORF；Aa3hnr基因全长为1 356 bp，编码了267个氨基酸，含有2个大小分别为51 bp和49 bp的内含子，有17个ORF；进化分析表明，Aa4hnr和Aa3hnr与Ophiobolus disseminans和Alternaria arborescens分别具有较高的一致性，同时Aa4hnr和Aa3hnr编码的蛋白均含有NAD (P)结合域，属于短链脱氢酶/还原酶(short-chain dehydrogenase/reductase，SDR)家族。药理学结果表明，三环唑处理显著降低了A.alternata DHN黑色素的生物合成，抑制了疏水性诱导的A.alternata侵染结构的形成；进一步分析Aa4hnr和Aa3hnr在疏水表面诱导的A.alternata孢子萌发阶段(2 h)、附着胞形成阶段(6 h)、侵染菌丝形成阶段(8 h)的基因表达量，Aa4hnr的基因表达量在A.alternata侵染结构分化的各个时期均发生下调，Aa3hnr在附着胞形成阶段(6 h)表达量下调，然而在侵染菌丝形成阶段(8 h)显著上调表达。【结论】Aa4hnr和Aa3hnr对梨果黑斑病菌侵染具有一定的调控作用。

Abstract:

[Background] Hydroxynaphthalene reductase (HNR), a key enzyme in the biosynthesis of 1,8-dihydroxynaphthalene (DHN) melanin, is involved in fungal melanin synthesis and has regulatory effect on the fungal growth and pathogenicity. However, the regulatory role of HNR in the infection structure differentiation of fungal pathogens remains unknown. [Objective] On the basis of the cloning and bioinformatics analysis of the hnr genes of Alternaria alternata, we preliminarily evaluated the regulatory role of HNR in the growth and infection structure differentiation of A. alternata through pharmacological methods, aiming to provide a theoretical basis for revealing the molecular mechanism of HNR regulating the infection structure differentiation of A. alternata. [Methods] The hnr genes were cloned by homologous cloning method, and the nucleotide and amino acid sequences were analyzed via bioinformatics tools including gene structure display server, open reading frame Finder, conserved domain search. The HNR-specific inhibitor tricyclazole was used for the study about the regulatory role of hnr in the growth and development, melanin synthesis, and infection structure formation of A. alternata. Real-time quantitative PCR (RT-qPCR) was employed to determine the expression levels of hnr genes at the spore germination (2 h), appressorium formation (6 h), and hypha formation (8 h) stages throughout the infection structure differentiation of A. alternata. [Results] The full-length coding sequences (CDSs) of two hnr genes were cloned from A. alternata and designated as Aa4hnr and Aa3hnr. Aa4hnr had a length of 1 266 bp, encoded 268 amino acid residues, and contained 9 ORFs and no intron. Aa3hnr, with a length of 1 356 bp, encoded 267 residues and contained 2 introns (51 bp and 49 bp) and 17 ORFs. The phylogenetic analysis showed that Aa4hnr and Aa3hnr shared high homology with the hnrs from Ophiobolus disseminans and Alternaria arborescens, respectively. The presence of NAD(P) binding domain indicated that Aa4hnr and Aa3hnr belonged to the short-chain dehydrogenase/reductase (SDR) family. Tricyclazole treatment significantly reduced the DHN melanin synthesis in A. alternata and inhibited the infection structure formation of A. alternata on hydrophobic surface. The expression of Aa4hnr was down-regulated at all the stages of infection structure differentiation, and that of Aa3hnr was down-regulated at the appressorium formation stage (6 h) while significantly up-regulated at the hypha formation stage (8 h). [Conclusion] Aa4hnr and Aa3hnr have some regulatory effects on infection of A. alternata.