玉米大斑病菌小柱孢酮脱水酶基因(scd)的克隆与功能分析

Cloning and functional analysis of scytalone dehytratase gene in Exserohilum turcicum

摘要:

根据已知植物病原真菌黑色素生物合成相关基因scd(scytalone dehydratase)氨基酸序列保守区域设计简并引物,分别以玉米大斑病菌基因组DNA和cDNA为模板,通过PCR技术获得scd基因的同源片段,利用SMART-RACE技术和3′RACE技术获得了scd的cDNA全长序列。并根据scd基因cDNA全长序列设计基因特异性引物扩增玉米大斑病菌基因组DNA获得了该基因DNA全长。通过DNA序列和cDNA序列对比分析发现scd基因编码一个180个氨基酸的开放阅读框架,DNA序列含有两个分别为50bp和78bp的内含子。生物信息学分析表明其氨基酸序列与水稻胡麻叶斑病菌的scd基因的相似性很高。DHN黑色素生物合成途径特异性抑制剂—Carpropamid处理玉米大斑病菌,在12~24h之内可以抑制病菌分生孢子的萌发和附着胞的产生,但随着处理时间的延长抑制剂的抑制作用变弱,并且经过抑制剂处理的病菌不能侵入寄主组织或不能在寄主组织内扩展。初步明确了scd与玉米大斑病菌黑色合成途径及致病性的关系。

Abstract:

Genemonic DNA and cDNA homologous fragments of the scd(scytalone dehydratase) gene were obtained by polymerase chain reaction（PCR）amplification from degenerated primer sets designed on the basis of the conserved amino acid regions of scytalone dehydratase and polyketide synthase domains from others fungis. The completed cDNA sequence of scd in E. turcica was obtained by the method of SMART-RACE and 3′RACE. There is one open reading frame composed of 181 codons and two deduced introns of 50 and 78 nucleotides in the scd gene. The deduced amino acid sequence of the scd showed high similarity to the amino acid sequence of scytalone dehydratase from Bipolaris oryzae. Carpropamid，a specific inhibitor，could inhibit the conidial germination and appressorium production of E. turcica within 24h treatment but no evident inhibitory effect after 24h . The experimental results also suggested that E. turcica could not penetrate the surface of corn tissue or increase in the corn tissue. It was conclusion that scd gene might play an important role in melanin biosynthetic pathway and pathogenicity of E. turcica.