桑椹菌核病菌(Scleromitrula shiraiana)黑色素生物合成相关基因的克隆与功能分析

Cloning and functional analysis of the melanin biosynthesis gene in Scleromitrula shiraiana

小柱孢酮脱水酶(scytalone dehydratase,SCD)及羟基萘还原酶(hydroxynaphthalene reductase,HNR)是真菌多聚二羟奈类(DHN)黑色素生物合成途径中的关键酶。根据已知真菌的小柱孢酮脱水酶及羟基萘还原酶的保守结构域设计兼并引物并利用RACE技术,获得桑椹菌核病菌(核地仗菌,Scleromitrula shiraiana)SsSCD1和Ss4HNR1的DNA和c DNA序列。SsSCD1和Ss4HNR1均含2个内含子和3个外显子,分别编码169和263个氨基酸残基。进化分析表明SsSCD1和Ss4HNR1与灰葡萄孢和核盘菌中小柱孢酮脱水酶和四羟基萘还原酶基因的亲缘关系最近。DHN黑色素合成特异性抑制剂三环唑处理核地仗菌,结果显示三环唑可抑制核地仗菌菌丝生长和黑色素合成,对SsSCD1的表达无显著影响,但Ss4HNR1的表达水平显著提高。这些结果表明三环唑能够特异性的抑制四羟基萘还原酶,且DHN黑色素是核地仗菌生长发育的重要产物。

Scytalone dehydratase and hydroxynaphthalene reductase are the important enzymes in fungal DHN melanin biosynthesis. Degenerated PCR primers were designed based on the consensus sequences from other fun- gi. Then the full genomic sequences of SsSCD1 and Ss4HNR1 were obtained by the method of RACE-PCR in Scleromitrula shiraiana. Both genes contain two introns and three exons with deduced 169 and 263 amino acid residues, respectively. Sequences of the two genes showed high similarity to SCD and 4HNR genes from Sclero- tinia sclerotiorum and Botryotinia cinerea T4, respectively. As a melanin synthesis inhibitor, tricyclazole inhibi- ted hyphal growth and DHN melanin biosynthesis. Also, Ss4HNR1 expression was significantly improved by tri- cyclazole treatment, but not SsSCD1. These results indicated that tricyclazole represses the activity of HN reduc- tase, and DHN melanin is a key product for the growth and development of S. shiraiana.