草菇PGAM基因克隆、结构及其在同核、异核菌株中的表达量分析

Cloning and Structural Analysis of Phosphoglycerate Mutas Gene and Its Expression Levels in Homokaryon and Heterokaryon of Volvariella volvacea

以福建农林大学菌物研究中心保存的草菇单孢菌株PYd21、PYd15及二者的杂交配对异核菌株H15-21为研究材料,利用本中心草菇的基因组、转录组及表达谱测序数据,对草菇磷酸甘油酸变位酶(PGAM)基因进行基因克隆,并对其结构与表达量进行研究。克隆测序了PYd21与PYd15中PGAM基因并进行比对,比对结果表明2个菌株的PGAM基因序列一致。利用转录组数据对PGAM基因结构进行分析,分析结果显示:该基因全长2 118bp,含有10个外显子、9个内含子,其中6个内含子存在内含子保留的可变剪切类型;开放阅读框(ORF)全长1 611bp、编码536个氨基酸。数字基因表达谱分析结果表明,PGAM基因在PYd21、PYd15及H15-21中的标准表达量分别为35.29、127.91、302.89TPM(Transcript Per Million Clean Tags),表达量依次升高,与菌丝生长速度显著正相关,且异核菌株H15-21中的表达量显著高于2个单孢菌株,暗示了异核体H15-21中两种不同的细胞核存在相互作用,影响了PGAM基因的表达。采用实时荧光定量PCR技术对基因表达量进行验证,与表达谱数据相符合。

Two single spore isolates named PYd21 and PYd15 respectively and their hybrid heterokaryon named H15-21 were studied as materials in this paper.Using sequencing data of genome,transcriptome and digital gene expression profiles of Volvariella volvacea,Phosphoglycerate mutas（PGAM） were cloned and its structure and expression were studied.Alignment of PGAM genes from PYd21 and PYd15 revealed that both sequences to be identical.Structural analysis of PGAM gene revealed that it consisted of 2118bp with an open reading frame（ORF） of 1611bp,encoding a polypeptide of 536 amino acids.PGAM gene contained 10 exons and 9 introns.One type of alternative splicing and 6 alternative splicing sites were identified during RNA processing.TPM（Transcript Per Million Clean Tags） values for PYd21,PYd15 and H15-21 were 35.29,127.91 and 302.89 respectively,which had significant positive correlation to the growth rate of mycelium.The expression levels of PGAM gene in PYd21,PYd15 and H15-21 indicated that two differently type of nucleus in the cell of H1521 interacted each other which affected expression level of PGAM gene in H15-21.The expression levels of PGAM gene were confirmed by real-time quantitative PCR.