草菇磷酸果糖激酶(PFK)基因克隆、结构及不同菌株中表达量分析

Cloning, structural analyses and expression levels of phosphofructokinase gene in different strains of Volvariella volvacea

从福建主栽品种屏优一号PY分离到2个不能正常出菇的单孢菌株PYd21、PYd15,二者配对杂交得到可正常出菇异核菌株H15-21。用solexa测序技术对PYd21基因组从头测序(de novo sequencing),对PYd15、PYd21、H15-21的菌丝体分别进行数字基因表达谱测序,对8个样品(PYd15、PYd21、H15-21的菌丝体,H15-21出菇后的原基、钮扣期菌柄、蛋形期菌柄、伸长期菌柄和成熟期菌柄)mRNA等量混合物进行转录组测序。克隆测序了PYd21与PYd15中磷酸果糖激酶(PFK)基因,比对结果表明二者序列一致。利用转录组数据对PFK基因结构进行分析,结果表明:该基因全长3,494bp,有12个外显子、11个内含子;开放阅读框(ORF)全长2,457bp,编码818个氨基酸,5′端非翻译区(5′UTR)长度281bp,3′端非翻译区(3′UTR)全长103bp;RNA在加工过程中存在1种可变剪切类型、6个可变剪切位点。数字基因表达谱分析结果表明,PFK基因在PYd21、PYd15及H15-21中的标准表达量分别为71.08、120.61、251.85(transcriptper million cleantags,TPM),表达量依次升高,与菌丝生长速度显著正相关。并且异核菌株H15-21表达量高于2个单孢菌株之和,基因表达存在协同增效作用。采用实时荧光定量PCR技术对基因表达量进行验证,表达谱数据切实可靠。

Two single spore unfruitful isolates, PYd21 and PYd15, were obtained from Volvariella volvacea CV. PY in FujianProvince. A hybrid heterokaryon, H15-21, was obtained by mating PYd21 and PYdl5, and it could fruit normally. Using solexa genome analyzer platform, the genome of PYd21 was de novo sequencing; digital gene expression profiles of the mycelium including PYd21, PYdl 5 and H15-21 were sequenced respectively; a transcriptome containing equivalent mRNA of 8 samples including the mycelium samples of PYd21, PYdl5 and H15-21, fruiting body samples of ＂pinhead＂ stage, and stipes of＂button＂, ＂egg＂, ＂elongation＂ and ＂mature＂ stages from H15-21 were sequenced. Phosphofructokinase （PFK） genes were cloned and sequenced from both PYd21 and PYdl5, and their structures were analyzed through paired-end mapping of transcriptional reads. Sequence alignment revealed both sequences to be identical, and PFK consists of 3,494bp with an open reading frame （ORF） of 2,457bp, encoding a polypeptide of 818 amino acids. PFK contained 12 exons and 11 introns, and the 5qJTR and 3＇UTR were 281bp and 103bp long, respectively. One type of alternative splicing and 6 alternative splicing sites were identified during RNA processing. TPM （transcripts per million tags） values for PYd21, PYd 15 and H 15-21 were 71.08, 120.61 and 251.85, respectively, showing significant positive correlation to the growth rate of mycelium. The data indicated that the expression level of PFK gene in H15-21 had synergistic effect. The expression levels of PFK gene were confirmed by real-time quantitative PCR.