假禾谷镰孢细胞凋亡基因FpTatD的鉴定与表达分析

摘要

【目的】鉴定和克隆假禾谷镰孢(Fusarium pseudograminearum)细胞凋亡相关基因FpTatD，分析FpTatD在假禾谷镰孢菌丝、分生孢子和侵染不同时期的表达，为探索细胞凋亡在假禾谷镰孢侵染过程中的功能提供理论依据。【方法】从GenBank获得模式物种已知的TatD氨基酸序列，利用BLASTP的方法在镰孢中鉴定TatD同源蛋白，并构建进化树；分别以假禾谷镰孢的基因组DNA和cDNA为模板，通过PCR方法克隆假禾谷镰孢FpTatD的基因序列和开放阅读框（ORF）序列；利用实时荧光定量PCR方法分析FpTatD在假禾谷镰孢生长、产孢及侵染不同时期的表达；利用转录组测序方法分析FpTatD在假禾谷镰孢与感病小麦和抗病小麦互作中的表达差异。【结果】镰刀菌中有4个保守的TatD同源蛋白，与其他物种的TatD蛋白构建系统进化树，发现TatD在进化上分成两个大的分支，第一个分支的TatD在所有物种中都非常保守，包括镰刀菌的TatD1和TatD2，第二个分支的TatD蛋白属于植物和真菌特有的，包括镰刀菌的TatD3和TatD4；克隆假禾谷镰孢的FpTatD1、FpTatD2、FpTatD3、FpTatD4基因长度分别为993、1331、1227、1176 bp，ORF区长度为993、1182、1227、1176 bp。FpTatD25′端包含两段长度为94和55 bp的非编码序列，FpTatD1、FpTatD3和FpTatD4均不含非编码序列。4个FpTatD编码蛋白质的分子量大小分别为36.37、43.13、45.59、44.25 kD。蛋白结构和序列分析显示FpTatD蛋白均具有明显的DNase结构域以及大部分保守的氨基酸位点；实时荧光定量PCR分析显示，FpTatD1和FpTatD2在假禾谷镰孢中的表达量高，且在侵染初期阶段明显诱导表达，尤其是FpTatD1在侵染36 h和3 d时分别上调表达8.8倍和7.6倍；而FpTatD3和FpTatD4表达量非常低，且在不同阶段的表达量无明显变化，说明FpTatD1和FpTatD2在假禾谷镰孢中起主要作用；通过分析假禾谷镰孢与感病小麦国麦301和抗病小麦周麦24互作的转录组数据，与实时荧光定量PCR结果一致，FpTatD1和FpTatD2表达量高，并在侵染阶段上调表达，而且相对于假禾谷镰孢与感病小麦的亲和互作中，其与抗病小麦的非亲和互作中，FpTatD诱导表达倍数更高。【结论】假禾谷镰孢细胞凋亡相关基因FpTatD可能参与病原菌与寄主的互作过程。%Objective]The objective of this study is to investigate the potential biological functions of apoptosis-related genes FpTatD inFusarium pseudograminearum, for this purpose,FpTatD genes were identified and cloned inF. pseudograminearum, and the expression of these genes were examined at mycelia, conidia and infection stages.[Method] The known TatD proteins were obtained from GenBank, and four TatD candidates in fusariums were identified by BlastP.FpTatD genes and open reading frames (ORF) were amplified from genome DNA and cDNA by PCR, and the transcription levels in mycelia, conidia and infection were examined by qRT-PCR. The expression levels ofFpTatD genes in the affinity interaction and non affinity interaction were assayed by RNA-seq.[Result]Four TatD candidates were identified inFusariums, which divided into two significant branches by the phylogenetic tree. TatD1 and TatD2 belong to an ancient family that is conversed in almost all eukaryotic, while TatD3 and TatD4 seem to come from a unique TatD family that only exists in plants and fungi. FourFpTatD candidates, which identified inF. pseudograminearum were cloned, and the full-length sequences ofFpTatD1,FpTatD2,FpTatD3 andFpTatD4 were 993, 1 331, 1 227 and 1 176 bp.FpTatD2 contains an intact open reading frame with 94 and 55 bp non-coding sequences in 5′ terminal, while FpTatD1,FpTatD3 andFpTatD4 consist continuous open reading frames. The four clones encode 36.37, 43.13, 45.59 and 44.25 kD proteins. All FpTatD contained the conserved DNase domain and most conserved amino acid residues. qRT-PCR analysis revealed thatFpTatD1 andFpTatD2 were highly expressed inF.pseudograminearum, and both were highly up-regulated at early stages of infection. Especially, the expression level ofFpTatD1 escalated to 8.8 and 7.6 times in 36 h and 3 d. However,FpTatD3 andFpTatD4 were poorly expressed at all stages. Thus, FpTatD1 and FpTatD2 might play important roles inF. pseudograminearum. RNA-seq analysis was consistent with qRT-PCR results thatFpTatD1 andFpTatD2 were highly expressed and up-regulated at early stages of infection. In addition, Compared to the affinity interaction betweenF.pseudograminearum and GM301,FpTatD genes showed higher expression levels in the non affinity interaction betweenF.pseudograminearum and ZM24.[Conclusion]Apoptosis-related geneFpTatD might play an important role in the interaction betweenF.pseudograminearum and its host.