松材线虫热激转录因子Bx-HSF-1基因的克隆及表达分析

Cloning and Expression Analysis of the Heat Shock Transcription Factor Bx-HSF-1 Gene in Bursaphelenchus xylophilus (Aphelenchida: Aphelenchoididae)

【目的】通过对热激转录因子基因Bx-HSF-1的克隆和表达分析,为深入研究c GMP、TGFβ-like和Insulin/IGF等热激抗逆代谢路径提供理论基础。【方法】根据松材线虫基因组数据设计引物克隆松材线虫Bx-HSF-1基因。进一步对Bx-HSF-1进行Gene Ontology基因功能预测、系统发育分析、保守结构域分析等生物信息学分析。应用JASPAR预测Bx-HSF-1靶位点DNA序列,根据松材线虫基因组数据筛选含有该序列的靶基因,并通过蛋白质三维结构分析和分子对接验证。应用荧光定量q-PCR检测Bx-HSF-1及其靶基因在热激和低温处理条件下4个时间点的表达量,结合转录组数据对Bx-HSF-1及其靶基因的表达量相关性进行分析。采用原位杂交对Bx-HSF-1基因在松材线虫体内的表达部位进行了分析。【结果】根据基因组数据,克隆到松材线虫热激转录因子Bx-HSF-1基因长度为1 404 bp的涵盖完整蛋白编码区基因序列。结构域分析表明Bx-HSF-1基因编码蛋白质的氨基酸序列含有DNA结合结构域和亮氨酸拉链结构域。GO分析表明该基因参与延长线虫寿命、正向调控繁殖率、运动、热激反应、抗逆态幼虫发育和抗逆反应等生理过程,并具有特异DNA序列结合的分子功能。氨基酸疏水性分析表明该DNA结合结构域由3个α螺旋和4个反向平行的β折叠构成一个疏水核心,该疏水核心可以与热激元件靶位点互作。对松材线虫基因组进行BLAST比对筛选出Bx-DAF-7是Bx-HSF-1的靶基因,Bx-DAF-7基因TATA盒上游序列具有Bx-HSF-1靶位点序列。荧光定量q-PCR结果表明热激和低温处理同样促进Bx-HSF-1转录、抑制Bx-DAF-7转录,Bx-HSF-1与Bx-DAF-7的表达量负相关。原位杂交结果表明Bx-HSF-1基因在正常培养线虫体内各细胞均有微量表达,热激培养线虫前体部信号显著,峡部和肠区体壁信号较显著。【结论】本研究表明Bx-HSF-1具有特异DNA序列结合的分子功能。预测抗逆态幼虫形成基因Bx-DAF-7是Bx-HSF-1靶基因。转录组测序及q-PCR结果表明Bx-HSF-1与Bx-DAF-7的表达量负相关。Bx-HSF-1基因受温度刺激后多数体细胞内均有较高表达,特别是神经元、体壁肌肉细胞和皮下细胞中表达显著。

【Objective】The thermotolerance transcription factor HSF-1 adjusts various cellular processes in nematode,such as the c GMP,TGFβ-like and Insulin / IGF signaling pathways,in response to heat stress. The cloning and expression analysis of the heat shock transcription factor Bx-HSF-1 would contribute to a deep understanding of the molecular mechanism of thermotolerance in Bursaphelenchus xylophilus. 【Method 】 The gene of Bx-HSF-1 was cloned with the method of PCR based on the genomic data of B. xylophilus. The bioinformatics analyses,such as Gene Ontology,phylogenetic and conserved domain analysis,were conducted to the amino acid sequence of Bx-HSF-1. The protein 3D structure and docking were analyzed to predict the target gene. The q-PCR was used to analyze the gene expression of BxHSF-1 and target gene. The gene expression location of Bx-HSF-1 was carried out by the method of in-situ hybridization.【Result】The CDS of Bx-HSF-1,which was 1 404 bp,was cloned based on the genomic data of B. xylophilus. The amino acid sequence of Bx-HSF-1 had a DNA binding domain and a b Zip domain. The results suggested that Bx-HSF-1 would be involved in the biological process of response to heat,dauer larval development and defense response,and had the molecular function of sequence-specific DNA binding. The hydrophobicity analysis indicated that the DNA binding domain was a hydrophobic core,which was constructed by 3 α helices and 4 antiparallel β folds. Bx-DAF-7 was selected as the target gene of Bx-HSF-1,based on the genomic data. The data of transcriptome and Q-PCR indicated that heat shock or hypothermia promoted the expression of Bx-HSF-1,and suppressed the expression of Bx-DAF-7. The expression of BxHSF-1 was negatively correlated with the expression of Bx-DAF-7. The in-situ hybridization showed clear signals of BxHSF-1 at the isthmus and intestine. 【Conclusion 】 This study showed that Bx-HSF-1 with a specific DNA sequence binding molecules function. Target gene prediction indicated that the dauer larval forming gene Bx-DAF-7 was the target gene of Bx-HSF-1. Transcriptome sequencing and q-PCR results showed that Bx-HSF-1 was negatively correlated with the expression of Bx-DAF-7. The expression of Bx-HSF-1 was located at neurons,body wall muscle cells and hypodermal cells.