Genomic Organization and Transcriptional Regulation of Two Homologous Fucolectin Genes AjFTL-1 and AjFTL-2 in Apostichopus japonicus
Genomic Organization and Transcriptional Regulation of Two Homologous Fucolectin Genes AjFTL-1 and AjFTL-2 in Apostichopus japonicus
复制标题
刺参两个同源岩藻凝集素基因AjFTL-1和AjFTL-2的基因组结构和转录调控
DOI：
10.1007/s11802-021-4515-8
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发表时间：
2021-02-01
影响因子：
1.6
通讯作者：
Li Chenghua，Chunsheng
中科院分区：
文献类型：
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作者：
Shao Yina，Yina;Wang Jiahao，jiahao;Li Chenghua，Chunsheng
F-型凝集素（Fucolectins）是一类糖结合蛋白，在天然免疫应答中起重要作用。在前期的研究中，我们发现两个同源的岩藻凝集素基因AjFTL-1和AjFTL-2在脂多糖（LPS）刺激下表现出不同的表达谱。然而，这两个基因的转录调控机制在很大程度上仍然未知。本研究克隆了AjFTL-1和AjFTL-2基因的5′端侧翼序列，并研究了其启动子在鲤丘疹上皮瘤（epithelioma papulosum cyprinid，EPC）细胞中的活性。首先，计算机模拟分析表明，这两个启动子都含有许多推定的转录因子结合位点，包括NF-κB、CREB B和CREBP 1，并且都含有TATA盒。荧光素酶检测和5′端截短分析表明，AjFTL-1和AjFTL-2在EPC细胞中均具有较高的启动子活性。AjFTL-1启动子的荧光素酶活性受肽聚糖（PGN）和甘露聚糖（MAN）的调控，而AjFTL-2启动子的荧光素酶活性受LPS和MAN的调控，表明AjFTL-1和AjFTL-2基因在不同的免疫刺激下表现出不同的转录调控模式。更重要的是，对功能启动子区域的分析揭示了AjFTL-1中存在两个潜在的NF-κB结合位点（−769 bp至−761 bp，−185 bp至− 172 bp），AjFTL-2中存在一个潜在的结合位点（−530至− 517 bp）。不同截短的报告载体和表达载体共转染显示，转录因子NF-κB/Rel可显著提高AjFTL-2启动子活性，但对AjFTL-1启动子活性无明显影响。这些结果表明，在海洋无脊椎动物中，不同的岩藻凝集素成员的转录调控和表达模式不同，可能在病原体感染的免疫防御中发挥不同的作用。
F-type lectins (Fucolectins) are carbohydrate-binding proteins and play important roles in innate immune responses against pathogenic microbial invasion. In our previous research, we found that two homologous Fucolectin genes, AjFTL-1 and AjFTL-2 , exhibited different expression profiles after lipopolysaccharides (LPS) challenge in Apostichopus japonicus . However, the transcriptional regulation mechanism of these two genes remains largely unknown. In this study, the 5′ flanking regions of AjFTL-1 and AjFTL-2 genes were cloned and the promoter activities were studied in epithelioma papulosum cyprinid (EPC) cell system. First, in silico analysis indicated that these two promoters both contain numerous putative transcription factor binding sites including NF-κB, CREB, and CREBP1, and both contain a TATA box. Additionally, luciferase assay and progressive 5′ truncation analysis revealed that AjFTL-1 and AjFTL-2 both possess high promoter activities in EPC cells. Moreover, the luciferase activity of AjFTL-1 promoter was significantly regulated by peptidoglycan (PGN) and mannan (MAN), while AjFTL-2 promoter was prominently regulated by LPS and MAN, indicating AjFTL-1 and AjFTL-2 genes showed different transcriptional regulation pattern under different immune stimulation. More importantly, analyses of the functional promoter regions revealed the presence of two potential NF-κB binding sites (−769 bp to −761 bp, −185 bp to −172bp) in AjFTL-1 and one potential binding site (−530 to −517bp) in AjFTL-2 . Different truncated reporter vectors and expression vector co-transfection revealed that transcription factor NF-κB/Rel could significantly increase the AjFTL-2 promoter activity, but not AjFTL-1 promoter activity. These findings indicated that in marine invertebrates, different Fucolectin members differ in transcription regulations and expression patterns, and might play different roles in immune defenses during pathogen infection.