引用本文: 陈明, 周谢菲, 高建军, 朱庭耀, 彭佳诚, 徐海圣. 2022. 中华鳖 STING 基因的克隆及功能分析. 水生生物学报, 46(3): 324-331. doi: 10.7541/2022.2021.133 Citation: Ming CHEN, Xie-Fei ZHOU, Jian-Jun GAO, Ting-Yao ZHU, Jia-Cheng PENG, Hai-Sheng XU. 2022. CLONING AND FUNCTIONAL ANALYSIS OF STING IN CHINESE SOFT-SHELLED TURTLE ( PELODISCUS SINENSIS ). ACTA HYDROBIOLOGICA SINICA , 46(3): 324-331. doi: 10.7541/2022.2021.133 为了研究干扰素基因刺激因子 STING 在中华鳖( Pelodiscus sinensis )先天免疫中的作用 , 克隆了中华鳖 STING 基因 (PsSTING )的cDNA序列, 其全长为2145 bp, 包含1152 bp的开放阅读框, 编码383个氨基酸。氨基酸序列比对发现, PsSTING 蛋白N端含有4个跨膜区和2个RXR内质网滞留基序, C端有1个解旋酶结构域。qRT-PCR结果显示, PsSTING 在中华鳖心脏、肝脏、脾脏、肺、肾脏、小肠、胃、皮肤、肌肉和血细胞等组织中均能表达, 其中在脾脏中的表达量最高, 其次是肝脏、小肠和肺, 而在血细胞中的表达量最低; 用嗜水气单胞菌、LPS和poly(I:C)刺激后, PsSTING 基因呈现出先上调后下调的趋势, 在刺激12h 时显著高于对照组( P ＜0.05), 24h达到最高值, 在48h后逐渐恢复到初始水平, 其蛋白表达量在脾脏和小肠中明显增加。体内RNA干扰后, 小肠中 PsSTING 的表达量显著下调( P ＜0.05), IFN信号通路中 TBK1 、 IRF3 、 IRF7、STAT6 和 IFN-β 等基因表达水平显著下调( P ＜0.05)。在HEK293T细胞中过表达 PsSTING 基因, 能够显著激活pIFN-β-luc的启动子。研究结果表明 PsSTING 基因参与调控了中华鳖的先天免疫反应。 干扰素基因刺激因子 Abstract: Stimulator of interferon gene ( STING ) is an important adaptor protein in the innate immune signaling pathway, which mediates the production of type I IFNs. To study the role of STING in the innate immunity of Chinese soft-shelled turtle, Pelodiscus sinensis, the PsSTING gene cDNA was cloned. The result showed that the sequence was 2145 bp in full length, containing 1152 bp open reading frame and encoding 383 amino acids. The amino acid sequence alignment showed that, the N-terminal sequence of PsSTING protein contained 4 transmembrane regions and 2 endoplasmic reticulum retention motifs and the C-terminal sequence of PsSTING protein contained 1 helicase domain. The homology comparison and the phylogenetic tree showed that the PsSTING protein had a higher homology with the other species in the order Testudines, such as Platysternon megacephalum, followed by the homology with the Crocodilia and Avian, the lowest origin compared with invertebrates. The qRT-PCR results indicated that PsSTING was expressed in the all examined tissues including heart, liver, spleen, lung, kidney, small intestine, stomach, skin, muscle and blood cells in the Chinese soft-shelled turtle, and it was expressed at the highest level in the spleen tissue, followed by the liver, small intestine and lungs, with the lowest expression in blood cells. Compared with the control group, PsSTING was significantly up-regulated and then down-regulated after stimulation with Aeromonas hydrophila , LPS and poly(I:C). It was significantly higher than that in the control group at 12 hours post-infection (hpi), and reached the highest value at 24 hpi, and then gradually returned to the initial level after 48 hpi. Similarly, the protein expression of PsSTING was significantly increased in the spleen and the small intestine. The siRNA interference result demonstrated that the expression of PsSTING was significantly down-regulated in the small intestine ( P ＜0.05) and the expression levels of its downstream genes TBK1 , IRF3 , IRF7 , STAT6 and IFN-β decreased significantly too after the interference of PsSTING . Its overexpression in HEK293T cells could significantly activate the promoter of pIFN-β-luc. The results of the study indicate that PsSTING participates in the regulation of the innate immunity of the Chinese soft-shelled turtle. Key words: Stimulator of interferon genes Pelodiscus sinensis Innate immunity Gene cloning Function 引物
Primer引物序列
Primer sequence (5′－3′)用途
Usage STING -3′F outerGCCATGTCCCAGGACGAAAGCGRACE STING -3′F innerTACCTGGGCCTCTGCGGAGCGACGGRACE STING -5′R outerAGGTACAGGATAAAGACGCAGACGRACE STING -5′R innerGGTGGATTTGGACGTTGGGTCTTTCRACEUPM LongTAATACGACTCACTATAGGGCAAGCAGTGGTATCAACGCAGAGTRACEUPM ShortCTAATACGACTCACTATAGGGCRACEβ-actin qFGAGACCTGACAGACTACCTqRT-PCRβ-actin qRAGGATGATGAAGCAGCAGTqRT-PCR STING -qF
STING -qR
STING -qsiF
STING -qsiRAGGAGAGAGGCTCGTACCAG
TGAGGACTCGGGAGACAGCAACA
CCGGCAGCAGCACCTGGAGGAATA
TCAGAAGCCGTCGCTCCGCAGAGGCqRT-PCRTBK1-qFTGCCAGTGCTTTGCTGCTTTACqRT-PCRTBK1-qRATGACAACCTCTGTTTTTTTGTGCAqRT-PCRIRF3-qFGCGCCCTCCGTCTGGAAACGCAACTqRT-PCRIRF3-qRGATCTCATAGACCTTGTGGGGGTCGqRT-PCRIRF7-qFGACATCACCACTAATGACTACAAAAqRT-PCRIRF7-qRGAGTTTGGGCTGATGATTTGATqRT-PCRSTAT6-qFTTTGACGGGGTCCTCGACCTCACCAAqRT-PCRSTAT6-qRCGGATGACGTGGGCGATGGTGATGCqRT-PCRIFN-β-qFAAGAGTTTAGAGCACCTGGAGAAqRT-PCRIFN-β-qRATTCCTTTATGGAAGTCCCATCCCAqRT-PCRNC-sense
NC-antisenseUUCUCCGAACGUGUCACGUTT
ACGUGACACGUUCGGAGAATTRNAi STING -senseCCGUCAUCAUGAUCAGCAATTRNAi STING -antisenseUUGCUGAUCAUGAUGACGGTTRNAi STING -petFAATGGGTCGCGGATCCTGGAAACTGCATATTCTGCTGCCGCTProtein expression STING -petRGGTGGTGGTGCTCGAGGCACGGTTTATGCGGGCCCGCCGGCProtein expression Figure 3. Protein immunofluorescence results of PsSTING and its relative immunofluorescence intensity in the liver, spleen and intestine of P. sinensis stimulated by exogenous substances Figure 4. Analysis of siRNA interference results and IFN-β dual luciferase test results