马氏珠母贝TLR6基因的克隆、序列分析与表达

Cloning, sequence analysis and expression studies on PmTLR6 gene from Pinctada fucata martensii

  • 摘要: 为了探究TLR6 (Toll like receptor 6)在马氏珠母贝免疫反应中的作用,实验采用cDNA末端快速扩增(RACE)技术获得了马氏珠母贝TLR6基因(Pm-TLR6)cDNA全长序列,并且运用实时荧光定量PCR(qRT-PCR)技术检测了Pm-TLR6在马氏珠母贝各组织中的表达情况、哈维氏弧菌刺激后和植核移植后血淋巴中的表达模式。结果显示:Pm-TLR6 cDNA全长2295 bp,其中5′非编码区(UTR)长94 bp,3′UTR长89 bp,开放阅读框(ORF)长2112 bp,编码703个氨基酸;多序列比对结果表明物种间TLR6具有较高的保守性;Pm-TLR6具有跨膜域、富含亮氨酸的重复序列(LRRs)和TIR域,符合TLRs家族特征。qRT-PCR数据分析表明,Pm-TLR6在马氏珠母贝肝胰脏、血细胞、鳃、性腺、闭壳肌、外套膜中均有表达,其中肝胰脏中表达量最高;哈维氏弧菌刺激后,Pm-TLR6在2 h表达上调,约为对照组(0 h)的9倍,随后的6 h恢复至正常水平,直至16 h表达水平开始回升并于24 h达到最大表达量,是对照组的29.4倍,具有显著性差异;植核移植实验结果显示Pm-TLR6在植核后5和10 d表达水平没有显著性变化,15和20 d表达量出现上升趋势,但差异不显著,最后于30 d达到最大值,约为空白对照组(0 d)的5倍,具有显著性差异。研究表明,Pm-TLR6可能在马氏珠母贝免疫防御反应中担任着重要的角色。

     

    Abstract: TLR6 (Toll like receptor 6) is a kind of pattern recognition receptors and plays an important role in resisting microorganism infection. To study the function of TLR6 in the immune response of P. fucata martensii, in this study, a full length of Pm-TLR6 was obtained using rapid amplification of cDNA ends (RACE) technology from P. fucata martensii. The expression patterns of Pm-TLR6 in all tissues and its sequential expression in the hemolymph after Vibrio harveyi stimulation and nucleus insertion operation were further detected by Quantitative Real-Time PCR technology. Results showed that the total length of Pm-TLR6 cDNA was 2295 bp, including a 5′ UTR of 94 bp, a 3′ UTR of 89 bp and an open reading frame (ORF) of 2112 bp which encodes 703 amino acids. Multiple sequence alignment indicated that TLR6 was highly conservative among species. The protein encoded by Pm-TLR6 has a transmembrane domain, several leucine rich repeats(LRRs) and a TIR domain, conforming to the characteristics of TLRs family. qRT-PCR data revealed that Pm-TLR6 was expressed in all tested tissues, including hepatopancreas, hemocytes, gill, gonads, adductor muscle and mantle, with the highest expression in hepatopancreas (P<0.05). AfterVibrio harveyi injection, the expression level of Pm-TLR6 increased at 2 h (9 fold vs. control), then dropped to normal levels at 6 h and began to increase at 16 h, with the highest level of expression appearing at 24 h (29.4 fold vs. control, p<0.05). The result of nucleus insertion surgery showed thatPm-TLR6 expression level was not significantly changed at 5 d and 10 d, and began to increase at 15 d and 20 d (P>0.05). Its expression reached the maximum level at 30 d with significant difference (5 foldvs. control, p<0.05). These results indicated thatPm-TLR6 may play an important role in the immune defense reaction of P. fucata martensii.

     

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