斑石鲷TGF-β1基因克隆和表达分析

Molecular Cloning and Expression Pattern Analysis of TGF-β1 in Spotted Knifejaw (Oplegnathus punctatus)

中文关键词: 斑石鲷  虹彩病毒  TGF-β1

英文关键词: Oplegnathus punctatus  Iridovirus  TGF-β1

中文摘要:

      虹彩病毒是造成斑石鲷(Oplegnathus punctatus)工厂化养殖中大规模死亡的主要病原，其感染力极强，感染后的斑石鲷死亡率高，严重影响了斑石鲷养殖产业发展。转化生长因子TGF-β1是一种重要的免疫调节因子，在病毒免疫应答中发挥重要作用。为研究TGF-β1在斑石鲷被虹彩病毒感染过程中发挥的作用，运用RACE和实时荧光定量qRT-PCR技术对TGF-β1进行了基因克隆，并对其进行在不同组织、不同时间点相对表达量的差异分析。结果显示，斑石鲷TGF-β1基因cDNA序列全长为3157 bp，5非编码区长712 bp，3非编码区长1278 bp，开放性阅读框长1167 bp，编码388个氨基酸，基因组包含6个外显子和5个内含子。同源分析发现，TGF-β1和鱼类相似度较高，与半滑舌鳎(Cynoglossus semilaevis)的同源性最高，为76.67%。TGF-β1在斑石鲷健康组织(肝脏、脾脏、肾脏、头肾、心脏、鳃、胃、肠和皮肤)中均有表达，在头肾、肠、肝脏和皮肤组织中表达量较高，而在脾脏和肾脏组织表达量较低。为进一步研究TGF-β1在病毒感染过程中相对表达量的变化，对健康斑石鲷注射虹彩病毒进行刺激，随后比较了TGF-β1在脾脏、肝脏、肾脏、头肾4个不同组织、不同时间点的相对表达量的差异，在头肾、脾脏和肝脏中，病毒刺激后TGF-β1的表达量均出现升高，但在脾脏和肝脏中，峰值出现在刺激后第4天，而在头肾中峰值出现在感染后的第10天。在肾脏中，病毒刺激后的TGF-β1的表达呈现下降趋势，0 d表达量最高，4、7 d依次降低，7 d降至最低，10 d有所恢复。以上研究表明，TGF-β1可能响应了虹彩病毒对机体的刺激，可能在对病毒免疫应答中发挥作用。而病毒感染后不同组织中TGF-β1相对表达量的差异，则值得进一步研究。

英文摘要:

      Iridovirus has a wide infection spectrum in aquatic animals, which is harmful to aquaculture. In spotted knifejaw (Oplegnathus punctatus), iridovirus is the major factor causing large-scale mortality. Iridovirus infection results in high mortality rate and thus limits the development of the spotted knifejaw industry. As an important immunoregulatory factor, TGF-β1 plays a vital role in viral immune response. In order to study its role in the process of iridovirus infection, we have cloned this gene and studied its spatiotemporal expression patterns. The total length of TGF-β1 gene is 3157 bp, including 5′-untranslated region (UTR) of 712 bp, 3′-UTR of 1278 bp. The open reading frame length is 1167 bp that encodes 388 amino acids, including 6 exons and 5 introns. Homologous alignment showed that the amino acid sequence of TGF-β1 in spotted knifejaw had a close identity to Cynoglossus semilaevis, of about 76.67%. TGF-β1 is widely distributed in tissues, and high expressed in the head kidney, intestine, liver, skin, but shows a relatively low expression in the spleen and kidney. To further illustrate the expression patterns of TGF-β1 during viral infection, we have analyzed the expression patterns of TGF-β1 in the spleen, liver, kidney, and head kidney at different time points after iridovirus infection. In head kidney, spleen and liver, the expression of TGF-β1 was increased post infection. The peak level in the spleen and liver appeared 4 days post infection (dpi), and in the head kidney the peak appeared at 10 dpi. In the kidney, viral infection seemed to downregulate TGF-β1 expression. The highest expression was observed at day 0, then decreased at 4 dpi and reach the lowest level at 7 dpi. At 10 dpi, the expression had recovered but still lower than level of day 0. These results suggest that TGF-β1 could respond to iridovirus infection and may play an important role in the process of viral immunity. However, the different expression pattern of TGF-β1 in different tissues after viral infection requires further study.