Characterization and expression analysis of ho1 from Sander lucioperca under acute hypoxia stress
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摘要: 梭鲈 (Sander lucioperca) 对低氧极敏感，在集约化养殖和苗种运输过程中易发生低氧应激和死亡现象。为探究血红素加氧酶1 (Heme oxygenase 1, HO1) 在梭鲈响应低氧过程中的调节作用，通过RACE (Rapid amplification of cDNA ends) 技术克隆了梭鲈ho1基因，其cDNA全长为1 256 bp，包含840 bp的开放阅读框 (Open reading frame, ORF)、162 bp的5'非编码区 (Untranslated region, 5'-UTR) 和254 bp的3'-UTR，编码279个氨基酸。多重序列比对显示，梭鲈HO1与翘嘴鳜 (Siniperca chuatsi)、舌齿鲈 (Dicentrarchus labrax) 和大口黑鲈 (Micropterus salmoides) 的氨基酸序列相似性分别为91.84%、88.69%和88.11%。实时荧光定量结果显示，ho1基因在所有检测组织中均有表达，其中在脑组织中高表达，其次是肾、肝、鳃等组织。低氧刺激前3 h，梭鲈ho1主要在皮肤、鳃中响应；低氧胁迫3 h后，ho1主要在心、肝、肾中发挥转录调控作用。复氧12 h，除肝脏外，梭鲈其他组织ho1的相对表达量均可恢复正常，低氧刺激对肝组织ho1的表达产生了较大影响。研究表明，ho1基因参与梭鲈响应低氧的分子调节机制并在其中发挥着重要的生物学功能，可为深入了解梭鲈低氧胁迫遗传机制提供理论参考。Abstract: Sander lucioperca is extremely sensitive to hypoxia, and is prone to hypoxia stress and death during intensive breeding and seedling transportation. In order to investigate the regulating effect of heme oxygenase 1 (HO1) in the response to hypoxia of S. lucioperca, we cloned the full-length cDNA sequence of ho1 gene by RACE (Rapid amplification of cDNA ends) technology. The results indicate that the cDNA length was 1 256 bp (840 bp ORF, 162 bp 5'-UTR and 254 bp 3'-UTR), encoding 279 amino acids. The multiple sequence alignment shows that the similarity of HO1 with Siniperca chuatsi, Dicentrarchus labrax and Micropterus salmoides was 91.84%, 88.69% and 88.11%, respectively. Real-time quantitative PCR discloses that ho1 was expressed in all the tested tissues, with the highest concentration in the brain, followed by the kidney, liver and gills. During the first 3-hour hypoxic stimulation of Pikeperch, ho1 primarily responded in the skin and gills, but mainly played transcriptional regulatory roles in the heart, liver and kidney after 3 h of hypoxic stress. At 12th hour of reoxygenation, the expression levels of ho1 in all the tissues except the liver returned to a normal level, and hypoxia stress had an enormous effect on the expression of ho1 in the liver. The study reveals that ho1 gene is involved in the molecular regulation mechanisms of S. lucioperca in response to hypoxia and plays an important biological role, which provides theoretical references for understanding the genetic mechanism of hypoxic stress.
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Key words:
- Sander lucioperca /
- ho1 /
- Gene clone /
- Hypoxia stress /
- Gene expression
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图 2 梭鲈ho1基因PCR扩增产物电泳图
Figure 2. Electrophoresis of PCR products of ho1 in S. lucioperca
图 3 梭鲈ho1基因cDNA全长序列及推导的氨基酸序列
Figure 3. Nucleotide and predicted protein sequences of ho1 of S. lucioperca
图 4 梭鲈与其他物种HO1氨基酸同源性比对
Figure 4. Multiple alignment of HO1 amino acid sequences between S. lucioperca and other species
图 5 基于不同物种HO1氨基酸序列构建的系统发育进化树 (NJ法)
Figure 5. Phylogenetic tree based on HO1 amino acid sequences of different species (Neighbor-Joining method)
图 6 梭鲈ho1在各组织的相对表达量
注：图中数值为“平均值±标准差”(N=3)，不同字母表示组间差异极显著(P<0.01)，后图同此。
Figure 6. Relative expression of ho1 in different tissues of S. lucioperca
Note: The values are "$ { {\overline X } \pm {\minifont \rm{SD}}}$" (N=3). Different letters indicate extremely significant differences among the tissues (P<0.01). The same below.
图 7 急性低氧胁迫与常氧恢复下梭鲈ho1在组织中表达变化
Figure 7. Expression of ho1 in S. lucioperca under acute hypoxia stress and normoxia recovery
表 1 引物序列
Table 1. Primers used in this study
基因
Gene目的
Purpose引物
Primer引物序列 (5'—3')
Primer sequence (5'–3')退火温度
Annealing temperature/℃ho1 ORF扩增 ho1-F1 GGAGCCAGAGAAGAAGACTCAG 59.8 ho1-R1 TGCAGCTCGTTTTCAGTGAC 60.2 ho1 RACE扩增 5'GSP ACACCGGGGAAGGCGAAGAATGAC 72.0 5'nGSP CACTGGGGTGGTTGGAGTTCCTGTC 70.9 3'GSP GAGGGCAGGTCCTGGGTCGAATC 71.2 3'nGSP ATGGGGCTAAAGGGCAGCGAAGGTC 73.2 ho1 RT-PCR ho1-F1 CTGTGCTCGCTGTATGAGGT 59.1 ho1-R1 CCAGTCCTGGCCATAGAAGT 59.2 gapdh RT-PCR gapdh1-F ATGTTCGTCATGGGCGTCAA 60.0 gapdh1-R CAGGCCCTCAATGATGACGA 60.0 
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