低温诱导型凡纳滨对虾DEAD-box RNA解旋酶基因的克隆与表达分析
CLONING, CHARACTERIZATION AND EXPRESSION ANALYSIS OF A COLDINDUCIBLE DEAD-BOX RNA HELICASE GENE IN LITOPENAEUS VANNAMEI
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摘要: 为研究凡纳滨对虾耐寒性状的分子基础, 克隆鉴定了凡纳滨对虾DEAD-box RNA解旋酶的同源基因。根据差减文库中筛选到的一个低温上调表达的DEAD-box RNA解旋酶基因片段, 通过RACE PCR扩增获得两条高度相似的cDNA全长序列, 两条cDNA均为1430 bp, 包含139 bp 5'UTR、79 bp 3'UTR和1212 bp 开放阅读框, 编码403个氨基酸残基。Blast比对结果显示, 两条cDNA与其他物种的DDX5相似性最高, 推测为凡纳滨对虾DDX5基因的两个变异体, 分别命名为LvDDX5variant A (LvDDX5A)和LvDDX5variant B (LvDDX5B), 在系统进化树中, LvDDX5A和LvDDX5B聚为最近的一支, 并与虾类DDX5、文昌鱼DDX、贝类的DDX5距离较近。Real-time PCR分析结果显示, LvDDX5A和LvDDX5B都呈多组织遍在表达, 在精巢、鳃、肠、肌肉和卵巢中,LvDDX5B表达量高于LvDDX5A, 而在肝胰腺中LvDDX5A表达量高于LvDDX5B。在低温胁迫对虾肝胰腺和心中, LvDDX5A呈上调表达而LvDDX5B表达量无明显变化。进一步对LvDDX5A在不同低温条件胁迫对虾的肝胰腺中表达变化进行了分析。结果显示, LvDDX5A在18℃、15℃、13℃和11℃低温胁迫36h均被诱导表达,并随着15℃和13℃低温胁迫时间的增加呈先上升后下降的表达模式, 在48h达到峰值, LvDDX5A的低温诱导表达模式暗示其可能是在凡纳滨对虾低温适应中发挥作用的剪接体。Abstract: Cold-inducible DEAD-box RNA helicases are conservative regulator of cold tolerance in archaea, prokaryote and eukaryote. We have got a cold-inducible DEAD-box RNA helicase EST from cold tolerant shrimps. In this study, two cDNA sequence of the DEAD-box RNA helicase gene were obtained by RACR PCR. The two cDNAs were 1430 bp in length including a 139 bp 5'UTR, a 79 bp 3'UTR and a 1212 bp open reading frame, encoding a peptide of 403 aa. Blast analysis showed that the two cDNAs had top hit of DDX homologous, thus named LvDDX5 variant A(LvDDX5A)and LvDDX5 variant B(LvDDX5B). Phylogenetic analysis revealed that LvDDX was related closely to shrimp DDX5,Branchiostoma floridae DDX and Aplysia californica DDX5. LvDDX5A and LvDDX5B were widely expressed, and LvDDX5B was higher in testis, gill, intestine, muscle and ovary, whereas LvDDX5A was higher in hepatopancreas. LvDDX5A but not LvDDX5B was up-regulated in hepatopancreas and heart by cold induction. LvDDX5A can be induced by various low temperatures including 18, 15, 13 and 11 ℃. With different time treatment at 15 and 13 ℃, LvDDX5A increased firstly, reached to the peak at 48h, and decreased afterward. These results suggest that LvDDX5A may play an important role in cold tolerance of Litopenaeus vannamei.
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Keywords:
- Litopenaeus vannamei /
- Cold-inducible /
- DEAD-box /
- RNA helicases /
- Cold tolerant
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