Molecular cloning, characterization and recombinant expression of crustacean hyperglycemic hormone in white shrimp Litopenaeus vannamei
Molecular cloning, characterization and recombinant expression of crustacean hyperglycemic hormone in white shrimp Litopenaeus vannamei
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凡纳滨对虾甲壳类高血糖激素的分子克隆、鉴定及重组表达
DOI：
10.1016/j.peptides.2013.07.030
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发表时间：
2014-03-01
期刊：
影响因子：
3
通讯作者：
Zheng, Debin
中科院分区：
文献类型：
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作者：
Liu, Maoqi;Pan, Luqing;Zheng, Debin
甲壳动物高血糖激素（CHH）在甲壳动物体内起着重要作用。本研究利用RACE技术首次从凡纳滨对虾眼柄组织中克隆了CHH基因的全长cDNA。LvCHH的cDNA全长为846 bp，其中5'端非翻译区（UTR）为65 bp，3'端非翻译区（UTR）为436 bp，含有一个典型的多聚腺苷酸化信号序列AATAA和一个poly（A）尾，开放阅读框（ORF）为345 bp。该ORF编码114个氨基酸的多肽，包括24个氨基酸的信号肽。成熟蛋白（74个氨基酸）的计算分子量为8.76 kDa，估计pI为6.78。LvCHH的序列以登录号HM 748790.2提交到NCBI GenBank中。系统发育分析表明，LvCHH与其他甲壳类的CHH聚在一起。组织分布分析表明，LvCHH mRNA在除鳃外的其它组织中均有表达，且在心脏中表达量最高。根据已获得的LvCHH基因的ORF序列和表达载体pET-32a（+）的克隆位点，分别设计含有Xho I和BamH I酶切位点的特异性引物。重组质粒LyCHH-pET 32a转化大肠杆菌BL 21（DE 3）。经SDS-PAGE和western blot分析，LvCHH得到了成功表达。结果表明：注射rLvCHH蛋白后，各实验组鳃Na+/K +-ATP酶活性在0-6 h内均出现峰值变化，且各处理组的峰值均出现在1h内。20和30 μ g/虾（-1）组在注射后1小时表现出显著的增加（P< 0.05）。L.将凡纳滨对虾暴露于低盐和高盐环境中96 h。低盐胁迫12 h和24 h时，CHH mRNA表达和鳃Na+/K+-ATP酶活性显著升高（P< 0.05），24 h时CHH mRNA表达下降，48 h时恢复到对照组水平，72 h后Na+/K+-ATP酶活性趋于稳定，高于对照组。高盐组与低盐组的mRNA表达趋势相同。Na+/K+-ATPase活性在低盐胁迫下无显著变化。这些结果表明，LvCHH是一种重要的激素，参与了游泳虾的渗透反应，为进一步研究甲壳动物的渗透调节生理机制提供了依据。(c)2013 Elsevier Inc. All rights reserved.
Crustacean hyperglycemic hormone (CHH) plays an important role in crustacean. In the present study, a full-length cDNA of CHH was cloned from the eyestalk of Litopenaeus vannamei by RACE approach for the first time. The full-length cDNA of LvCHH was 846 bp, containing a 5' untranslated region (UTR) of 65 bp, a 3' UTR of 436 bp with a canonical polyadenylation signal-sequence AATAA and a poly (A) tail, and an open reading frame (ORF) of 345 bp. The ORF encoded a polypeptide of 114 amino acids including a 24 amino acid signal peptide. The calculated molecular mass of the mature protein (74 amino acids) was 8.76 kDa with an estimated pI of 6.78. The sequence of LvCHH was submitted in NCBI GenBank under the accession number HM748790.2. Phylogenetic analysis revealed that LvCHH was clustered with CHH of other crustaceans. Tissue distribution analysis revealed that the expression of LvCHH mRNA was observed in all tissues but gill, and was highest in heart. Specific primers containing Xho I and BamH I restriction sites respectively, were designed based on the obtained ORF sequence of LvCHH gene and the cloning sites of expression vector pET-32a (+). The recombinant plasmid LyCHH-pET32a, was used to transform Escherichia coli BL21 (DE3). LvCHH was successfully expressed by means of SDS-PAGE and western blot analysis. We detected gill Na+/K+-ATPase activity after rLvCHH protein injection and found that All the experimental group Na+/K+-ATPase activity presented peak change among 0-6 h, and the peaks of all treated groups occurred in 1 h. 20 and 30 mu g/shrimp(-1) groups showed significant increase (P< 0.05) in 1 h post-injection. L. vannamei were exposed for 96 h to hypo-and hyper-salinity challenge. Hypo-salinity caused a significant rise (P< 0.05) in the mRNA expression of CHH and gill Na+/K+-ATPase activity at 12 h and 24 h respectively, then the CHH mRNA expression declining by 24 h, and returned to control group level by 48 h, and the Na+/K+-ATPase activity tended to be stable after 72 h, and higher than that of control. The hyper-salinity challenge had the same trend at mRNA expression with the hypo-salinity group. The Na+/K+-ATPase activity had no significant change under the low salinity challenge. All these results indicate that LvCHH is an important hormone involved in the osmosis responses of swimming shrimps, and can provide further information of crustacean osmoregulation physiological mechanism. (c) 2013 Elsevier Inc. All rights reserved.