锯缘青蟹蜕皮抑制激素cDNA的分子克隆及其表达分析
cDNA cloning and expression analysis of molt-inhibiting hormone in the mud crab Scylla serrata
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摘要: 根据近缘种类同源序列设计简并引物,采用逆转录聚合酶链式反应(RT-PCR)技术,首次扩增获得锯缘青蟹(Scylla serrata)眼柄组织中编码蜕皮抑制激素(MIH)成熟肽全长cDNA序列及部分信号肽序列.将该序列克隆到pUCm-T载体上进行序列测定.结果表明,编码锯缘青蟹MIH成熟肽的cDNA由234个碱基组成,由此推测MIH成熟肽含78个氨基酸残基.该序列不仅与其它短尾类甲壳动物的MIH氨基酸序列具有高度的同源性(79%~91%),还与该激素同一家族的性腺抑制激素、大颚器抑制激素的氨基酸序列具有较高的相似性.RNA斑点杂交结果显示,MIH基因在眼柄神经节及脑组织中均有表达,而在肌肉、中肠腺中不表达.Abstract: In the present study, cDNA encoding molt..inhibiting hormone ( MIH) was amplified from the total RNA of eyestalk neural ganglia of the mud crab, Scylla serrata, by means of reverse transcription polymerase chain reaction( RT..PCR) with degenerate oligonucleotide primers based upon the homology DNA sequence of MIH from other Brachyuraspecies. Then the fragments were cloned with pUCm..T vector and sequenced. The total length of 316bp sequence was obtained that encoded full..length mature peptide of 78 amino acids and partial signal pept ide. According to the search results of GenBank databases, the cDNA fragment and the deduced amino acid sequence of mature peptide share 79% ~ 91% identity with that of other species in the Brachyura. Expression of MIH was detected in the eyestalk, brain, midgut gland and muscle tissues examined by RNA dot hybridization. The results indicated that the MIH was expressed only in the eyestalk and brain not in other tissues.
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