日本沼虾表皮几丁质结合蛋白基因的克隆以及KK-42对其表达的影响

为探讨KK-42缩短日本沼虾蜕皮周期的可能机制,首次从日本沼虾步足表皮组织克隆了编码几丁质结合蛋白的一个新基因——MnCBP-1部分cDNA序列,对其序列进行生物信息学分析,qRT-PCR技术检测MnCBP-1基因的表达水平以及KK-42的影响.结果表明,MnCBP-1基因部分cDNA序列为1396bp,含有特征性的几丁质结合结构域(chitin-bind-4),经BLAST比对,MnCBP-1与蚤状溞相似性为44%.qRT-PCR结果显示,步足表皮组织内MnCBP-1基因表达量随着蜕皮前期(D0-2)的开始逐渐增多.KK-42处理后,蜕皮间期(C)幼虾内MnCBP-1基因表达水平分别在6、12、24和48h比对照组显著升高,而在D0-2期和D3-4期MnCBP-1基因转录物仅在个别时间点有所增多.研究表明,MnCBP-1基因表达与蜕皮周期有关,KK-42处理可显著诱导MnCBP-1基因表达,且表达的高峰提前至C期,这可能是KK-42缩短蜕皮周期的机制之一.

Cloning and the Effect of KK-42 on Chitin-Binding Proteins Genes from the Cuticle of Macrobrachium Nipponense

In order to investigate the possible molecular mechanism of KK-42 treatment shortening the duration of molting cycle of the juvenile prawn Macrobrachium nipponense,the part sequence of a chitin-binding protein gene-MnCBP-1was firstly cloned from pereiopod cuticular tissue of M.nipponense and the bioinformatics analysis of the sequence was done.The expression level of the MnCBP-1as well as the effect of KK-42 on the gene expression were investigated usingqRT-PCR.Results showed that the part sequence of the MnCBP-1contained 1396 bp in which the characteristic chitin-binding domain(chitin-bind-4)was included.The similarity of the deduced amino acid sequence for MnCBP-1 was 44 % to Daphnia pulex.In pereiopod cuticular tissue,qRT-PCR analysis revealed that the expression level of MnCBP-1increased continuously from stage D0-2to stage D3-4.The MnCBP-1mRNA transcripts in stage C significantly rose at 6~48hafter KK-42 treatment,but that in premolt increased only at some time points following KK-42 administration.In conclusion,our present results display that the change of the MnCBP-1expression is related to the molting cycle.The gene expression not only can be significantly up-regulated but also peak stage C in advance after KK-42 treatment,which may be one of the molecular mechanisms of KK-42 shortening the duration of molting cycle of the juvenile prawn M.nipponense.