摘要
为了解日本沼虾几丁质合成酶(Chs)基因在蜕皮过程中的作用,本实验采用RACE技术首次从表皮中克隆了几丁质合成酶基因(Mn Chs)c DNA全长,并用生物软件对其序列进行生物信息学分析,RT-PCR技术检测该基因的时空表达模式。结果表明,其c DNA全长5 133bp,5'UTR为283 bp,3'UTR为159 bp,开放阅读框(ORF)长度为4 701 bp,编码1 566个氨基酸,分子量为179.57 ku,理论等电点为6.09,包含2个几丁质合成酶的标签序列EDR和QRRRW及Chitin-synth-C结构域。经BLAST比对,与日本仿长额虾、淡水枝角水蚤Chs相似性分别为89%和63%。RT-PCR结果显示,在蜕皮周期各阶段,不同组织Mn Chs的表达量差异显著:头胸甲在A期达到最高,胃肠在D0和D4期均较高,尾扇和肌肉在D4期最高,肝胰腺则普遍较低。结果表明,Mn Chs基因转录物不仅存在于表皮,在其他组织中也有分布,且mRNA水平的变化与蜕皮周期有关,作为几丁质生物合成的关键酶,推测该基因的表达在新外表皮和内表皮的形成中发挥重要作用。
In order to study the role of chitin synthase（ Chs） gene from Macrobrachium nipponense in the molting cycle,the M. nipponense chitin synthase（ Mn Chs） of the cuticular tissue was first cloned using rapid amplification of c DNA ends（ RACE） method,and its sequence was analyzed with a biological software.Spatio-temporal expression of the Mn Chs was determined by RT-PCR. The full-length contains 5 133 bp with a 283 bp of 5＇-untranslated region（ UTR）,a 159 bp of 3＇ UTR and a 4 701 bp of open reading frame encoding a putative Mn Chs protein of 1566 amino acids with a predicted molecular mass of 179. 57 ku and p I of 6. 09. It includes two specific tag sequence of Chs（ EDR,QRRRW） and a Chitin-synth-C domain.Sequence comparison shows that the Mn Chs deduced amino acid sequence shares an overall similarity of89% to Pandalopsis japonica and of 63% to Daphnia pulex. The significant difference of Mn Chs mRNA levels among tissues is observed during molt stages. The high level occurs at stage A in carapace,at stage D0 and D4in gastrointestine,at stage D4 in tail fan and muscle. However,the level from hepatopancreas is relatively lower during molt stages. In conclusion,our present results showthat Mn Chs is not only expressed in cuticle,but also in other tissues,and its level change is related to the molting cycle. As a key enzyme catalyzing chitin synthesis,Mn Chs expression may be involved in the formation of newexocuticle and endocuticle.
出处
《水产学报》
CAS
CSCD
北大核心
2015年第10期1450-1458,共9页
Journal of Fisheries of China
基金
国家自然科学基金(30940008)
河南省基础与前沿技术研究(142300410021)
关键词
日本沼虾
几丁质合成酶
克隆
序列分析
表达
Macrobrachium nipponense
chitin synthase
clone
sequence analysis
expression
