显微介导中国对虾基因鲤肌肉蛋白差异表达分析

Differential Expression Analysis of Muscle Protein From the Common Carp(Cyprinus carpio)Micro-injected With Total DNA of Chinese Shrimp(Fenneropenaeus chinensis)

本文通过显微注射方法,将中国明对虾(Fenneropenaeus chinensis)总DNA直接导入鲤(Cyprinus carpio L.)受精卵内,获得显微介导中国对虾基因鲤。为了研究显微介导中国对虾基因鲤蛋白质的表达变化,利用iTRAQ技术定量蛋白质测序分析了未进行显微介导和显微介导中国明对虾基因的2种鲤的肌肉组织,寻找与氨基酸和脂肪酸相关的差异表达蛋白,并分析了差异表达蛋白GO功能注释和KEGG代谢通路。结果共鉴定蛋白质999个,表达差异蛋白92个,其中上调蛋白58个,下调蛋白34个。差异蛋白Pathway富集分析表明,显著富集于脂肪酸和氨基酸等代谢通路上的差异蛋白有13个,其中在精氨酸和脯氨酸代谢通路中发现的上调7.51倍的肌酸激酶(creatine kinase)有促进鱼类肌肉发育的作用;而在脂肪酸代谢通路中发现的下调0.59倍的线粒体三功能蛋白(mitochondrial trifunctional protein),有提高鱼体内多种饱和与不饱和脂肪酸含量的作用。同时对筛选到的两个蛋白进行实时荧光定量PCR验证,证实了蛋白质组的分析结果。

In this study,transgenic common carp(Cyprinus carpio)offsprings were obtained by micro-injection of the total DNA of Chinese shrimp(Fenneropenaeus chinensis)into the fertilized eggs of common carp.In order to probe into the protein expression pattern of transgenic common carp,the muscle tissues of transgenic and non transgenic common carp were quantitatively sequenced by isobaric tags for relative and absolute quantitation(iTRAQ)technology,and the differentially expressed proteins related to amino acids and fatty acids were identified.Gene ontology and KEGG metabolic pathway analysis were conducted and all the differentially expressed proteins were classified into different categories.Proteins of 999 in total were identified,in which,92 proteins were found to be differentially expressed in transgenic common carp,including 58 up-regulated proteins and 34 down regulated protein.Pathway enrichment analysis of differential proteins showed that there were 13 differential proteins significantly enriched in fatty acid and amino acid metabolic pathways.In these genes,creatine kinase was expressed up-regulated by 7.51 times in arginine and proline metabolic pathways,which could promote muscle development in fish.The 0.59 fold down regulated mitochondrial trifunctional protein were found in the fatty acid metabolism pathway which may play a role in increasing the content of various saturated and unsaturated fatty acids in fish.The expression patterns of creatine kinase and mitochondrial trifunctional protein were then validated by quantitative real-time PCR(qRT-PCR),suggesting the reliability of protein analysis results.