鸭腺苷琥珀酸裂解酶基因序列特征及表达与肌肉肌苷酸含量的相关性分析

doi:10.3864/j.issn.0578-1752.2012.04.019

摘要/Abstract

摘要： 【目的】克隆鸭腺苷琥珀酸裂解酶（adenylosuccinate lyase，ADSL）基因的完整编码区（coding sequences，CDS），对其进行核酸和蛋白序列分析，研究该基因在各品种肌肉组织mRNA表达水平；对该基因mRNA表达水平与肌肉组织肌苷酸（inosine monophosphate acid，IMP）含量进行相关性分析。为揭示中国优良地方鸭种的风味基因结构、研究其生物学功能以及与重要风味物质的关系奠定理论基础。【方法】通过PCR扩增和克隆测序获得鸭ADSL基因部分cDNA序列，运用生物信息学方法分析鸭ADSL基因编码区序列、蛋白结构和进化关系，利用real-time-PCR技术检测鸭肌肉组织中ADSL基因mRNA表达水平，利用高效液相色谱法测定肌肉组织IMP含量。【结果】①鸭ADSL基因CDS全长均为1 380 bp，编码459个氨基酸，与鸡的同源性为94.77%。②ADSL蛋白为偏碱性，有较为强烈的信号肽和细胞内跨膜结构，蛋白二级结构主要由α-螺旋构成。③鸭和鸡首先聚为一类，后与人、鼠等哺乳动物聚为一类，而与两栖、鱼类相聚较远。④ADSL基因表达量品种间差异极显著（P＜0.01）；各品种内雌性表达量极显著高于雄性（P＜0.01）；同一性别内胸肌表达量极显著高于腿肌（P＜0.01）（樱桃谷鸭除外，为P＞0.05）。⑤IMP含量在品种间差异极显著（P＜0.01）；各品种内雌性含量显著高于雄性（P＜0.05）；同一性别内胸肌含量显著高于腿肌（P＜0.05）。【结论】鸭ADSL基因CDS序列与鸡的同源性极高，在肌肉组织中该基因的表达量及对应IMP含量表现为不同品种、同一品种不同性别、同一性别不同部位间极显著或显著差异，两者并显现出显著正相关。

关键词: 鸭, ADSL, 克隆, mRNA表达, 肌苷酸, 高效液相色谱法

Abstract: 【Objective】The CDS (coding sequences) of duck ADSL gene was cloned. The study was aimed to analyze gene sequences, protein structure, relative expression levels of ADSL gene in muscle and the correlation between gene expression and inosine monophosphate (IMP) content in ducks. This research revealed the ADSL gene structure of excellent native ducks of China, established a foundation for further research of its biological function and relationship with flavor substances. 【Method】 Reverse transcriptase PCR was employed to clone the cDNA of ADSL gene. The coding region sequences, protein structure and phylogenetic relationships were analyzed by bioinformatics software. real-time-PCR was employed to examine the relative expression levels of ADSL gene in muscle of ducks. IMP content in breast and leg muscle was detected by HPLC. 【Result】 The CDS of ADSL gene in ducks had 1380 bp in length, encoded 459 amino acid residues and showed 94.77% sequence identity with chicken. The physical and chemical properties of duck ADSL indicated that ADSL is a basic protein. And the protein has more strongly signal peptide and transmembrane segments in cells. The secondary structure of duck ADSL was mainly made by α helixes. Phylogenetic analysis showed that duck and chicken were in the same clustering, and then clustered with human, mice and other mammals, both of which had a distant relationship with fish and amphibian. The expression levels of ADSL gene among different varieties were extremely different (P＜0.01), that between female and male were extremely different (P＜0.01), and that among different locations were different (P＜0.05) except those in cherry valley ducks (P＞0.05). IMP content among different varieties were extremely different (P＜0.01), and among different sexes and locations were very different (P＜0.05). 【Conclusion】 The CDS of ADSL gene in ducks showed a high sequence identity with chicken. The expression levels of ADSL gene and the IMP content of relative locations were extremely different (P＜0.01) or different (P＜0.05) among different varieties, sexes and locations, respectively. ADSL gene relative expression and IMP content were significantly correlated.

Key words: duck, adenylosuccinate lyase, cloning, mRNA expression, IMP, high-performance liquid chromatography

徐善金, 虞德兵, 汪峰, 何宗亮, 张剑锋, 于海龙, 杜文兴. 鸭腺苷琥珀酸裂解酶基因序列特征及表达与肌肉肌苷酸含量的相关性分析[J]. 中国农业科学, 2012, 45(4): 774-785.

XU Shan-Jin, YU De-Bing, WANG Feng, HE Zong-Liang, ZHANG Jian-Feng, YU Hai-Long, DU Wen-Xing. Analysis of Sequence Characters of ADSL Gene and Correlation between Gene Expression and IMP Content in Duck[J]. Scientia Agricultura Sinica, 2012, 45(4): 774-785.

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