【作者】 张红；

【导师】 韩英；

【作者基本信息】 东北农业大学 ， 动物遗传育种与繁殖， 2015， 硕士

【摘要】 翘嘴鲌(Culter alburnus)是中国名贵的鲤科(Cyprinidae)淡水鱼类,具有很高的经济价值。兴凯湖翘嘴鲌体银白色,肉质细嫩,其优良的肉质性状受到广泛关注。但由于过度捕捞,导致其天然资源日益减少,市场需求日趋紧张。因此,翘嘴鲌的人工养殖前景较好。鱼肉受消费者欢迎主要由于其营养丰富,肉质及风味良好。当前,国内外对体肌中肌苷酸(Inosine-5’-Monophosphate,IMP)水平的研究较多,从分子水平展开对鱼类肉质相关基因的研究也越来越多,越来越多的鱼类风味候选基因被逐步发掘出来,但不同储藏温度下IMP含量变化规律、不同生长时期IMP含量差异以及与IMP生成代谢密切相关的腺苷一磷酸脱氨酶(Adenosine5’-monophosphate deaminase,AMPD1)基因的研究还很少。本研究运用高效液相色谱技术测定翘嘴鲌2龄、3龄、4龄养殖群体及4龄野生群体,在(20±1)℃、4℃、-20℃贮存条件下肌肉组织中肌苷酸(IMP)含量及其变化规律。根据Genbank中已知的鲤科鱼类AMPD1基因序列设计引物,采用PCR技术和RACE技术首先克隆出翘嘴鲌腺苷一磷酸脱氨酶(AMPD1)的部分c DNA序列,再利用获得的序列设计引物扩增出包含3’UTR和5’UTR的其他部分的cDNA序列,将所得序列进行拼接,得到全序列;采用荧光定量PCR技术(Real-time PCR)检测AMPD1基因在兴凯湖翘嘴鲌肌肉组织中的表达量。试验结果如下:(1)2龄未经贮存(0h)肌肉组织IMP含量为3.4297mg/g,2龄和3龄群体IMP含量显著高于4龄IMP含量(p<0.05)。在3个贮存条件下,肌肉组织IMP含量在温度较高条件下降解速度更快,而冷冻保存肌肉组织测得结果显示IMP含量均较高,且降解缓慢。不考虑年龄和温度变化的影响,IMP含量与肌肉组织的贮存时间也有关系:IMP含量达到峰值后,贮存时间与IMP含量呈负相关。建议保存温度为低温冷冻,且7d内食用最佳。(2)获得了翘嘴鲌AMPD1基因c DNA序列全长2592bp,Genbank登录号为KP735961。其中包括完全的CDS区序列长度为2154bp,完整的3’UTR区片段大小为351bp,5’UTR区片段大小为87bp。预测编码717个氨基酸和一个终止密码子。(3)通过Blast比对显示翘嘴鲌AMPD1基因核苷酸序列CDS区与斑马鱼(Danio rerio)的同源性高达89%;而其氨基酸同源性比对结果显示与欧洲川鲽(Platichthys flesus)同源性为84%;与斑马鱼(Danio rerio)氨基酸同源性较高为93%,且编码的氨基酸数目与斑马鱼相同,均编码717个氨基酸。比对兴凯湖翘嘴鲌AMPD1基因的核苷酸序列和氨基酸序列均显示,其与鲤科鱼类的同源性更高。(4)对兴凯湖翘嘴鲌2龄、3龄、4龄养殖群体和4龄野生群体肌肉组织进行AMPD1基因表达分析。Real-time PCR结果表明,2龄翘嘴鲌AMPD1基因mRNA表达量最高,3龄表达量次之,显著高于野生和养殖群体4龄肌肉组织表达量(p<0.05);4龄野生群体肌肉组织表达量显著高于4龄养殖群体肌肉组织表达量(p<0.05)。AMPD1基因表达量随年龄递减。AMPD1表达量与IMP含量呈正相关。更多还原

【Abstract】 Alburnus(Culter alburnus) is one kind of rare Chinese carps(Cyprinidae) freshwater fish, with high economic value. Xingkaihu alburnus own silvery white body, tender meat and delicious taste, its trait of excellent meat quality attracts widespread attention. However, the overfishing leads to natural resources dwindling and much tighter market demand. Therefore, alburnus predict a good prospect for artificial breeding. The quality, flavor and taste of fish meat are significant factors affecting the popularity of consumers. At present, there are more and more researches about inosine monophosphate(Inosine-5’-Monophosphate, IMP) content in muscle tissue from the molecular level. Many fish flavor candidate genes have been gradually uncovered, but few researches focus on variation of IMP content under different storage temperatures, IMP content during different growth stages and adenosine monophosphate deaminase(Adenosine 5’-monophosphate deaminase, AMPD1) gene related to IMP metabolism.In this study, by high performance liquid chromatography, under(20±1)℃, 4℃and-20℃ storage conditions, inosine monophosphate(IMP) content and its variation of age 2, 3, 4th instar Xingkaihu alburnus muscle tissue have been measured. According to Genbank known carps AMPD1 gene sequence, gene primers were designed. By using RT-PCR and RACE, partial c DNA sequence of AMPD1 was cloned AMPD1, and then we use the obtained sequence to design primers for amplifying other parts of c DNA sequence contained 3’UTR and 5’UTR, and the resulting sequences were spliced to obtain the complete sequence; by using quantitative PCR(Real-time PCR), we detected the expression of AMPD1 gene in Xingkaihu alburnus muscle tissue. Test results are as follows:(1) IMP content of 2-year-old age group was 3.4297mg/g, 2 and 3 years old age group were significantly higher(p <0.05). Under different three storage temperatures, IMP content of muscle tissue degraded faster under storage conditions of(20±1)℃; the IMP contents of specimens stored at-20℃ even maintained higher level and degraded slower than the fresh muscle tissues in specimens. Regardless of the influence of age and temperature change, IMP content also had relationship with storage time of muscle tissue. After reaching peak level, IMP content negatively correlated with storage time. It was recommended in low storage temperature and edible within 7 d.(2) Obtained the CDS region of alburnus AMPD1 gene and unknown area of the gene 3 ’end and the 5’ end by using RACE technology. Obtained 2592 bp c DNA sequence after sequence assembled, including encoding 717 amino acids and a termination codon of complete CDS sequences(2154bp), complete 3’UTR region(351bp) and 5’UTR region(87bp). Genbank accession number was KP735961.(3) Blast comparison displayed that the CDS region between alburnus AMPD1 gene and zebrafish(Danio rerio) showed a high homology of 89%. Amino acid homology comparison between AMPD1 gene and European flounder(Platichthys flesus) showed a high homology of 84%; with zebrafish(Danio rerio) showed a high homology of 93%, and the number of encoded amino acids was the same as zebra fish, both of them encoded 717 amino acids.(4) Analyzed the expression of AMPD1 gene in muscle tissues between 2, 3, and 4 age instar breeding Xingkaihu alburnus groups and 4 age instar wild groups. Real-time PCR results showed that the mRNA expression of 2 age alburnus AMPD1 gene was the highest, followed by the expression of the 3 age instar group, the former two were significantly higher than 4 age instar muscle tissues both the wild and breeding groups(p <0.05); between 4 age instar groups, the muscle tissue expression of wild groups was significantly higher than that of breeding groups(p <0.05). Consistent with existing research, AMPD1 gene expression decreased with age. AMPD1 expression quantity and IMP content were positively correlated.更多还原