【作者】 蒋克勇；

【导师】 王雷；

【作者基本信息】 中国科学院研究生院（海洋研究所） ， 水产养殖， 2012， 博士

【摘要】 现代水产养殖业在片面追求高产量、高生长速度的同时，水产动物的肉质风味却在急剧下降，而且这一矛盾愈加尖锐。本研究针对这一现实问题，首先，采用营养学调控手段和高效添加剂使用策略来改善水产养殖鱼虾的品质，提高其肌肉出肉率和蛋白质含量，增加其呈味氨基酸含量；降低脂肪含量，改变脂肪酸组成和比例，提高不饱和脂肪酸，尤其是多不饱和脂肪酸的含量；提高肌肉主要鲜味物质IMP的含量；综合改善养殖水产品的色泽、口感和风味等，初步建立了水产动物肉质风味改良的技术方法和评价指标。其次，采用生化和分子生物学的方法，利用RT-PCR和RACE等技术，克隆与牙鲆肌肉IMP和IMF形成密切相关的AMPD1基因和LPL基因，对基因的结构和功能进行解析；采用Real Time PCR技术，从mRNA水平研究上述基因在牙鲆生长发育阶段的表达时向性，组织特异性，表达丰度等；以及结合HPLC法测定IMP含量，索式抽提法测定IMF含量和组织相关酶活；探索基因表达与酶活、风味物质含量之间的相关关系，从分子水平对IMP和IMF的形成和变化规律给予初步解释。主要结果如下：实验组罗非鱼的鱼片出肉率高于对照组2.65%；肌肉氨基酸总量、必需氨基酸含量以及呈味氨基酸含量也均高于对照组，呈味氨基酸含量提高1.41-1.7%；实验组罗非鱼肌肉多不饱和脂肪酸含量相比对照组提高明显，其中EPA+DHA含量显著提高，EPA含量提高2倍以上，∑n-3/∑n-6比例比对照组也有明显提高；而实验组罗非鱼肌肉主要鲜味物质IMP含量显著高于对照组，提高达5-10倍以上。实验组凡纳滨对虾肌肉中必需氨基酸总量相对于对照组提高不明显，而鲜味氨基酸总量提高明显，其中Asp，Glu和Gly显著提升；实验组凡纳滨对虾肌肉多不饱和脂肪酸总量相比对照组提高显著， EPA+DHA含量显著提高，∑n-3/∑n-6比例差异不明显；实验组凡纳滨对虾肌肉主要鲜味物质IMP含量比对照组提高3倍以上。通过感官评定，肉质风味改良组水产动物的体形、体表色泽、肌肉口感、致密度、鲜度和气味等指标相比对照组均有不同程度的改善。本研究首次克隆到与牙鲆IMP和IMF代谢相关的2个基因，从牙鲆肌肉中克隆得到AMPD1基因(Genebank登录号：JF323023.1)，其cDNA全长2526bp，开放阅读框(ORF)长2211bp，编码736个氨基酸；从牙鲆肝脏中克隆得到LPL基因（Genebank登录号：HQ850701.1），其cDNA全长2003bp， ORF长1645bp，编码514个氨基酸。牙鲆9种组织的RT-PCR结果表明，AMPD1特异性高效在肌肉中表达，在其余组织中不表达，属于M型AMP脱氨酶；qRT-PCR分析表明LPL在牙鲆各组织中普遍表达，在腹腔肠系膜组织、肝脏和肾脏中表达丰度最高，在其余组织中表达较低。牙鲆成鱼(750±40g)肌肉AMPD1基因表达量明显高于幼鱼(7.5±2g)(p<0.05)；成鱼肌肉IMP含量(3.35±0.21mg/g)也明显高于幼鱼(1.08±0.04mg/g)(p<0.05)，肌肉AMPD1基因表达和IMP含量之间表现正相关，揭示AMPD1基因对IMP形成的重要调控作用。无论是牙鲆幼鱼还是成鱼，其肌肉LPL基因表达量、酶活力均明显低于肝脏LPL基因表达量、酶活力；幼鱼与成鱼肌肉LPL基因表达和IMF含量均未呈现明显差异；推测LPL基因可能不是鱼类IMF形成的唯一主效基因，可能更多的是参与调控鱼类肝脏中脂肪的蓄积和分配，而肝脏是鱼类最重要的贮脂性调节器官。更多还原

【Abstract】 As the high-yield and high speed growth in aquaculture industry, the meat quality ofaquatic animals dramatically dropped down. To solve this contradiction，firstly, nutritionalregulation and feed additive formulation method were used to improve the meat quality ofaquatic animals, fish fillet ratio, protein content, and delicious amino acid content wereincreased; fat content, fatty acid composition and proportion were changed, especiallyPUFA content was increased; the main flavor substance IMP content was enhancedsignificantly; the color, taste, and flavor of aquatic animals were all improved in differentdegrees. Secondly, RT-PCR and RACE methods were used to clone the IMP and IMFmetabolism related AMPD1gene and LPL gene, genes structure and function werecharacterized; Real Time PCR method was used to study the mRNA expression indifferent tissues and developmental stages; combined with IMP content by HPLC, IMFcontent by Soxlet extraction, and enzyme activities; relationships among gene expression,enzyme activity, and flavor substances content were established; the metabolicmechanisms of IMP and IMF were explained primarily at molecular level. The mainresults are as follows:In tilapia, fish fillet ratio of experimental group was higher by2.65%than control;the amounts of muscle total amino acids, EAA, and flavor amino acids were all higherthan control, flavor amino acids content was increased by1.41-1.7%; PUFA content ofexperimental group was significantly higher than control, total EPA+DHA were increasedsignificantly, EPA content was increased2times,∑n-3/∑n-6was also higher than control;IMP content of experiment group was increased5-10times than control. In Litopenaeusvannamei, EAA content of experimental group was not improved significantly thancontrol, but flavor amino acids content, including Asp, Glu, and Gly, was increasedsignificantly; PUFA content of experimental group was significantly higher than control, total EPA+DHA were increased significantly,∑n-3/∑n-6was no obvious changes; IMPcontent of experiment group was increased3times than control. Sensory analysis showedthat the shape, color, muscle texture, density, fresh, and odor of experimental animals wereimproved in various degrees than control.Two IMP and IMF metabolism related genes were cloned from Japanese flounder.AMPD1gene was cloned from muscle of flounder (Genebank accession number:JF323023), the full-length cDNA was of2526bp, open reading frame (ORF) was2211bpin length, encoding736AA; LPL gene was cloned from liver of flounder (Genebankaccession number: HQ850701), the full-length cDNA was of2003bp, ORF was1645bp inlength, encoding514AA. Nine tissues were separated from flounder, RT-PCR showed thatthe AMPD1gene was specifically expressed in muscle, whereas not detected in resttissues; LPL gene was ubiquitously expressed in various tissues, highest expressed in theabdominal mesentery tissue, liver, and kidney, and lower in other tissues. AMPD1geneexpression level in muscle of adult fish (750±40g) was significantly higher than juvenile’s(7.5±2g)(p<0.05); IMP content in muscle of adult fish (3.35±0.21mg/g) was also higherthan juvenile’s(1.08±0.04mg/g)(p<0.05), revealing the important regulatory role ofAMPD1gene in the formation of IMP metabolism. LPL gene expression level and enzymeactivities of both adult and juvenile flounder were lower in muscle than that in liver; LPLgene expression level and IMF content in muscle of juvenile and adult fish showed nosignificant difference; revealing LPL might not be the only major gene for IMFmetabolism in fish muscle, implicating the more function of LPL for participating theregulation of lipids metabolism in fish liver.更多还原