【作者】 邓瑞鹏；

【导师】 韩雅莉；

【作者基本信息】 汕头大学 ， 生物化学与分子生物学， 2005， 硕士

【摘要】 大量事实证明,有机锡污染是导致海洋腹足类动物发生性畸变的主要原因,但有机锡致性畸变的机制尚未完全清楚。多数人认为,有机锡抑制了正常螺体内细胞色素P450芳香化酶的活性而导致性畸变现象产生。结合细胞色素P450酶系在生物体内所起的重要作用,本文决定以此为切入点,以分布最广、性畸变现象最普遍、特征最明显的疣荔枝螺为研究对象,对其细胞色素P450酶系进行生化水平的部分研究,同时也深入到分子水平进行探索,以期在更深的层次对性畸变现象做出阐释。 首先,本实验从雄性、雌性和性畸变疣荔枝螺内脏团制备得到微粒体酶液,对细胞色素P450酶系三个主要成分:细胞色素P450、细胞色素b5、NADPH-细胞色素P450还原酶进行测定,结果发现性畸变螺的细胞色素b₅含量与雌、雄螺存在显著差异,通过比较还发现性畸变螺的细胞色素P450含量、NADPH-细胞色素P450还原酶活性与雄螺更接近。说明性畸变螺不但在表型与雄螺相象,而且体内P450酶系的部分成分也更接近于雄螺。 其次,通过生物软件分析出CYP19的氨基酸保守序列,自行设计简并引物,分别以雌疣荔枝螺总RNA和基因组DNA为模板,进行简并RT-PCR及简并PCR,将所得产物进行克隆、测序,结合生物信息学中BLAST序列比对,对结果进行分析。结果显示RNA及基因组DNA均含有一长约746bp的未曾报道过的序列,它与Genbank现已公布的细胞色素P450相关性较小,而与部分海洋鱼类反转录酶相关性较大,且与斑马鱼DNA序列相似。另外,还根据昆虫CYP4家族的特异性氨基酸序列及P450超家族的共同特征序列,采用另一对简并引物,同样进行有关研究,结果显示:疣荔枝螺不同组织的RNA中都含有一长468bp左右的序列,这同样是一段新序列,与部分物种的CYP4存在一定的相关性,可能在螺体内起一定重要的作用,但具体功能有待进一步研究。展望通过相应的分子生物学技术,如在此基础上用cDNA末端快速扩增法（RACE）获得其全长cDNA序列,再进行表达实验等,可以对其功能进行深入研究。 细胞色素P450酶系是一种普遍存在于动、植物中的代谢酶系,催化多种反应,在生物体内的生理生化功能中发挥着重要作用。本文在生化水平对疣荔枝螺细胞色素P450酶系进行研究,所得结果能为从细胞色素P450角度解释疣荔枝螺性畸变现象提供基础。同时也从疣荔枝螺RNA及基因组DNA得到有关的基因序列,为进一步探讨该基因在螺体内的作用打下基础,也为海洋软体动物的分子生物学研究提供参考。更多还原

【Abstract】 A large number of studies have demonstrated that imposex in marine gastropods is induced mostly by organotin pollutants. But the mechanism of imposex has not been identified. It has been accepted that the inhibition of cytochrome P450 aromatase by organotin compounds will result in the imposex phenomena. In this thesis, Marine gastropod, Thais clavigera, was chosen as the experimental material, because they are widespread, sensitive to organotin and have high imposex frequency. Considering P450 enzymes catalyze many reactions that are important in organism, cytochrome P450 enzymes and its genes in T. clavigera were studied. The results may contribute to further explain the imposex phenomena.Firstly, microsomes were respectively prepared from the visceral masses of male, female and imposex samples of T. clavigeras. The main components including cytochrome P450, cytochrome b₅ and NADPH-cytochrome P450 reductase in microsome cytochrome P450 enzymes were determined by spectrophotometry. The results indicated that the content of cytochrome b₅ in male and female was twice as that in imposex T. clavigeras, and the differences among them were significantly. Compared among the male, normal female and imposexed female snails, the content of cytochrome P450 and the activity of NADPH-cytochrome P450 reductase in imposex snails were more close to that in males. The result hinted that imposex snails were similar to males not only in morphology but also in some biochemical properties.Secondly, CYP genes were studied. CYP19 conserved regions were derived by aligning amino acid sequences by Clustal X. Degenerate primers were designed with Primer Premier 5. Genomic DNA and total RNA were isolated from T. clavigeras. The corresponding sequences were amplified by degenerate PCR and degenerate RT-PCR. Then the amplified fragments were ligated into pUCm-T vector and cloned. Their nucleotide sequences were determined. The result showed that a same fragment of 746bp was amplified from genomic DNA and total RNA. The BLAST programs were used for searching protein and DNA databases for sequence similarities. The BLAST result showed that it was a new sequence and similar to zebra fish DNA sequence and some fish reverse transcriptase. But it has small homology of CYP 19 gene sequences that have been in Genbank.In addition, sequences were amplified by RT-PCR with using degenerate primers that weredesigned against CYP4 sequences. However, the result of sequence analysis revealed that there was a 468bp fragment that existed in pleopod and viscera of T. clavigeras. It is a new sequence and its function is unknown. But it shows some sequence homology with the corresponding regions of insect CYP4. Its potential function needs further studies, such as RACE, blotting, gene expression to demonstrate.It is well-known that cytochrome P450 enzymes can be found in many types of tissues and organisms. They are very important to organisms. They play an essential role in drug metabolism, catalyze many physiological reactions such as carbon source assimilation, biosynthesis of hormones, and so on. In this paper, the experimental results of P450 enzymes were helpful to further understand the mechanism of imposex that caused by organotin compounds in T. clavigeras. And what we have studied on CYP genes could make foundation for further study on mollusks on the level of molecular biology.更多还原