斑节对虾金属硫蛋白基因cDNA克隆与表达分析

采用RACE-PCR方法从斑节对虾(Penaeus monodon)总RNA反转录产物中获得了502 bp的金属硫蛋白(Pm-MT)基因cDNA序列。该序列包含69 bp 的5非编码区(UTR)和256 bp 的3非编码区(UTR)以及177 bp的开放阅读框(ORF), 可编码58个氨基酸。在其编码的氨基酸序列中半胱氨酸含量丰富, 不含芳香族氨基酸, 存在有无脊椎动物金属硫蛋白的特征序列CKCXXXCXCX, 预测的分子量约为6.05 kD, 理论等电点7.75。序列比对分析表明Pm-MT与北美淡水蟹(Pacifastacus leniusculus)的相似性高达91.4%, 与美国龙虾(Homarus americanus)的同源性最高为84.5%。实时定量PCR显示在所检测组织中, Pm-MT的mRNA在卵巢的表达量最高, 其次为胃、肠、心脏、脑和肝胰腺; Pm-MT的mRNA在斑节对虾6个不同发育期的卵巢中的表达量都很高, 其中在Ⅱ期卵巢中的表达量最高。实验所得结果以期为Pm-MT进一步在卵巢发育的功能研究提供基础材料。  

MOLECULAR CLONING AND EXPRESSION ANALYSIS OF METALLOTHIONEIN FROM PENAEUS MONODON

Metallothionein (MT) is a family of cysteine-rich, low molecular weight proteins (MW ranging from 500 to 14000 Da), which is the only kind of metal metabolism in a conspicuous role of small proteins in vivo. MT is concerned with trace elements in the storage, transport and metabolism, body growth and development, aging and certain diseases. In addition, it also has free radical scavenging, antagonism of ionizing radiation, toxic heavy metals and other unique biological functions. The MT widely distributes in the cells, where it exists almost tissues except the connective tissue. In the normal female animal reproductive system, the expression amount of MT might go change with the level of hor-mones of physiological cycles. In the aspect of the MT of vertebrates research reports showed, the mammals, like homo sapiens, guineas and Felis silvestris catus, the structure of gene, even the function of reproduction, were well studied. It revealed that Pm-MT might regulate the process of reproduction and afterborn, while in the aspect of Invertebrates, the research focus on its performance of detoxication of heavy metal. By so far, yet to see on black tiger shrimp (Penaeus monodon) MT(Pm-MT)gene structure and function of the coverage. We have cloned the full sequence of Penaeus monodon Metallothionein gene (Pm-MT) in this experiment. Firstly, we constructed a cDNA library by using the ovary and eyes stalks of mature Penaeus monodon as raw materials, through general T3 primer sequencing and BLAST analysis, we obtained the Penaeus monodon EST library. And then by the apply of the techniques of Rapid Amplification of cDNA ends, we cloned the Pm-MT gene, which complete gene sequence was consisting of 502 nucleotides, encoding corresponding 58 amino acids. The sequence of amino acids was rich in cysteine, free of aromatic amino acids. The molecular weight and theoretical isoelectric point of Pm-MT was Predicted about 6.05 KD and 7.75 respectively. Pm-MT owned a characteristic sequence of invertebrate metallothionein CKCXXXCXCX, and its N-terminal sequence shared a high conserved structure with other crustacean. Sequence alignment analysis showed that the Pm-MT and the North American freshwater crab (Pacifastacus leniusculus) shared a similarity of 91.4%, and the homology of Pm-MT and U.S. lobster (Homarus americanus) was up to 84.5%. The quantification result of Pm-MT mRNA in 6 tissues of Penaeus monodon showed that, in ovary the highest levels, followed by stomach, intestines, heart, brain and hepatopancreas. Furthermore, the Pm-MT expression was found to be high level in the six ovarian stages of development and the expression of ovarian growth levels in the second growth phase was significantly higher than that of others, while the lowest in the first growth phase. To conclude, the results and data above, grounded on the experiment of Pm-MT, might offer essential information and material in development and research about ovarian function.