斑馬魚pycr1a基因分析

Abstract

早衰綜合症（PS）是一種罕見的加速生理衰老的遺傳性疾病，使患病的個體看起來比正常情況下更加衰老。與HGPS不同，ARCLII是一種鮮為人知的早衰綜合症，其特徵是皮膚褶皺或者皮膚鬆弛以及與早衰相關的骨質流失。其一部分病因（22名患者）是由PYCR1基因的突變引起的，PYCR1編碼吡咯啉-5-羧酸還原酶1，其在涉及NADP（+）和ATP生成的脯氨酸合成中起到一定的作用。在這項研究中，我在斑馬魚中註釋了pycr1a基因並分析其表達。根據RT-PCR分析，pycr1a 的RNA水平在受精後後12小時之內很高，而且在眼睛和卵巢中含量很高。原位雜交顯示pycr1a出現在胚胎早期發育階段的眼睛和大腦中。此外西方墨點法顯示，其可以有效結合Pycr1a羧基末端结构域GST融合蛋白的Pycr1a抗體，並可染到斑馬魚线粒体蛋白中之Pycr1a。在表型上，突變雌魚的體重較低，長度較野生型斑馬魚更短，而突變型和野生型雄魚之間沒有明顯的差異。實驗中關於pycr1a分子上和物理上的特徵將為未來斑馬魚早衰綜合症的研究提供基礎性支持。

Progeroid syndromes (PS) are a group of rare genetic disorders which speed up physiological aging, making affected individuals appear to be older than they are. Unlike the HGPS (Hutchinson–Gilford progeria syndrome), ARCLII (autosomal recessive cutis laxa type II) is a less known progeria syndrome characterized by wrinkly skin or cutis laxa and bone loss associated with premature aging. Some cases (22 patients) resulted from mutations of PYCR1 gene-encode pyrroline-5-carboxylate reductase 1-which play a role in proline production involving NADP (+) and ATP generation. In this study, I annotated a pycr1a gene in zebrafish and performed its expression analysis. According to RT-PCR, pycr1a RNA level is high before 12 h post fertilization (hpf) stages and has the highest levels in eyes and ovary. In-situ hybridization shows that pycr1a locate in eyes and brain in early developmental embryos. Furthermore, I generated a Pycr1a antibody, which effectively bind to GST fusion protein of the pycr1a carboxyterminal domains and zebrafish Pycr1a mitochondria protein. Phenotypically, mutant female fish have a lower body weight and s shorter length than wide type fish while there is no significant physical difference between male fish. The molecular and physical features of pycr1a will provide basic support for future study of progeria syndromes in zebrafish.