| 李新佳,陈婉姨,罗素兰,等.大理石芋螺Fosmid文库的构建及鉴定[J].中国海洋药物,2021,40(2):55-61. |
| 大理石芋螺Fosmid文库的构建及鉴定 |
| Construction and Identification of the Fosmid Library for Conus marmoreus |
| 投稿时间：2020-07-09 修订日期：2020-09-06 |
| DOI： |
| 中文关键词: 大理石芋螺 Fosmid 基因组文库构建 鉴定 |
| English Keywords:Conus marmoreus Fosmid Genomic library construction Identification |
| Fund Project: |
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| 中文摘要: |
| 目的 芋螺毒素(芋螺肽)具有特异结合各种离子通道和受体的特殊功能,拥有巨大的新药开发潜力。但因海洋生态环境的破坏和无序采集,芋螺的种类和数量在不断减少,在国际上已被列为濒危保护动物。所以,对芋螺遗传资源和基因信息的长期保存具有非常重要的科学和实际意义。方法 研究针对中国南海产大理石芋螺(Conus marmoreus),利用磁珠法提取其高质量的基因组DNA,经过末端修复和胶回收、连接、包装、侵染等步骤,构建大理石芋螺基因组Fosmid文库。结果 经鉴定该文库包括242,500个克隆；克隆片段长度平均约为40kb；文库覆盖了大理石芋螺基因组的4.61倍。经过传代培养,第0代和第100代的Fosmid克隆的质粒酶切图谱无明显差异。该基因组Fosmid文库,对基因组的覆盖率高,稳定性好,对某一基因被克隆筛选的概率达到了99.01%。本文还利用该文库克隆出了一个新的α-芋螺毒素前体基因。结论 大理石芋螺Fosmid文库的构建有利于该种芋螺的基因组资源长期保存而不会因物种濒危被丢失,将为大理石芋螺毒素基因的进一步发掘、基因序列结构和功能的研究等奠定重要基础。 |
| English Summary: |
| Objective Conotoxins (Conopeptides) could target various ion channels and receptors specifically, which display huge potential for new drug development. However, due to the destruction of the marine ecological environment and disorderly collection within these decades, the species and quantities of Conus (cone snails) are decreasing, which has been listed as endangered animals in the world. As a consequence, it is important and significant to protect and preserve genetic resources and gene information of Conus. Methods Here, Conus marmoreus native to South China Sea was chosen for its genomic library construction. Magnetic bead method was used to extract high-quality genomic DNA of C. marmoreus. Through a series of end reparation and gel extraction, ligation, packaging and infection steps, the genomic Fosmid library of C. marmoreus was constructed. Results This library was identified to contain 242,500 clones, which covers the genome up to 4.61-fold. The average length of the cloned fragment is about 40 kb. After subculturing, the plasmid restriction map of the library showed no significant difference between the 0 and 100th generation. The genomic Fosmid library of C. marmoreus possess a high rate of genome coverage and great stability, in which probability of screening a target gene is 99.01% in theory. A novel α-conotoxin precursor gene was cloned and identified from this library. Conclusion The library would be beneficial for long-term preservation of C. marmoreus genomic resources and gene information, which will not be lost in the future although its endangered status. This research would lay an important foundation for further discovery of conotoxin genes, structure and function investigation of related genes from Conus marmoreus genome. |
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