Gene mining and sequence analysis of purine nucleosidase based on RNA-Seq
Gene mining and sequence analysis of purine nucleosidase based on RNA-Seq
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基于RNA-Seq的嘌呤核苷酶基因挖掘与序列分析
DOI：
10.3791/61561
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发表时间：
2020
期刊：
影响因子：
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通讯作者：
Shan Lin
中科院分区：
文献类型：
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作者：
Fenfang Wu;Shiping Hu;Yun Ran;Xiaoni Chen;Shan Lin
冬虫夏草（Ophiocorphinensis）是一种名贵真菌类中药，含有丰富的腺苷等活性成分。腺苷被认为是一种生物有效成分，具有多种抗肿瘤和免疫调节活性。为了进一步阐明嘌呤核苷酶（PN）在腺苷生物合成中的作用机制，基于冬虫夏草RNA-Seq数据库，成功挖掘并分析了一个编码PN的基因。PN的cDNA全长为855 bp，编码284个氨基酸。BLAST分析表明，该基因与NCBI中的核苷水解酶基因同源性最高，为85.06%。ProtProm分析表明，相对分子质量为30.69 kDa，等电点为11.55。用Predict Protein软件对PN的二级结构进行了预测，结果表明：α螺旋结构占28.17%，链结构占11.97%，环结构占59.86%。进一步从转录组中克隆PN基因，并通过琼脂糖凝胶电泳进行验证。本研究为真菌类中药腺苷生物合成的遗传调控提供了更充分的科学依据和新的思路。
Caterpillar fungus (Ophiocordyceps sinensis) is one of the most valued fungal Traditional Chinese medicine (TCM), and it contains plenty of active ingredients such as adenosine. Adenosine is considered as a biologically effective ingredient that has a variety of anti-tumor and immunomodulatory activities. In order to further elucidate the mechanism of purine nucleosidase (PN) in adenosine biosynthesis, a gene encoding PN was successfully mined and further analyzed based on the RNA-Seq database of caterpillar fungus. The full-length cDNA of PN was 855 bp, which encoded 284 amino acids. BLAST analysis showed the highest homology of 85.06% with nucleoside hydrolase in NCBI. ProtProm analysis showed that the relative molecular weight was 30.69 kDa and the isoelectric point was 11.55. The secondary structure of PN was predicted by Predict Protein; the results showed that alpha helix structure accounted for 28.17%, strand structure accounted for 11.97%, and loop structure accounted for 59.86%. Moreover, PN gene was further cloned from transcriptome and detected by agarose gel electrophoresis for verification. This study provides more sufficient scientific basis and new ideas for the genetic regulation of adenosine biosynthesis in fungal TCM.