Isolation And Identification Of Small RNA In Ganoderma Lucidum

Ganoderma, Fungus Ganoderma lucidum of Polyporaceae, is a kind of preciously medicinal and edible Fungus, belongs to the treasure house of Tradition Chinese Medicine(TCM). Micro RNA(mi RNA) is a type of endogenous non-protein-coding small RNA of 18-26 nucleotide(nt), which plays an important role in the negative regulation of gene expression. According to research r EPorts, Ganoderma lucidum contains many biologically active ingredients, including polysaccharides, alkaloids, proteins, fatty acids, sterols, amino acids, triterpenoids and trace elements. Study of those components has been very thorough, but none of small RNA in Ganoderma lucidum, especially the r EPort of mi RNA. This study aims to preliminary explore Ganoderma lucidum mi RNA—isolating and identificating Ganoderma lucidum mi RNA, lays the foundation for the study on the mechanisms of Ganoderma lucidum mi RNA in the field of medicine, opens new road for the study on Ganoderma lucidum in edible value.Fresh Ganoderma lucidum sporocarp and Ganoderma lucidum spores were collected, then used for total RNA extraction by portable method and Direct-zolTM RNA Minipr EP method, followed by the concentration and purity test of total RNA to select a better material of Ganoderma lucidum and better method; Illumina sequencing platform was applied for high-throughput sequencing of Ganoderma lucidum small RNA, followed by bioinformatics to analysis small RNA in-d EPth, simultaneously novel putative candidate mi RNA(PC mi RNA) was predicred; 4 known mi RNA and 3 PC mi RNA were selected to be verified using q RT-PCR and Northern blotting. The main results are as follows:(1) After the st EP of total RNA extraction of Ganoderma lucidum by portable method and Direct-zolTM RNA Minipr EP method, concentration and purity of total RNA respectively are: portable method of Ganoderma lucidum sporocarp—2510 ng/?L, OD260/280 1.84; Direct-zolTM RNA Minipr EP method of Ganoderma lucidum sporocarp—2054 ng/?L, OD260/280 2.00; portable method of Ganoderma lucidum spores—420 ng/?L, OD260/280 1.85; Direct-zolTM RNA Minipr EP method of Ganoderma lucidum spores—270 ng/?L, OD260/280 1.99. It follows that it is better that to choose Ganoderma lucidum sporocarp as the material and Direct-zolTM RNA Minipr EP method as the extraction method in study of Ganoderma lucidum combining with electrophoresis.(2) Small RNA library of Ganoderma lucidum was constructed using Illumina high-thoughput sequencing, 10,127,291 high-quality sequences were obtained from it, 9,071,523 clean sequences(774,547 unique) remained after adapters and junk reads were removed. Statics of clean reads from small library was analyzed and statics of the effective length sequences(18-50 nt) was interc EPted, in which there are higher peaks respectively at the position of 30 nt(pi RNA), 43 nt and 50 nt. The proportion of small RNA of 18-26 nt(mainly are mi RNAs) in small RNA of 18-50 nt is nearly 50%.(3) Mi REvo software was used for analyzing clean reads, and 132 known mi RNA and 34 PC mi RNA were detected, new precursor mi RNA can form characteristic hairpin. 10436 sequences matched compared with Rfam database. Evolution situation of known mi RNA was analyzed using NCBI, mi RBase and Mage software.(4) 4 known mi RNA and 3 new PC mi RNA were selected to analyze expression and verify the results of high-throughput sequencing using q RT-PCR and Northern blotting. 6 sequences(glu-mi R9386, glu-mi R-125, glu-mi R-99, glu-mi R-99-1 and PC mi RNA 1, PC mi RNA 3) of All mi RNAs have a high expression in q RT-PCR, 5(glu-mi R9386, glu-mi R-125, glu-mi R-99, glu-mi R-99-1 and PC mi RNA 2, PC mi RNA 3) of them have a high expression in Northern blotting.After isolating, identifying and analyzing Ganoderma lucidum micro RNA, we found that: A large number of small RNA exist in Ganoderma lucidum, which indicates that there are complex gene expression regulation of post-transcriptional in Ganoderma lucidum; the present work firstly indentified the small RNAs in Ganoderma lucidum, amplifying the species of small RNAs and new micro RNAs for gene library, and laid the foundation for further study on the mechanisms of Ganoderma lucidum small RNA in the field of medicine, especially micro RNA.