| Polyponus umbellatus is a peculiar and wild grifola (Polyponus umbellatus (Pers.) Fr)species in Changbai Mountain.Polyponus umbellatus is a traditional fungi for medicinal,which have the function of clearing damp and promoting diuresis.In recent years,people pay more and more attention on Polyponus umbellatus,because we found that the Polyponus umbellatus can improved the antitumor and immunity function.At present,the domestic research mainly focused on determing the pharmacological active substances,the function of Polyponus umbellatus and so on.But the study about the functional genomics and pharmacological active substances of Polyponus umbellatus hasn’t been published.With Polyponus umbellatus mycelium as material,which is cultivated in different period,we established the methom of extracting the total RNA.Using Trizol method.Trizol improved method to extract the7,14,21and28days Polyponus umbellatus mycelium total RNA.its purity and integrity to meet the requirements of the following study.Mixture the7,14,21and28days Polyponus umbellatus mycelium total RNA.then adopt SMART technology to build the Full-length cDNA library of Polyponus umbellatus mycelium.The cDNA library’s vector is lambda Trip1Ex2.The titers of unamplified library is1.69X106pfu/mL,restructuring rate is97.06%,the target fragment are mainly distributed in the500～2000bp.The titers of amplified library is1.504×1010pfu/mL.We selected six ESTs to sequence,the length of the six ESTs are627bp,878bp,941bp,652bp,766bp and1293bp.It was proved that they were the full-length cDNA,and all had ORF.Using Blastn and Blastx analysis in Nr/Nt database,the ESTs and protein sequence of them have high identity.The results show that extracted the total RNA of polyponus umbellatus mycelium successfully,and constructed a high-quality full-length cDNA library of polyponus umbellatus mycelium.have provided theoretical foundation for the study of improving immunity and anti-tumor function. |
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