Identification And Expression Analysis Of MicroRNA During Seed Germination In Magnolia Sieboldii K.Koch

Magnolia sieboldii K.Koch seed is characterized by deep dormancy,Seed germination rate is low,and seedling is complex.Therefore,to research seed dormancy and germination on M.sieboldii have both academic value and practical significance.M.sieboldii seed as the research object of this paper.Based on the use of deep sequencing,we preliminary explored miRNA expression regulation in M.sieboldii seed germination in transcription,and combining selecting of reference gene,performing analysis of targets expression.The paper provided important information for subsequent research on miRNA molecular regulation in M.sieboldii seed germination.Main results of this st?dy were as follows:1.Isolation of total RNA from M.sieboldii seed is difficult.Lots of common method were not applicable to the RNA extraction.Through improvement of various methods,we found TRIzol combined with the CTAB method and an optimized modified TRIzol method can got high-quality total RNA with high purity and good integrity,which are suitable for transcriptome sequencing,small RNA sequencing,RT-PCR,qRT-PCR,and other downstream molecular applications.2.we selected and compared 6 novel candidate reference genes(rPS4,TM7,H2A,B5,ORMDL,Srbp1)and 6 traditionally used reference genes(ACT7,UBQ,GAPDH,ETELF,60S rRNA,?-TUB)based on Transcriptome Sequencing of M.sieboldii seed.Analysis of qRT-PCR showed that TMA7,and 60S rRNA in traditionally used reference are the most appropriate reference genes for different stages of seed germination,while Srbpl,and also 60S rRNA in traditionally used reference are the most appropriate reference genes for different tissues.3.A total of 74 miRNAs(9 conserved and 65 novel miRNA)were found in M.sieboldii seed via sRNA sequencing.And 64 miRNA were identified differentially expressed in germination.In addition,analysis of the miRNA abundance between libraries identified 28(12 increased,16 decreased),1(decreased),1(increased),22(12 increased,10 decreased),and 20(12 increased,8 decreased)significantly differentially expressed miRNAs after seed imbition,Seeds stratification 0d-30d,30d-60d,60d-75d and 75d-90d respectively,which suggest miRNA periodic expression in M.sieboldii seed germination.4.A total of 1653 target mRNAs were found for 74 miRNAs,and 472 of which exhibited functional annotation.GO annotation enrichment showed that the majority of target genes involved in major biological processes were enriched in metabolic process and cellular process and single-organism process.In the molecular function category,the target genes were associated with binding and catalytic activity.In the cellular component group,the target genes were associated with cell,cell part,organelle,and membrane.5.KEGG pathway analysis showed that a total of 4,0,0,5,and 5 different pathways enriched with.target genes of differentially expressed miRNAs were found after seed imbition,Seeds stratification 0d-30d,30d-60d,60d-75d and 75d-90d respectively.Which suggest Seed metabolic stability during stratification 0d-60d,and seed metabolic complex after imbition and stratification 60d-90d.KEGG annotation classification showed that starch and sucrose metabolism pathways was dominant in entire germination,and spliceosome was the most dominant before Seed coat cracking,while plant-pathogen interaction was more dominant after Seed coat cracking.6.Expression pattern of GAMYB,ABH1 and AFB2 maybe associated with miR159,MS-miR23 and MS-miR2 being predicted to target GAMYB,ABH1 and AFB2 respectively.However,whether the latter target the former or not still need further verification.