Transcriptome Analysis Of Ginkgo Biloba Seed During Dormancy

We applied light microscopy and transmission electron microscopy to investigate the cellular and subcellular changes that occur during dormancy and divided the process into different stages in Ginkgo biloba seeds.We also profile global mRNA expression in C with various dormancy statuses to explore the regulatory network of seed dormancy.The main results were as follows:1.We divided the Ginkgo biloba seeds dormancy processs according to the seed morphology and germination time,and then use the digital camera to record the different stages of seed germination.At the first stage,the embryo was small and its structure began to develop,which needed long time to germinate,about 150 days,we divided this stage into dormancy induction stage.At the second stage,the embryo grew and elongation rapidly,and radicle,hypocotyl,plumule and cotyledon began to form,but the cotyledon was not yet open,which needed shorten time to germinate,about 60 days,we divided this stage into to dormancy maintain stage.At the third stages,tissue differentiation was finished with the cotyledon expansion and radicle elongation,while the seeds began to germinate,we divided this stage into dormancy release stage.We performed morphological and anatomical observations to investigate the morphological and structural characteristics of embryo at dormancy induction stage,dormancy maintain stage and dormancy release stage in G.biloba,indicating that at dormancy induction stage,starch grains and other nutrients are accumulated in the embryo stem cells and embryonic stem cells present in large volume and cell gap.At dormancy maintain stage,embryo stem cells are disappearing,while leaf primordium differentiation is completed,and radicle,hypocotyl,plumule and cotyledon began to form,nutrients are accumulated in the embryo cells at the same time.At dormancy release stage,tissue differentiation with organ formation and the accumulation of nutrients in embryo are continuing,a large number of nutrients are accumulating in cotyledons as well.Further the embryos of Ginkgo seeds were observed using transmission electron microscopy,the results verified that the nutrients were starch grain..2.Based on Illumina HiSeqTM 2500 platform,Illumina RNA sequencing generated a total of 41,150,448,49,385,302,46,128,674,55,768,706,46,208,328,47,208,328 reads,and the Q20 valued up to 96.5%,indicating that the sequencing quality was great.Based on clean reads,the de novo assembly yielded 133,278 unigenes with a mean length of 664 bp,and the N50 of unigenes valued up to 1256 bp.These transcripts have enriched the transcriptome information of G.Biloba.All of the distinct unigene sequences were searched against six protein and nucleic acid databases by BLASTx,a total of 50,409 unigenes were annotated,accounting for 37.82%of all unigenes.Based on a comparison against the KEGG databases using BLASTx,the analysis of metabolic pathway showed that 12,590 unigenes were assigned to 19 main KEGG pathways.Among these pathways,we searched for 1154 putative unigenes which may involve in carbohydrate metabolism pathway,as well as 619 putative unigenes which related to lipid metabolism.Furthermore,we found 390 unigenes may involve in signal transduction.3.The significance of gene expression variations was analyzed by comparing six embryo dormancy libraries.A total of 15,155 DEGs were detected.Based on these significantly enriched KEGG pathways,most of the DEGs on plant hormone signal transduction,cell cycle,fatty acid metabolism and so on.4.The hormone plays an important role in regulating seed dormancy,therefore,we analysised DEGs in plant hormone signal transduction pathways through the KO enrichment,found a large number of genes in response to abscisic acid,gibberellic acid,ethylene and auxin,and they have different expression patterns in different stages of dormancy.Among them,the ABA receptor protein and GA decompose gene over expression at dormancy induction stage,and the expression level decreased to dormancy maintain and release stage.On the contrary,GA receptor and synthesis gene and negative regulation factor in ABA signal transduction,ETH signal transduction and IAA synthesis genes in the dormancy induction stage of low expression,and significantly up-regulated to the dormancy release stage.5.Plant seed need to undergo cell development process from dormancy to release,where cell development or cell division activity is the most basic physiological process.Thus,the DEGs involved in cell division or DNA replication were analyzed from the dormancy induction stage to dormancy release stage of G.biloba.Among them,the gene associated with cell division proteins CDC and DNA replication licensing factor MCM are highly expressed at the dormancy induction and maintain stages,and are significantly down-regulated at the dormancy release stage.6.During the process of seed dormancy,the storage material was transformed and used to provide nutrients for seed germination.The gene related to sucrose biosynthesis SPS was significantly higher at dormancy maintain and release stages than dormancy induction stage,while the genes related to sucrose degradation SUS and invertase STP13 began to large express at dormancy induction stage and significantly reduced at the dormancy release stage.Meanwhile a large number genes associated with amino acid synthesis and fatty acid metabolism were expression at dormancy induction and maintain stages,and reduced to dormancy release stage.Otherwise,the expression pattern of the E3 ubiquitin-protein ligase was on the contrary.7.Transcription factors(TFs)play an important role in the growth and development of embryos.A total of 1780 TFs were identified in the embryos of G.biloba.Among them,639 TFs were differently expressed,which belong to 69 TF families,including MADS-box,bHLH,AP2/EREBP,MYB and so on.And the largest number was AP2-EREBP,where 73 different TFs,otherwise bHLH,MYB,bZIP and other TF families were also much.Further analysis showed that compared to dormancy induction stage,the expression level of AP2-EREBP,bZIP,MYB and other TFs was increased gradually in dormancy maintain and release stages,while the MADS-box TF family members was increased at dormancy maintain stage and then decreased at dormancy release stage.8.To verify the expression profiles in different stages of embryo dormancy of genes identified through RNA-Seq,we further selected ten genes related to ABA response and signal transduction,GA response and biosynthesis,ETH response and signal transduction,and IAA response,and then examined by qRT-PCR.The results showed that the expression trends of RNA-Seq and qRT-PCR were basically the same,further indicated the expression level of hormone was different in G.biloba at different dormancy stages,meanwhile indicated that the results of RNA-Seq are reliable.Further compare the gene expression patterns in the embryo and endosperm,GA2ox expressed more differently in embryo,while the ETR and ARF expression patterns in the embryo and endosperm are basically identical,but the ARF are more significant differences in the endosperm.In the present study,these results provide a valuable resource for future molecules studies during G.biloba embryo dormancy,and will give fundamental basis for further study of the regulatory mechanisms underlying seeds dormancy in G.biloba.