Transcriptome Sequencing Of Sporophyll And Sporogenesis Gene Screening In Ophioglossum Vulgatum L.

Ophioglossum vulgatum L.belongs to Ophiolossaceae in Eusporangiopsida.It belongs to the primitive fern according to the evolutionary.It is also an important rare medicinal plant.The dominant plant body of O.vulgatum is the sporophyte.The sporophyte is composed of a vegetative leaf and a sporophyll formed by the specialization of sporangium spikelet,which makes it a model plant to study the development of sporophyte and sporangium.Sporangium development and spore formation are important biological events in the asexual generation of plants.In seed plants,the generation of pollen has been studied in detail.However,the molecular mechanism of regulating sporophyll development,especially the spore development in ferns is still unclear.In this paper,transcriptome sequencing technology was used to annotate the gene functions at different developmental stages of sporophyll of O.vulgatum,and the genes related to sporangia and spore development of O.vulgatum were screened out,and the functions and regulatory effects of these genes were analyzed.This study provides a reference basis for exploring the developmental regulation mechanism of sporangia and spores in O.vulgatum,and also has a certain significance for revealing the development and phylogenetic evolution of ferns.The main research results are as follows:A total of 36.82 G raw data reads were obtained by using RNA-seq technology.After data filtering and quality assessment,40,3588394 clean data reads sequences were obtained,and a total of 137,060 Unigenes were obtained by comparing them with protein functional database for functional annotation information.The gene expression analysis technology was used to construct the gene expression profile c DNA library of different developmental stages of the sporophyte,including shoot tip stage(S stage),sporophyte development stage(D stage)and sporophyte maturity stage(M stage).In this study,FPKM method was used and FDR < 0.05 and log2|FC| ? 1 as the standard measure of differentially expressed genes.A total of 6250 differentially expressed genes were obtained.Respectively,708,3683 and 1859 differentially expressed genes were screened in S vs D,D vs M and S vs M stages.The GO and KEGG functional enrichment analysis of differentially expressed genes showed significant enrichment in plant hormones,plastids,lipids,cell differentiation and cell wall related genes,which may play an important role in the development of sporophyte.Relative quantitative methods are often used to identify gene expression levels.In order to obtain reliable gene expression data under different developmental conditions in tissues,reference genes that can be stably expressed in different periods should be obtained as controls.Based on transcriptome sequencing and analysis software of Norm Finder,Best Keeper and Genorm,the stability of 8 candidate internal reference genes in Ophioglossum vulgatum L.was evaluated.The results showed that Op UBQ4 and Op ACT could be used as reference genes for the study of gene expression in different developmental stages of the sporophylls.According to the research progress of microsporogenesis in angiosperms,the genes that may be involved in the development of sporophyte were analyzed from three aspects.Seven regulatory genes related to cell differentiation and tapetum cell determination were identified from the shoot tip meristem(S phase)of the spores of Ophioglossum vulgatum L.Fourteen regulation genes related to spore wall formation and five transcription factors involved in spore development were identified from different genes at different stages of spore leaf development.The expression of 8 candidate genes significantly different in different periods was analyzed and verified by q RT-PCR.The expression levels of Op BAM,Op SERK,Op CHI and Op MYB genes were up-regulated during the differentiation and sporogenesis of sporophyte bud cells,and the expression of Op LEC and Op PP2 C genes were up-regulated during sporophyte and sporophyte maturation.The results showed that the expression of these genes in different stages of sporothyte development was consistent with the results of transcriptome expression analysis,which may be involved in the process of sporogenesis.In conclusion,this study provided detailed transcriptome information for the sporophyll and the molecular mechanism of sporogenesis of O.vulgatum L.and identified 26 regulatory genes involved in sporogenesis as well as stable reference genes.This study laid a foundation for the further study on the molecular mechanism of sporogenesis of O.vulgatum L.