MicroRNAs Involved In Age-related Flowering Pathway In Ginkgo Biloba Based On Transcriptome Sequencing

It is very important to reveal the mechanism of age regulation in Ginkgo biloba from the molecular level.In this study,the stem/flower bud of Ginkgo biloba in its infancy,one year of flowering and many years of flowering were used as experimental materials.The important genes and miRNAs regulating flowering in three age stages of Ginkgo biloba were identified and analyzed by high-throughput sequencing technology and bioinformatics technology,and the flowering network map of age pathway regulating flowering of Ginkgo biloba was constructed.The main results are as follows:In this study,we identified and analyzed the important genes and miRNAs regulating flowering in three age stages of Ginkgo biloba,and constructed the flowering network map of Ginkgo biloba by age pathway regulation,using the method of high-throughput sequencing and bioinformatics.The main results were summarized as follows:?1?Nine microRNA libraries were constructed and 151.46 M clean reads were obtained.Each sample was no less than 13.34 M clean reads and Q30 was about 98%.A total of 594 miRNAs were identified from the small RNA library,including 89 known miRNAs and 505 new predicted miRNAs,286 of which belong to 97 miRNA families.Target genes of miRNA were predicted by targetfinder software.The results showed that 12762 target genes were predicted and 11210 target genes were annotated.?2?The results of miRNA differential expression analysis showed that 30 miRNAs with significant expression differences were screened out.Using go enrichment analysis and pathway analysis,we classified the functions of DEGs among the target genes of differentially expressed miRNA.The results showed that 24 target genes were enriched in the biological regulatory pathway,13 target genes were enriched in the plant hormone signal transduction pathway,5 target genes were enriched in the reproductive process and 2 target genes were enriched in the plant growth pathway,suggesting that these pathways might be related to the flowering regulation of Ginkgo biloba.Further analysis of miRNA and target genes showed that miRNA156 and miRNA172 expression patterns were not obvious,while miR159,miR319,miR408,miR7127 and their negative regulatory target genes played a complex regulatory role in the regulation of Ginkgo biloba's age pathway in the flowering process.The results of qPCR showed that the expression trend was consistent with the transcriptome data.?3?A total of 57.45Gb of high quality data were obtained by transcriptome sequencing,and the percentage of Q30 base was 94.59%or more.Sequence alignment showed that the number of homologous genes of Ginkgo and Picea sitchensis was the highest?10816,31.18%?,followed by Amborella trichopoda(3,677,10.60%,followed by Nelumbo nucifera?2707,7.82%?;KEGG?Kyoto Encyclopedia of genes and Genomes?results showed that UniGene of Ginkgo biloba flower bud involved 126 metabolic pathways in three age stages,among which starch and sucrose metabolism?304 genes?,plant hormone signal transduction?302 genes?,ubiquitin mediated protein hydrolysis?222 genes?and plant circadian rhythm?72 genes?may play an important role in flower bud induction The function of being able or lacking.?4?The difference gene analysis showed that there were less up-regulated genes?37?and down-regulated genes?75?in young flower bud vs.one-year flower bud,while there were more up-regulated genes?592?and down-regulated genes?871?in young flower bud vs.one-year flower bud vs.961 and down-regulated genes?1203?in flower bud.Through further enrichment analysis of the differentially expressed genes with KEGG and GO,eleven candidate key genes related to flowering were screened out in three age stages of Ginkgo biloba:SPL?gene.Gb₂3 724?gene.Gb₀3922?,AP2?gene.Gb₀0766?,MADS-box gene?gene.Gb₀1886,gene.Gb 15398,gene.Gb 28337,Gingko_newGene₂213?,GA regulatory protein?gene.Gb 34467?gene.Gb₂8606?gene.Gb 33214?and DELLAs protein?gene.Gb₃4644?.?5?Through the joint analysis of transcriptome and small RNA sequencing,the flowering regulatory network of age pathway in Ginkgo biloba was constructed:one is the regulatory mode dominated by miRNA7127,SPL,AP2 and SOC1;the other is the regulatory pathway dominated by GA regulatory protein,DELLAs,miRNA159,MYB transcription factor and miRNA319;miRNA408 is involved in the regulation of flowering by participating in the regulation of plant biological clock.