Studies On Transcriptome And Digital Gene Expression Profiling In Ovules And Leaves Of Ginkgo Biloba L.

Ovules are an essential reproductive structure and can play a role in the life cycle and alternation of generations in all seed plants. At present, the molecular genetic basis of ovule development of angiosperms has been extensively studied, whereas limited information on related studies of ovules in gymnosperms is available. Ginkgo biloba L. is the one of the oldest gymnosperms in extant species, and its morphological and anatomic studies have indicated that the ovule of G. biloba may show similarities in structure and function with primitive ovules of early seed plants. In addition, morphological characteristic of G. biloba ovules is the foliar ovule-bearing organs (O-ha-tsuki, leaf-borne seeds) occasionally found in both extant G. biloba and Paleozoic Ginkgophytes. Therefore, systematic research based on the level of genome in G. biloba ovule will has important scientific values. Nowadays, although some molecular and genomic studies of G. biloba have focused on biosynthesis of secondary metabolites, phylogenetic and evolutionary aspects, there has be no systematic studies regarding genetic similarities and differential gene expression between the ovules and leaves of G. biloba. Therefore, in this study, we used the combined method of the Illumina next-generation sequencing HiSeqTM 2000 platform and bioinformatics to perform de novo transcriptome sequencing and analysis on G. biloba female leaves, and to conduct digital gene expression (DGE) profiling sequencing between the ovules and leaves, and finally identified numerous commonly and differentially expressed genes in ovules and leaves. Additionally, many homologous genes associated with floral development were expressed in ovules. The main results were summarized as follows:(1) Total RNA was successfully extracted from leaves and ovules of G. biloba using a modified CTAB, method, respectively. After the quality of the RNAs were tested qualified, the total RNAs from leaves and ovules were used for transcriptome sequencing and digital gene expression profiling sequencing.(2) Based on Illumina HiSeqTM 2000 platform, Illumina RNA sequencing generated a total of 56,341,460 raw reads. After stringent quality checks and data cleaning, we obtained 54,253,512 clean reads and the Q20 valued up to 97.21%, indicating that the sequencing quality was great. Based on these clean reads, the de novo assembly yielded 77,898 unigenes with a mean length of 829 bp, and the N50 of unigenes valued up to 1743 bp. These transcripts have enriched the transcriptome information of G. biloba.(3) All of the distinct unigene sequences were searched against six protein and nucleic acid databases(Nr, SwissProt, KEGG, COG, GO, Nt) by BLASTx, a total of 42,624 unigenes were annotated, accounting for 54.72% of all unigenes. Based on a comparison against the KEGG and Swiss-Prot databases using BLASTx, the analysis of metabolic pathway showed that 1859 unigenes were assigned to 18 main KEGG pathways regarding biosynthesis of secondary metabolites. Among these pathways, we searched for 90 putative unigenes encoding 11 known key enzymes which may involve in flavonoid biosynthesis pathway, as well as 61 putative unigenes encoding 17 known key enzymes which related to ginkgolide/bilobalide. Furthermore, we found that up to 855 unigenes encoding diverse transcription factors(16 kinds) may involve in multiple biological processes based on annotation of Swiss-Prot, including growing development and morphogenesis, biosynthesis of secondary metabolites and stress adaptation in G. biloba leaf. In addition, many a subset of candidate genes involved in development was identified in G. biloba.(4) We developed a de novo transcriptome assembly, combined with information from public genetic and protein databases, to generate an integrated transcriptomic resource(100124 transcripts) for DGE sequencing and reads mapping. We construct and sequenc two DGE libraries(leaf and ovule) by RNA-seq, which generated 11,597,234 and 12,459,128 clean reads after removal of the low-quality reads, respectively. Based on these clean reads, the number of sequences that could be mapped to the reference database, and then 80.21% and 74.48% were mapped to the reference database perfectly, respectively. Furthermore, the results of saturation analysis and randomness assessments were performed to evaluate DGE sequencing quality. Altogether, all these results indicating the sequencing quality and match quality were great.(5) Through the analysis of gene expression level, we found 42,149 genes commonly expressed in the leaf and ovule,14,679 and 6301 genes specifically expressed in the leaf and ovules, indicating a high percentage of common functional and conserved genes were expressed in both ovules and leaves. Many abundantly expressed genes encoding related proteins in the leaf were speculated that may involve in plant defense and photosynthesis. The abundantly expressed genes in the ovule might relate to cell wall biogenesis, secretion, and flavonoid biosynthesis. Importantly, according to the annotation of transcripts, we identified 121 homologous genes associated with ovule development.(6) We performed morphological and anatomical observations to investigate the morphological and structural characteristics of mature leaves and ovules in G. biloba, indicating that the leaves and ovules share a number of similar characteristics in overall morphology, presence of stomata, and chloroplast structure. On the basis, we identified many commonly expressed involved in stomatal development, chloroplast development and photosynthesis in both the leaf and ovule, and quantify their expression profiles of 11 randomly selected genes by quantitative real-time PCR. The qRT-PCR results were consistent with the results of DGE analysis pattern. Altogether, microscopic observations and functional genetic expression implied that the ovule was able to carry out chlorophyll biosynthesis, photosynthesis, and gas exchange in common with the functions of leaves.(7) The significance of gene expression variations was analyzed by comparing the leaf and ovule libraries. A total of 11,434 DEGs were detected, comprising 4575 up-regulated genes and 6859 down-regulated genes in leaves. According to GO function enrichment analysis, cell division (GO:0051301), cell differentiation (GO:0030154), DNA replication initiation (GO:0006270), DNA-dependent DNA replication (GO:0006261), hormone transport (GO:0009914), and floral organ formation (GO:0048449) were significantly enriched. In addition, up to 38 KEGG pathways were significantly enriched by mapping all of the DEGs to pathways in the KEGG database. Based on these significantly enriched GO terms and KEGG pathways, most of the DEGs were down-regulated in the leaves, which indicated that a closely correlation in the development processes of leaves and ovules.(8) Through performed cluster analysis and screening on the DEG library (|log2(ratio)|≥4) between leaves and ovules, we found many genes that showed significantly different expression, which associated with DNA replication, cell division and cell expansion, flavonoid biosynthesis, starch and sucrose metabolism, hormone signaling transduction, and development. Meanwhile, the qRT-PCR experiments were performed results to verify the expression profiles of genes in our sequencing analysis, and results showed qRT-PCR results were consistent with the results of DGE expression profiling. Totally, these results suggested that significantly differentially expressed genes between the leaf and ovule during the same developmental period, which contributed to regulate their process of growth and development.In the present study, these results provide a valuable resource for future genetic and genomic studies on G. biloba, and will give fundamental basis on diverse biological mechanisms and the molecular mechanisms of ovule development between ovules and leaves of Ginkgo biloba. In addition, it may provide a molecular insight into ovule evolution in early seed plants.