| Herb Genome Program includes a serial of project on genomics, proteomics, metabolomics, genetic metabolic engineering and molecular breeding for greatly valuable and/or endangered medicinal plants, which aims to obtain good-quality, high-yield and anti-resistant herbal medicine and establish modern biological engineering system of pharmaceutical components. Among the field of HGP, transcriptome is one of the most remarkable and popular studies. In Lingnan area, many herbal medicines, such as Nervilia fordii, encounter resource endangerment, and the processes in existence are incapable to eaze the plight. In additional, transcriptome analysis is still blank in herbal medicines from Lingnan area.Nervilia fordii, belonging to Orchidaceae family, is a famous and valuable herbal medicine in Lingnan Area. The whole plant or leaf was used as a traditional Chinese medicine named Qingtiankui, for its benefits to human, such as moistening the lung to stop the cough, clearing away heat and relieving toxin, promoting circulation to relieve pains, and significant effect in curing pediatric respiratory diseases. Wild resource of N. fordii has endangered due to human excessive harvest and its rigorous requirements of germination and growth. The species has been listed in "Catalogue of Rare and Endangered Plants Grown on Limestone in South China" and "International Trade Convention for Endangered Plants and Animals". In order to ease the plight, existing studies have been attempting to obtain N. fordii resource by domestic cultivation and tissue culture proliferation. However, these two methods encounter their bottleneck with seedling scarcity, seedling hardening, and cultivation expanding, respectively; the resource of N. fordii acquired from these approaches is far from satisfactory of the market and clinic demand. Under the circumstance that imbalance of supply and demand and, driven by economic interests, many adulterants are mistaken as N. fordii in the market. In addition, dry N. fordii are defined to three types according to different leaf size.In order to resolve species confusion of N. fordii and provide a scientific foundation for resource protection and biological research of N. fordii, DNA barcoding and transcriptome analysis were carried out in this thesis. N fordii was acquired that with accurately identification using DNA barcoding; and then, transcriptome features of N. fordii were explored, genes involved in synthesis of secondary metabolites and plant physiological processes were mined by high-throughput sequencing and bioinformatics’annotation, and the reliability of transcriptome features was validated using gene cloning and expression of EBP1gene, a key regulator functionally associated with plant organ size, as a sample. The methods and conclusions were summarized below.1Molecular identification of Nervilia fordii using DNA barcodesThree DNA sequences (ITS2, rbcL, matK) acquired from three types of N. fordii and their common adulterants. Nervilia plicata, Plantago asiatica, Centella asiatisa, Ipomoea batatas et al, were analyzed using MEGA software. The Results revealed that matK exhibited longest sequence while ITS2showed shortest, and ITS2contributed most average variation while rbcL contributed least. The interspecific divergence (0.088-1.138) between N. fordii and its adulterants was far higher than the intraspecific one in N. fordii (0.000-0.004). The NJ trees constructed based on three DNA barcoding sequence independently were capable to discriminate N. fordii and its adulterants intuitively.To further evaluate the feasibility of ITS2, rbcL, matK and LSU D1-D3between N. fordii and its congeneric species, corresponding universal primers were used to amplify and sequence four candidate DNA barcodes respectively. All sequence exhibited well universality in Nervilia and were suitable selection of candidate barcodes in Nervilia. At intraspecific level, none variation were found in matK and LSUD1-D3regions, however,0.4%,7.2%of divergence of ITS2within N. aragoana, N. mackinnonii, and0.2%of variation of rbcL within N. fordii were detected. ITS2contributed most average interspecific variation (22.9%), followed by LSU D1-D3(4.6%), matK (3.7%) and rbcL (2.0%). Similar results were showed by wilcoxon test. However, matK gene exhibited significant barcoding gap between distribution of intra-and inter-specific variation and identified all Nervilia species used in this study. The phylogenetic tree constructed by matK gene illustrated the genetic relationship among Nervilia species.Therefore, matK gene can served as standard DNA sequence for authentication of N. fordii and its adulterants and congeneric species, and as a potential DNA barcode for Nervilia.2Transcriptome signature of Nervilia fordiiA total of142,220Unigenes were assembled from transcriptome sequencing data of N. fordii leaf and corm. A total of5,684SSR motifs were screened out from5,223Unigenes in the database, which were composed of di-nucleotide, tri-nucleotide, tetra-nucleotide, penta-nucleotide and hexa-nucleotide, and the di-nucleotide motif AG/CT repeat most. By means of bioinformatics annotation,38,640Unigenes were classified into25function categories of COG database, and110,029Unigenes were sorted into three main categories biological process, cellular component and molecular function of GO database, and28,970Unigenes distributed in281pathways in KEGG data library. Gene expression analysis of leaf and corm revealed that62,205Unigenes exhibited remarkable diversity (FDR(?)0.001,|Log2(Corm_RPKM/Leaf_RPKM)|(?)1), including31,488up-regulated genes and31,117down-regulated ones. In additional, expression trend and tissues specific of10genes encoding enzymes related to flavonoid biosynthesis,18genes encoding key enzymes involved in terpene backbone biosynthesis, and27functional genes participating plant physiological processes were excavated and illustrated in N. fordii from transcriptome data.3Cloning and analysis of plant organ size regulator EBP1gene from N. fordiiAn1185-bp Unigene encoding EBP I gene was selected from transcriptome database, and then cloned from N. fordii using RACE-PCR. The acquired gene was named as NfEBP1, with an ORF contained1188base pairs and encoded395amino acids, which shared99%identity with the Unigene. Bioinformatics analysis revealed that molecular weight of NfEBPl protein was43.5kDa and the isoelectric point was6.12, and its overall structure exhibited hydrophilic; NfEBP1contains none of the signal peptide, chloroplast transit peptide, mitochondrial targeting peptide, and located entirely outside cell membrane. The predicted secondary structure of NfEBP1was composed of21α-helixes,21extend strands,18β-turners and28random coils. The putative model of three-dimensional structure exhibited high homology with the crystal structure of human EBP1. NfEBPl also showed high homology with EBP1protein from other plants, and possessed a functional domain PA2G4.For expression analysis of NfEBP1, five common reference genes (18s rRNA, Actin, Ubiquitin, EF-1α,β-tubulin) were compared in different tissues (leaf, corm and petiole) of N. fordii. The stability of the candidate reference genes were evaluated by Ct value using GeNorm and NormFinder software. The analysis of GeNorm and NormFinder revealed that β-tubulin was more stable than others and could be served as reference gene for the normalization of gene expression in different tissues of N. fordii using real-time fluorescence PCR. The relative expression of NfEBP1in three tissues was calculated by formula F=2-ΔΔCt. NfEPB1was ubiquitously expressed in all tissues of N. fordii, and the relative expression level reached the highest in leaf, and the lowest in petiole, which was coincident with the trait of NfEBP1in transcriptome. The results validated the feasibility of transcriptome database in the subsequent study of N. fordii.Manipulating organ size regulator NfEBPl acqured from this study would provided an approach for promoting organ size and yield of N. fordii.In conclusion, the transcriptome profile of N. fordii with quick and accurate identification using DNA barcoding was demonstrated, and several key genes participated in synthesis of secondary metabolites and plant physiological processes were found and their expression trends were illustrated, which provide a firm foundation for variety breeding and resource protection of N. fordii by means of molecular biological approaches, and a novel and considerable research way for other endangered medicinal plants in Lingnan area. |
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