Transcriptome, Chloroplast Genome And Related Studies Of Ginkgo Biloba And Magnolia

As the increase to the demand of herbal medicine, serious destruction of ecological environment, and long-term unplanned development of Chinese medicine resource, a large number of populations and species are likely to become extinct this century. Molecular biology and biotechnology provide important opportunity of development for studying for medical plants and traditional Chinese herbs modernization and help to resolve the problems above. Based on the high throughput sequencing technology, studies have focused on the transcriptome analysis, medicinal natural product biosynthesis, the key enzyme gene cloning and identification, and producing area suitability, respectively, using Ginkgo biloba L. and 5 species of Magnolia as object of research. Based on the results of experimental research, the main results are as follows:1. Ginkgo biloba L. is monotypic species native to China and has old, dioecious,medicinally important characteristics. The functional genes related to these characteristics have not been effectively explored due to a limited number of expressed sequence tags (ESTs) from Ginkgo. To discover novel functional genes efficiently and to understand development of a living fossil tree, Ginkgo, we used massive parallel pyrosequencing on the Roche 454 GS FLX Titanium platform to generate 64057 ESTs. The ESTs combined with the 21590 Ginkgo ESTs in GenBank were assembled into 22304 unique putative transcripts, in which 13922 novel unique putative transcripts were identified by 454 sequencing. After being assigned to putative functions with Gene Ontology (GO) terms, a detailed view of the Ginkgo biological systems was displayed, including characterisation of unique putative transcripts with homology to known key enzymes and transcription factors involved in ginkgolide/bilobalide and flavonoid biosynthetic pathways, as well as unique putative transcripts related to development, response to disease and defence. The fact that three full-length Ginkgo genes encoding key enzymes were found and cloned, suggests that high-throughput sequencing technology is superior to traditional gene-by-gene approach in discovery of genes. Additionally, a total of 204 simple sequence repeat (SSR) motifs were detected. Our study not only lays the foundations for transcriptome-led studies in biosynthetic mechanisms, but also contributes significantly to the understanding of functional genomics and development in non-model plants. 2. Chloroplast genome sequences are a versatile tool for molecular systematic, species identification and genetic engineering, but taxon sampling remains sporadic and chloroplast genetic transformation encounters the developing bottleneck because of the great effort to acquire full-length sequences. Pure chloroplast DNA extraction and cost-effective sequencing strategy are the decisive factors. Here, we demonstrate a protocol to sequence more complete chloroplast genomes simultaneously for plant identification using the 454/Roche platform by parallel tagged sequencing. The procedure could be employed routinely for chloroplast genome sequencing of most land plant species. Our study reports the complete chloroplast genome of Ginkgo, with the length of 156982 bp. It harbors a pair of inverted repeat of 17732 bp (IRa and IRb) separated by 22267-bp-long SSC and 99251-bp-long LSC. The genome content and the relative positions of 129 genes (including 43 tRNA genes,4 rRNA genes, and 6 ycfs). Ycf2 is located in LSC rather than IR in previous report. A total of 10 stable intraSNPs and 20 cp-SSR loci with length of more than 30 bp are detected. Another part of our relative chloroplast study produced 9 chloroplast genomes (5 species of Magnolia, Magnoliaceae) including a new complete sequences (the others not showed) made a phylogenetic analysis in species level combined with existing one complete genome sequences as outgroup. We proved that chloroplast genome sequences may be a universal single-locus DNA marker for interspecies identification. The discovery of cpSSR of Ginkgo and 5 species of Magnolia will help to develop the noval markers and the distribution can help to further understanding of cpDNA. And the reported complete choloroplast genomes are facilitate to chloroplast development of molecular mechanism and provides new opportunities in across a wide array of fields, such as increase productivity, improve quality and biopharmaceuticals.3. We present CpGAVES, a web server that allows the annotation of a chloroplast genome sequence, and the visualization and editing of the annotation results in an interactive and real-time manner. CpGAVES accepts a completely sequenced chloroplast genome as input. First, it accurately predicts protein-coding sequences and exon-intron structures on the basis of interspecies mapping of full-length cDNA. Second, tRNA genes and inverted repeats are identified using tRNAscan and vmatch. Using 109 chloroplast genome sequences as test, we show that CpGAVES outperforms pervious similar applications. Third, the annotation results are presented as a circular map that includes the name, location, orientation, and length of the various features. A Flex-based module is implemented, allowing the users to add, delete, and edit the features on-line. Users also have options to download the annotation results in GFF3 format for further analysis in other third party software tools or in JPEG format for publication. Finally, CpGAVES can be used to create a Sequin file for GenBank submission of the annotated genome sequence. CpGAVES is freely available at http://CpGAVES-plantidit.dnsalias.org, and it will significantly facilitate research work on chloroplast genomes. The web CpGAVES, to a large extent, is facilitate to analyze chloroplast sequences efficiently.