Dataset Information

De novo transcriptome assembly of transgenic tobacco (Nicotiana tabacum NC89) with early senescence characteristic.

ABSTRACT: The enzyme, α-farnesene synthase (AFS), which synthesizes α-farnesene, is the final enzyme in α-farnesene synthesis pathway. We overexpressed the α-farnesene synthase gene (previously cloned in our lab from apple peel) and ectopically expressed it in tobacco (Nicotiana tabacum NC89). Then, the transgenic plants showed an accelerated developmental process and bloomed about 7 weeks earlier than the control plants. We anticipate that de novo transcriptomic analyses of N. tabacum may provide useful information on isoprenoid biosynthesis, growth, and development. We generated 318,925,338 bp sequencing data using Illumina paired-end sequencing from the cDNA library of the apical buds of transgenic line and the wild-type line. We annotated and functionally classified the unigenes in a nucleotide and protein database. Differentially expressed unigenes may be involved in carbohydrate metabolism, nitrogen metabolism, transporter activity, hormone signal transduction, antioxidant systems and transcription regulator activity particularly related to senescence. Moreover, we analyzed eight genes related to terpenoid biosynthesis using qRT-PCR to study the changes in growth and development patterns in the transgenic plants. Our study shows that transgenic plants show premature senescence.

Supplementary information

The online version contains supplementary material available at 10.1007/s12298-021-00953-z.

SUBMITTER: Liu H

PROVIDER: S-EPMC7907299 | biostudies-literature | 2021 Feb

REPOSITORIES: biostudies-literature

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De novo transcriptome assembly of transgenic tobacco (Nicotiana tabacum NC89) with early senescence characteristic.

Liu Heng H Liu Yu Y Cheng Nini N Zhang Yuanhu Y

Physiology and molecular biology of plants : an international journal of functional plant biology 20210219 2

The enzyme, α-farnesene synthase (AFS), which synthesizes α-farnesene, is the final enzyme in α-farnesene synthesis pathway. We overexpressed the α-farnesene synthase gene (previously cloned in our lab from apple peel) and ectopically expressed it in tobacco (Nicotiana tabacum NC89). Then, the transgenic plants showed an accelerated developmental process and bloomed about 7 weeks earlier than the control plants. We anticipate that de novo transcriptomic analyses of N. tabacum may p ...[more]

PMID: 33707866

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Project description:BackgroundPollen tube growth is essential for plant reproduction and represents a widely employed model to investigate polarized cell expansion, a process important for plant morphogenesis and development. Cellular and regulatory mechanisms underlying pollen tube elongation are under intense investigation, which stands to greatly benefit from a comprehensive understanding of global gene expression profiles in pollen and pollen tubes. Here, RNA sequencing technology was applied to de novo assemble a Nicotiana tabacum male gametophytic transcriptome and to compare transcriptome profiles at two different stages of gametophyte development: mature pollen grains (MPG) and pollen tubes grown for six hours in vitro (PT6).ResultsDe novo assembly of data obtained by 454 sequencing of a normalized cDNA library representing tobacco pollen and pollen tube mRNA (pooled mRNA isolated from mature pollen grains [MPG] and from pollen tubes grown in vitro for 3 [PT3] or 6 [PT6] hours) resulted in the identification of 78,364 unigenes. Among these unigenes, which mapped to 24,933 entries in the Sol Genomics Network (SGN) N. tabacum unigene database, 24,672 were predicted to represent full length cDNAs. In addition, quantitative analyses of data obtained by Illumina sequencing of two separate non-normalized MPG and PT6 cDNA libraries showed that 8979 unigenes were differentially expressed (differentially expressed unigenes: DEGs) between these two developmental stages at a FDR q-value of <0.0001. Interestingly, whereas most of these DEGs were downregulated in PT6, the minor fraction of DEGs upregulated in PT6 was enriched for GO (gene ontology) functions in pollen tube growth or fertilization.ConclusionsA major output of our study is the development of two different high-quality databases representing the tobacco male gametophytic transcriptome and containing encompassing information about global changes in gene expression after pollen germination. Quantitative analyses of these databases 1) indicated that roughly 30% of all tobacco genes are expressed in the male gametophyte, and 2) support previous observations suggesting a global reduction of transcription after pollen germination. Interestingly, a small number of genes, many of which predicted to function in pollen tube growth or fertilization, were found to be upregulated in elongating pollen tubes despite globally reduced transcription.

Project description:BackgroundNicotiana benthamiana has been widely used in laboratories around the world for studying plant-pathogen interactions and posttranscriptional gene expression silencing. Yet the exploration of its transcriptome has lagged behind due to the lack of both adequate sequence information and genome-wide analysis tools, such as DNA microarrays. Despite the increasing use of high-throughput sequencing technologies, the DNA microarrays still remain a popular gene expression tool, because they are cheaper and less demanding regarding bioinformatics skills and computational effort.ResultsWe designed a gene expression microarray with 103,747 60-mer probes, based on two recently published versions of N. benthamiana transcriptome (v.3 and v.5). Both versions were reconstructed from RNA-Seq data of non-strand-specific pooled-tissue libraries, so we defined the sense strand of the contigs prior to designing the probe. To accomplish this, we combined a homology search against Arabidopsis thaliana proteins and hybridization to a test 244k microarray containing pairs of probes, which represented individual contigs. We identified the sense strand in 106,684 transcriptome contigs and used this information to design an Nb-105k microarray on an Agilent eArray platform. Following hybridization of RNA samples from N. benthamiana roots and leaves we demonstrated that the new microarray had high specificity and sensitivity for detection of differentially expressed transcripts. We also showed that the data generated with the Nb-105k microarray may be used to identify incorrectly assembled contigs in the v.5 transcriptome, by detecting inconsistency in the gene expression profiles, which is indicated using multiple microarray probes that match the same v.5 primary transcripts.ConclusionsWe provided a complete design of an oligonucleotide microarray that may be applied to the research of N. benthamiana transcriptome. This, in turn, will allow the N. benthamiana research community to take full advantage of microarray capabilities for studying gene expression in this plant. Additionally, by defining the sense orientation of over 106,000 contigs, we substantially improved the functional information on the N. benthamiana transcriptome. The simple hybridization-based approach for detecting the sense orientation of computationally assembled sequences can be used for updating the transcriptomes of other non-model organisms, including cases where no significant homology to known proteins exists.

Project description:BackgroundNicotiana benthamiana has been widely used for transient gene expression assays and as a model plant in the study of plant-microbe interactions, lipid engineering and RNA silencing pathways. Assembling the sequence of its transcriptome provides information that, in conjunction with the genome sequence, will facilitate gaining insight into the plant's capacity for high-level transient transgene expression, generation of mobile gene silencing signals, and hyper-susceptibility to viral infection.Methodology/resultsRNA-seq libraries from 9 different tissues were deep sequenced and assembled, de novo, into a representation of the transcriptome. The assembly, of 16GB of sequence, yielded 237,340 contigs, clustering into 119,014 transcripts (unigenes). Between 80 and 85% of reads from all tissues could be mapped back to the full transcriptome. Approximately 63% of the unigenes exhibited a match to the Solgenomics tomato predicted proteins database. Approximately 94% of the Solgenomics N. benthamiana unigene set (16,024 sequences) matched our unigene set (119,014 sequences). Using homology searches we identified 31 homologues that are involved in RNAi-associated pathways in Arabidopsis thaliana, and show that they possess the domains characteristic of these proteins. Of these genes, the RNA dependent RNA polymerase gene, Rdr1, is transcribed but has a 72 nt insertion in exon1 that would cause premature termination of translation. Dicer-like 3 (DCL3) appears to lack both the DEAD helicase motif and second dsRNA binding motif, and DCL2 and AGO4b have unexpectedly high levels of transcription.ConclusionsThe assembled and annotated representation of the transcriptome and list of RNAi-associated sequences are accessible at www.benthgenome.com alongside a draft genome assembly. These genomic resources will be very useful for further study of the developmental, metabolic and defense pathways of N. benthamiana and in understanding the mechanisms behind the features which have made it such a well-used model plant.

Dataset Information

De novo transcriptome assembly of transgenic tobacco (Nicotiana tabacum NC89) with early senescence characteristic.

Supplementary information

Publications

De novo transcriptome assembly of transgenic tobacco (<i>Nicotiana tabacum</i> NC89) with early senescence characteristic.

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