Molecular Tools Developed for Nicotiana attenuata and Solanum nigrum

The molecular tools for the ecological expression system developed in our Department comprise tools for the identification of ecologically relevant genes (SAGE libraries, 300 ESTs from differential methods such as DDR-RTPCR and Subtractive Hybridization, and more recently RNAseq), genomic and transcriptomic databases, tools for the transcriptional analysis of the identified genes (qPCR and microarrays) and transformation systems for studying of the ecological relevance of the identified genes (stable transformation systems for posttranscriptional gene silencing or overexpression of genes, VIGS based methods). A microsatellite based method for the identification of different N. attenuata populations, ecotypes and individuals has also been developed.

SAGE (Serial Analysis of Gene Expression)
We have generated in collaboration with GenXPro GmbH (http://www.genxpro.info/) two SAGE libraries with more than 350,000 total tags from N. attenuata wounded and FAC (Fatty Acid Conjugates) elicited leaves. These tags represent ca. 23,000 unique transcripts and approx. 500 of these tags are statistically > 2-fold up-regulated after FAC treatment compared to wounding whereas 250 are > 2-fold down-regulated. Some of these tags have been selected for gene function characterization in N. attenuata using VIGS and RNAi gene-specific silencing techniques. Consistent with the high sensitivity of the SAGE technique to detect very low abundant transcripts (potentially encoding for regulatory factors), a large number of differentially expressed tags in FAC treated samples correspond to low copy number tags, and preliminary sequence alignment analysis has classified them as encoding for potential regulatory components (e.g., transcription factors, protein kinases, protein phosphatases, calcium binding proteins) (Fig. 1).

Genomic databases
In an ongoing project with the Max-Planck-Institute for Molecular Genetics the whole N. attenuata genome is being sequenced. A first draft version has been established and is available on our department´s internal BLAST server. In addition, the genome of the related species Nicotiana obtusifolia has been sequenced with lower coverage. The availability of both sequences will allow studies on the evolution of ecologically relevant traits in the genus Nicotiana.

Transcriptomic databases
The sequences of the whole transcriptomes of N. attenuata and of the related species N. obtusifolia are also available. To establish normalized whole transcriptome sequence libraries, tissues from different organs or growth stages (lamina, midrips, stems, flowers, roots, seedlings) of one species (both uninduced and induced from different time points) was collected and mixed in equal fractions. In cooperation with vertis Biotechnologie AG (www.vertis-biotech.com) mRNA was isolated, normalized and sequenced using the 454 technology.
mRNA from different tissues of N. attenuata collected at different time points after elicitations, was isolated, labeled and sequenced using Illumina technology.  From these data the nearly complete transcriptome of N. attenuata was assembled de novo. This project also included sequencing of induced and uninduced leaf samples of an additional N. attenuata ecotype.  Sequence comparisons between both ecotypes will give insight in intraspecific genome variations and adaptation. All next generations sequencing transcriptome assemblies can be analyzed on our group internal BLAST server.

Microarrays
The 43,533 N. attenuata contigs from 454 sequencing with the best coverage were used to design 60mer oligonucleotides and to create a 44k microarray design for the Agilent Technologies microarray platform (http://www.genomics.agilent.com/). In cooperation with the department of Entomology we use at present slides with this microarray for the study of gene regulation. In the near future an improved Agilent 60k array covering the complete N. attenuata transcriptome as assembled from the latest Illumina sequencing data will be available for transcriptional studies. Moreover, different microarrays for the study of gene regulation in N. attenuata and S. nigrum have been developed and produced in our group. We extensively used an array consisting of 1404 different 50mer oligos spotted in 4 replica (5616 spots) on epoxy coated glass slides. Spotting is performed on a VersArray Chip Writer Compact System (Fig. 2). Sample preparation, hybridization and gaining of microarray raw data on a GMS 418 Array Scanner (Fig.3) are performed in our department as well. Statistical tools for the evaluation of the microarray data have been developed. A database with the results from hundreds of hybridizations from our ecological experiments is available.

qPCR
Quantitative PCR can be performed on an Stratagene MX3005P cycler (Fig. 4)  For transcriptional studies, primers and probes for many ecologically important genes are available.

Stable Transformation System
Since N. attenuata is a native species, the standard transformation approaches developed for cultivated tobacco could not be applied. A new Agrobacterium based transformation procedure with new transformation vectors optimized for it was developed (See transformation facility). This transformation system was successfully adapted to S. nigrum. We constructed around 200 binary plant transformation vectors each containing an inverted repeat sequence of a gene to be silenced and around 35 vectors for the ectopic expression of full length genes. Our latest cloning vectors for these types of constructs are the pRESC8/pRESC9 plasmids (Fig. 5) with a hygromycin plant selectable marker and with the nptII gene as bacterial selectable marker. More than 300 different transgenic plant lines have been generated and we continue to produce new lines at about 50 per year. Current efforts are directed toward the development of the highly sensitive LhGR/DEX – pOp6-promoter inducible system. The implementation of this system into our N. attenuata transformation system will allow us to ask completely new research questions.

VIGS (Virus induce Gene Silencing)
A virus-induced gene silencing system (VIGS) based on a tobacco rattle virus containing vector has been adapted for N. attenuata (Saedler and Baldwin, 2004) and S. nigrum (Hartl et al., 2008) which allows the rapid silencing of endogenous genes (Fig. 6) in plants in a matter of weeks. Currently 250 different constructs for VIGS are available in the department.

Genotyping
Primer pairs amplifying N. tabacum microsatellite markers (Bindler et al. TAG, 2007) were screened with N. attenuata gDNA from different populations for amplification of polymorphic loci. By using fluorescent-labeled primers, size determination of the identified PCR fragments (Fig. 7) on an ABI 3100 sequencer (Fig. 8) confirmed that 6 primer pairs are suitable for genotyping. These will allow us to genotype individual N. attenuata plants that we work with in natural habitats. The initial plant material grinding for the HTP gDNA isolation procedure is performed in a SPEX SamplePrep 2000 Geno/Grinder® (Fig. 9).

Molecular Biology Standard Lab equipment
Our lab is well equipped with standard molecular biology instrumentation. Here's an overview of the equipment.

Modification and Preparation of DNA and RNA:

  • 6 PCR-Machines (Polymerase chain reaction)
  • 8 Incubators
  • 1 Concentrator
  • 6 Micro-Centrifuges
  • 3 Table-Centrifuges (refrigerated)
  • 1 Gel Documentation System

Cultivation of Bacteria:

  • 3 Table Shaking Incubator
  • 1 Horizontal Shaking Incubator

Analysis of the DNA and RNA:

  • 1 Aligent 2100 Bioanalyzer (Fig.10)
  • 1 Tecan Infinite 200
  • 1 Tecan Microplate Reader
  • 2 Photometer
  • 1 ABI Genetic Analyzer 3100
  • 1 Isotope Imaging System (Phosphoimager)
  • 1 Stratagene qPCR Machine Mx3005P
  • 1 ABI Sequence Detector System SDS 7700
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