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Team:Cambridge-JIC/Marchantia/Promoter
From 2014.igem.org
One of the key aims for our project is to introduce Marchantia polymorpha to iGEM with a toolset that enables future teams to develop it further and capitalise on its benefits. So given the limited knowledge about its genetic makeup at present, we have sought to find possible inducible promoters in Marchantia that could be used for parts.
Method
To start off, we hypothesised that inducible promoters would be associated with responses to environmental cues or pressures and that, given their importance, related genetic motifs would be largely conserved in the evolution of land plants. So as Marchantia polymorpha is an early land plant[1], we thought it was likely that many homologues to its genes could still be found in later plants and that these genes could be used to find promoters.
We reviewed research papers to create a shortlist of inducible plant promoters for which we might find homologues in the marchantia genome. We narrowed our search to promoters regulated under limiting supply of nutrients nitrates, sulphates, phosphates (although light variation, circadian rhythm, metabolism and development related inducers were also considered initially). For the majority of our analyses, we selected Arabidopsis thaliana as a model organism from which to identify target genes given the quality of genetic information that is available for the plant. However, we also used genes from the following organisms were data was available: B. nigra, L. esculentum, B. napus, C. reinhardtii, G. max, N. plumbaginifolia, P. patens.
We identified a shortlist of 27 genes that might be regulated under limiting supply of the essential nutrients nitrates, sulphates, and phosphates.
We obtained the peptide sequences for these genes from the following online databases:
Thalemine - https://apps.araport.org/thalemine/begin.do [2]
GenBank - http://www.ncbi.nlm.nih.gov/genbank [3]
TAIR - http://www.arabidopsis.org/ [4]
UniProt - http://www.uniprot.org/ [5]
We used Geneious™ to run tblastn[6] and query the protein coding sequences against the nucleotide sequences of the m. polymorpha scaffolds. Our dataset was made of large gap read mapping transcripts obtained by mRNA sequencing conducted by the Haseloff Lab on the m. polymorpha Cam strain. Open Reading Frame (ORF) and Coding Sequence (CDS) predictions were made using the CLC bio Transcript Discovery plugin[7].
We selected the most convincing hits as those with a grading above 30% and got a shortlist by ranking them based on concurrence with an existing gene prediction as this made the selections more reliable. We isolated possible promoter regions as those 2kbp upstream from the start of the purported gene.
Hits for very short regions of homology were not selected. This generally corresponded to hits shorter than 5% of the sequence length of the predicted gene, although slightly shorter hits were noted as support for the reliability of a good match.
Results and Discussion
Out of the 27 genes that we shortlisted, we were able to obtain products from a PCR off extracted M. polymorpha DNA with a success rates of 50% - 70% on successive runs. We ran out of time to satisfactorily complete this stage and debug our difficulties with Gibson Assembly, but we think that we have good evidence that these candidates warrant further investigation.
Notes:
• In Figures 2 and 3 the predicted gene numbers are grouped according to the related essential nutrient: genes 603 – 11107 inclusive relate to nitrates; genes 3555 and 3556 relate to sulphates and 170-11169 inclusive relate to phosphates.
• The percentage overlaps in Figure 3 measure how much of a gene sequence length was covered by the hit sequence length, and how much of the hit sequence length was covered by an mRNA sequence length in a 1 -to-1 mapping between nucleotides.
The number of Open Reading Frames (ORFs) resulting from our initial predictions totalled 99 000, which seemed too large to be realistic, and half of these were only 100 amino acids (100 aa) long. By filtering the dataset using a threshold of 300 aa for candidate genes we obtained a distribution of lengths that seemed reasonable.
In the blastx output, the longest complete sequence match was 40%. This is small as expected given the phylogenetic distance between m.polymorpha and a. thaliana [3][4].
The Codon Usage table we obtained for m. polymorpha is not strikingly similar to that of a. thaliana, as expected from the 400 million years of evolutionary divergence between them [5].
However, there is a similarity in the slight preference for C over other bases at the end of codons and that for G-p-C sites, as can be seen in the codon table.
References
1. Wellman CH, Osterloff PL, Mohiuddin U. 2003. Fragments of the earliest land plants. Nature 425, pp. 282-285. back to top
2. Thalemine - https://apps.araport.org/thalemine/begin.do [Accessed: July – September 2014]back to top
3. GenBank - http://www.ncbi.nlm.nih.gov/genbank [Accessed: July – September 2014]back to top
4. TAIR - http://www.arabidopsis.org/ [Accessed: July – September 2014]back to top
5. UniProt - http://www.uniprot.org/ [Accessed: July – September 2014]back to top
6. NCBI Blast ®, http://blast.ncbi.nlm.nih.gov/Blast.cgi back to top
7. Qiagen, CLC bio Transcript Discovery ®, http://www.clcbio.com/clc-plugin/transcript-discovery/#description back to top
