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Previous published work has shown that in maize, the imprinted geneMaternally Ex-

pressed Gene1 (ZmMeg1) (GRMZM2G354335) is expressed exclusively in the Basal

Endosperm Transfer Layer (BETL) of the developing kernel (Gutierrez-Marcos et al., 2004). The gene product, MEG1 is a small CRP with a pivotal role in sugar partition- ing within the seed through regulation of BETL transfer cell development (Costaet al.

, 2012).

ZmMeg1 is a member of theMeg1family that forms a gene cluster on maize chromosome

7, identified by screening an endosperm cDNA library constructed from maize endo- sperm tissue at 7 DAP (Gutierrez-Marcoset al. , 2004; Hueroset al. , 1999a). Further analysis by screening subtracted cDNA libraries constructed from specific cell types, identified another gene on the periphery of theMeg1 cluster with homology at the pro- tein level, but differences in the promoter region (Leet al., 2005). From identified novel transcripts, homology searches highlighted an expressed sequence tag (EST) in the cent- ral cell, which has been termedMeg like protein1 (ZmMlp1) (GRMZM2G145466_T01). Previous chromosomal alignment work analysing maize chromosome 7 and 2, with sorghum chromosome 2, shows that while there have been many chromosomal rearrange- ments, there is a high level of cluster conservation (Figure 3.1). TheZmMeg1 family can be found on maize chromosome 7, but has been lost from maize chromosome 2. ZmMlp1

has been mapped to the end of the ZmMeg1 cluster and interestingly, is the only gene conserved in sorghum 2 (Figure 3.1). Further homology searches in the genomes of barley, wheat and rice found homologous genes forZmMlp1, but not forZmMeg1. This conservation pattern could be indicative of an ancestral origin for ZmMlp1, giving rise to the ZmMeg1 family through multiple gene duplication events in maize. Though

3 Discovery and Molecular Characterisation of the MEG-Like Protein 1 Gene pattern, gene product or function of ZmMlp1. As a first step towards identifying the function of ZmMlp1, the aim of this chapter is to characterise its expression in Zea

mays.

Maize 2

Sorghum 2

Maize 7

MLP1 MEG1 gene family

Maize 2

Sorghum 2

Maize 7

Meg1 gene family GRMZM2G145466_T01

Figure 3.1: Schematic diagram comparing maize chromosomes 7 and 2, with sorghum chromosome 2. Many chromosomal rearrangements are indicated, though a high level of cluster conservation remains. The Meg1 family can be found in maize 7, but has been lost from maize 2. GRMZM2G145466_T01 can be found at the end of theMeg1

cluster and is the only gene conserved in sorghum 2.

In order to characterise this novel gene, a number of different experimental techniques were employed. A GUS reporter system was used to spatially and temporally assess the activity of theZmMlp1 promoter in developing seeds. This is a classical technique that allows visualisation of promoter active tissues by the development of a blue pigment in the site of activity (Jefferson et al., 1987a). This technique is relatively simple to use, providing that stable transgenics can be generated, allowing the rapid processing of large numbers of samples in batch assays. Additionally, the analysis method is extremely simple, with visual screening achievable with the naked eye and imaging requiring only a light microscope. There are however, some disadvantages to this technique; the assay must be optimised in chemical composition and incubation time to avoid the pigment bleeding out into surrounding tissues. Furthermore, the sample is essentially fixed at the point of assay, a process that can cause tissue distortion. An alternative method

3 Discovery and Molecular Characterisation of the MEG-Like Protein 1 Gene could be to use a GFP reporter which would allow live imaging of promoter activity at a much higher resolution. However, the sample analysis techniques required for GFP imaging are a lot more laborious, requiring extensive confocal or epifluorescent microscopy. Luciferase is another reporter system that could have been used, though this does not give the optical resolution required for this analysis.

Expression of the gene itself was assessed through a combination semi-qPCR, qRT- PCR and in situ hybridisation. In situ hybridisations have the advantage of spatially assessing the gene expression at the cellular level, though the technique is extremely labour intensive and requires a great deal of care in sampling and fixation to ensure mRNA integrity. Semi q-PCR is a dilution based PCR technique that yields gel bands of varying intensity according to target sequence concentration, though quantitative analysis of gels can be unreliable. qRT-PCR is considered the gold-standard in expres- sion quantification, with unparalleled levels of sensitivity. However, great care must be taken with primer design, PCR condition optimisation and data analysis to ensure assay specificity and data reliability (Bustinet al. , 2009).

Protein localisation was carried out by both immunohistochemistry and GFP tagging approaches in order to gain the advantages and counter some of the disadvantages of both techniques. GFP labelling is an excellent technique for protein localisation stud- ies, producing high quality images through confocal microscopy. However, this requires expensive and time consuming generation of stable transgenic lines, relatively laborious analysis methods and potential issues with steric alteration of the target protein causing localisation abnormalities. Antibody labelling however is relatively quick, simple, high- throughput and can yield reasonable quality images when combined with high quality sample preparation. However, due to the nature of antibody-epitope interaction, there remains the possibility for cross-reaction with other proteins containing sequence sim- ilarities. The application of this technique to a novel protein requires the generation of

3 Discovery and Molecular Characterisation of the MEG-Like Protein 1 Gene a custom antibody and hence, acquisition of sufficient protein antigen for inoculation. To this aim, bacterial recombinant protein expression was employed, incorporating a His-tag for affinity purification. This system was chosen for its ease of use, high yield potential and low cost compared to the alternatives of yeast or plant based expression systems. However, the main disadvantage of using a bacterial expression system for a plant protein is the differences is protein folding machinery, leading to the potential for incorrectly folded product.

This chapter reports the combined use of these techniques to elucidate the expres- sion characteristics of a novel gene, ZmMlp1 and the biochemical characteristics of its product ZmMLP1 in the developing maize seed. This data is then considered in the context of the wider research area and used to drive functional based hypotheses and further research aims.

Results