USING LASER CAPTURE MICRODISSECTION (LCM) TO EXAMINE MADS-BOX GENE EXPRESSION IN THE UPPER AND LOWER FLORAL MERISTEMS OF MAIZE

ECU Author/Contributor (non-ECU co-authors, if there are any, appear on document)

Kate Adriana Nukunya (Creator)

Institution

East Carolina University (ECU )

Web Site: http://www.ecu.edu/lib/

Abstract: MADS-box transcription factors are important regulators of flower development in all flowering plants. In the grasses, flowers (called florets) are contained in spikelets. Maize spikelets contain two florets (the upper and lower florets) that are morphologically identical, although development of the lower floret is delayed compared to the upper floret. Floral meristems are groups of undifferentiated cells that give rise to floral organs. bearded-ear (bde) encodes a MADS-box transcription factor required for multiple aspects of floral development. bde mutants affect the upper and lower florets differently, suggesting the gene regulatory network in the upper and lower floral meristems are different. In addition, two other MADS-box transcription factors (zmm8 and zmm14), are expressed only in the upper floral meristem (UFM), but not in the lower floral meristem (LFM). Together, these data suggest that the gene regulatory networks in the UFM and LFM are distinct and some genes, including MADS-box genes are differentially expressed. The long-term goal of this research is to globally identify genes specifically expressed in the UFM and LFM. Floral meristems cannot be manually dissected, so we are using laser capture microdissection (LCM) to specifically isolate UFM and LFM. LCM allows specific cells to be isolated from fixed, sectioned tissue using a laser. This tissue can then be used for downstream applications, including RNA isolation. The goal of this project is to isolate UFM and LFM using LCM and test for the expression of maize MADS-box transcription factors using RT-PCR and qPCR. I have optimized fixation and RNA isolation protocols for LCM, and we have isolated RNA from sectioned tissue. In addition, we have successfully isolated UFM and LFM from sectioned tissue using LCM, and extracted RNA for amplification. We have tested for the expression of several control genes using quantitative RT-PCR (qRT-PCR). zmm8 and zmm14, which were initially thought to be expressed exclusively in the UFM, were observed in the LFM albeit at much lower levels. Other spikelet meristem genes like ids1 and bd1 were also expressed in the UFM and LFM. However, ra1 and ra2, which are expressed in the SPM and in the anlagen of the SM were not observed in the mixed meristems (MM) containing a mixture floral meristems and spikelet meristems or the UFM and LFM. Pepcase1 and zmTIP2-3, which are expressed in the leaves and roots respectively were not observed in any floral meristems. qRT-PCR results showed that expression of zmm8 and zmm14 in the LFM is much lower than in the UFM. zmMADS3 was observed in both UFM and LFM but there was no significant difference in levels of expression in the two floral meristems. Together, these data suggests differential gene expression in the UFM and LFM may be studied by looking at the expression level of genes in the two floral meristems and not simply by looking at the absence or presence of a gene.

Additional Information

Publication

Thesis

Language: English

Date: 2023

Subjects

Biology;Bearded-ear (bde);Differential gene expression;Floral meristems;Flower development;Laser capture microdissection (LCM);MADS-box transcription factors

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