Regulation of floury2-mRFP expression in response to long term selection for grain protein concentration in maize

Lucas, Christine J.

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https://hdl.handle.net/2142/18449 Copy

Description

Title

Regulation of floury2-mRFP expression in response to long term selection for grain protein concentration in maize

Author(s)

Lucas, Christine J.

Issue Date

2011-01-14T22:51:16Z

Director of Research (if dissertation) or Advisor (if thesis)

Moose, Stephen P.

Department of Study

Crop Sciences

Discipline

Crop Sciences

Degree Granting Institution

University of Illinois at Urbana-Champaign

Degree Name

M.S.

Degree Level

Thesis

Keyword(s)

• maize
• floury2
• selection
• Grain
• protein
• floury2-monomeric red fluorescent protein (mRFP)
• red fluorescent protein(RFP)
• genetics
• Illinois Long Term Selection Experiment

Abstract

The Illinois Long-term Selection Experiment is the longest running continuous genetics experiment in higher plants in the world. Seven hundred and twenty-one cycles of divergent selection for grain protein and oil concentration have produced populations with the known phenotypic extremes for this trait, and also illustrate the nature of responses to phenotypic selection. This report provides updates to the experiment, including the introduction of new chemical analytical procedures, assessment of methods for estimating genetic gain, and the recent initiation of reverse selection experiments from cycle 103 of Illinois High Protein (IHP). Prior genetic mapping studies suggest that the response to selection in this experiment is dependent upon the cumulative action of many genes with small phenotypic effects. An alternative theory explored here is that the response depends on quantitative expression variation of a few major regulators. Analysis of crosses between IHP and Illinois Reverse High Protein (IRHP) provides evidence that the response to selection in IRHP is due to one or a few loci with main effects, which is associated with biased frequencies for variant alleles of the ASPARAGINE SYNTHETASE3 gene. Long-term divergent selection for grain protein concentration in the Illinois Protein Strains has dramatically altered the accumulation of the 19-kD and 22-kD α-zeins. Known regulators of α-zein protein accumulation are Opaque2 (O2), the Prolamin-box Binding Factor (PBF), and factors influencing the folding of zeins into endosperm protein bodies. RNA analysis and measuring protein abundance are two effective approaches for studying the regulation of zein expression, but they are also expensive, destructive and laborious. Furthermore, study of individual zein genes is complicated by their high copy number and sequence similarity. An alternative inexpensive and nondestructive approach to investigate the regulation of zein expression is the use of transgenic Floury2-mRFP reporter lines (Dave Jackson’s lab at Cold Spring Harbor), where the expression of the readily visible monomeric red fluorescent protein (mRFP) is controlled by the genomic sequences encoding the Floury2 -zein. The FL2-mRFP transgene has been introgressed into inbred lines derived from the four Illinois Protein Strains (IPS), as well as the reference inbred B73. We found that FL2-mRFP expression not only correlates with grain protein concentration, but also follows known patterns of zein accumulation throughout development. At all developmental stages, RFP expression was strongest in Illinois High Protein (IHP), the lowest in Illinois Low Protein (ILP) and intermediate in Illinois Reverse High Protein (IRHP), Illinois Reverse Low Protein (IRLP), and B73. By crossing FL2-mRFP to an o2 mutant introgressed into IHP, we show that its expression is strongly activated by O2, illustrating that the FL2-mRFP transgene is regulated in the same manner as endogenous -zein genes. Future experiments will use the FL2-mRFP transgene as a tool for identifying regulators influencing protein concentration in ongoing genetic mapping studies.

Graduation Semester

2010-12

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http://hdl.handle.net/2142/18449

Copyright and License Information

Copyright 2010 Christine J. Lucas

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