Establishment of Cytosolic Small Heat Shock Protein Knockout Lines in Arabidopsis thaliana

Presenter: Haofeng Lin

Faculty Sponsor: Elizabeth Vierling

School: UMass Amherst

Research Area: Biochemistry and Molecular Biology

Session: Poster Session 5, 3:15 PM - 4:00 PM, Auditorium, A84

ABSTRACT

The goal of the project is to investigate the function of cytosolic small heat shock proteins (sHSPs) and their role in stress tolerance and normal development in plants. sHSPs are low molecular weight ATP-independent protein chaperones. They are highly expressed during elevated temperatures as they bind to denaturing proteins to prevent irreversible aggregation. They are also expressed during specific stage of seed development. In the model plant Arabidopsis thaliana, our studies focus on two types of sHSPs, Class I (CI) and Class II (CII). There is total of six Class I (Hsp17.4-I, 17.6A-I, 17.6B-I, 17.6C-I, 17.8-I, and 18.1-I) and two Class II (Hsp17.6-II, Hsp17.7-II) sHSPs in A. thaliana. Using CRISPR methodology, we have successfully generated knockout lines of all six CI sHSPs, including one double knockout line (17.6A-I and 17.6C-I), and we are combining these mutants by genetic crossing to study their mutant phenotypes. For CII, we generated double knock out mutants using an available17.6-II T-DNA mutant in which we used CRISPR to mutate 17.7-II, developing three double mutant alleles. Using one of the double CII mutant lines, we added back both CII genes, generating three independent complemented lines. Immunoblot analysis confirmed that the CII mutants and complemented lines show no CII sHSP expression or heat-induced sHSP expression, respectively. The CII sHSP mutants and complemented lines are now being tested for stress and developmental phenotypes.

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