## LSU Historical Dissertations and Theses

1994

Dissertation

#### Degree Name

Doctor of Philosophy (PhD)

#### Department

Plant Pathology and Crop Physiology

Norimoto Murai

#### Abstract

In many dicotyledons, the nutrient reserves are stored in the cotyledons. Phaseolin, 7S globulin, is the major seed protein from common bean Phaseolus vulgaris. In vitro transcription assays and gene transfer studies indicate that phaseolin gene expression is regulated primarily at the transcriptional level. In this dissertation, I studied cis- and trans-acting factors that may play a role in the transcriptional regulation of the $\beta$-phaseolin gene. Gel mobility shift and exonuclease III protection assays identified four distinct DNA binding proteins, CAN, AG-1, CA-1, and TATA-box binding protein. Three CANNTG motifs, CACGTG ($-$248/$-$243), CACCTG ($-$163/$-$158), and CATATG ($-$100/$-$95), were found to be preferred target sequences of CAN. The cis-activities of CAN and AG-1 binding sites were studied systematically by substitution mutations. The results indicate that the CACGTG (G-box) motif a major positive cis-element and acts synergistically with the CACCTG motif. The results also show that AG-I binding sites function as major positive ($-$191/$-$182) and negative ($-$376/$-$367,$-$356/$-$347) cis-elements. These results led me to hypothesize that CAN and AG-1 play a major role in the transcriptional regulation of the $\beta$-phaseolin gene. As a first step to understand the molecular nature of CAN, a bean seed cDNA library was constructed and screened for proteins capable of binding to oligonucleotides containjng the phaseolin G-box. Three positive clones were identified and further studied. DNA sequencing analyses indicate that the three cDNAs encode two homologous proteins that belong to the basic region/helix-loop-llelix (bHLH) protein family. Gel mobility shift assays with the proteins, PG1 and PG2, expressed in E. coli indicate that the two bHLH proteins preferentially bind to the G-box among the three phaseolin E-boxes. Northern blot analysis showed that PG1 is expressed constitutively in the plant and that PG2 is expressed primarily in the root. Based on these and other results, the transcriptional regulation of the $\beta$-phaseolin gene is discussed in relationship to the embryogenesis of common bean.

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