Doctoral Dissertations

Paul G. Twigg

Date of Award

12-1993

Degree Type

Dissertation

Degree Name

Doctor of Philosophy

Major

Botany

Major Professor

Beth C. Mullin

Committee Members

Peter M. Gresshoff, Leslie G. Hickok, Stuart Riggsby

Abstract

For some time, gene expression specific to the symbiotic nodules of legume species has been known. Until the present, similar studies of the symbiotic nodules of actinorhizal plants have been unsuccessful, due mainly to difficulties in extraction of intact and biologically active mRNA. The purpose of this study was to develop a reliable protocol for the extraction of intact and biologically active RNA from the roots and nodules of Alnus glutinosa, and subsequently to identify one or more cDNA clones encoding nodule-specific proteins.

In the course of this study, a dependable RNA extraction protocol was developed, from existing protocols, and cDNA libraries were prepared from poly(A) RNA of alder root and nodule tissue. A library subtraction technique was employed to generate a library enriched in nodule-specific, or enhanced sequences. The nodule-enriched cDNA library was screened with a labelled subtracted probe, prepared from nodule poly(A) RNA, to identify putative nodule-specific clones. In total, 197 areas hybridizing to the subtracted probe on colony lift blots of the nodule-enriched library were isolated. Plasmid DNA preparations from the bacteria isolated from these areas were used to prepare slot blots. The slot blots of the putative nodule-specific cDNA plasmids were screened individually with labelled cDNA generated from poly(A) RNA of alder roots and nodules. From this screen, two possible nodule-specific cDNA plasmids were identified. These clones were hybridized to Northern blots of LiCl-selected RNA from alder nodules, roots, and leaves. From this analysis, one of the clones, pAgNt84, was identified as nodule-specific, strongly hybridizing to alder nodule RNA with no detectable hybridization to RNA from other tissues. Subsequent Southern blot analysis confirmed that the pAgNt84 cDNA was of host plant origin.

Following sequence analysis of the pAgNt84 cDNA, it was found that the clone was composed of 681 bp, correlating well with the size of the transcript identified from Northern blot analysis. The cDNA was found to encode a putative open reading frame of 98 amino acids. The derived amino acid iv sequence of this open reading frame was found to be high in glycine and histidine, at 15.3% each. This characteristic indicated a possible relationship of this sequence to those of a plant glycine-rich protein family. Aligiunent of the putative pAgNt84-encoded amino acid sequence with several members of the plant glycine-rich protein family revealed the conserved nature of the N-terminal sequences purported to encode a signal peptide. This view of the N-terminal region as a signal peptide was supported by the characteristic hydrophobic nature revealed by secondary structure and hydrophobicity ancilysis of the pAgNt84 cDNA-derived amino acid sequence.

Nucleic acid comparisons revealed an interesting relationship of the pAgNt84 cDNA to a cDNA encoding a glycine-rich protein from Medicago sativa, which lacks a signal peptide. The similarity of these sequences begins only after the region encoding the putative signal peptide in the pAgNt84 cDNA ends. This fact indicates a common evolutionary origin, but with one sequence gaining or more likely the other losing the signal peptide encoding region.

Based upon the similarity of the putative signal peptide with that of the members of the plant glycine-rich protein family, a possible localization and function of the pAgNt84 cDNA-encoded protein product is proposed.

Download
Files over 3MB may be slow to open. For best results, right-click and select "save as..."

Share

COinS