Isolation of genes associated with adventitious root development in Populus

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Title: Isolation of genes associated with adventitious root development in Populus
Author: Wu, Qian
Advisors: Barry Goldfarb, Committee Chair
Abstract: Twenty-three gene and enhancer trap transformed poplar lines had been identified for study based on GUS expression during adventitious root formation. Histological analysis was conducted on wild-type stem cutting bases to determine the important stages of root initiation and development. Cell divisions were first observed two days after cutting preparation, leading to meristem organization in Day 4. In Day 5, the new root primordia emerged from the stem and root hairs formed on the new root in Day 6. We used TAIL-PCR to isolate genomic sequences flanking the insert in fourteen trap lines. The sequenced flanking regions were aligned with the genome sequence in the poplar genome database to find predicted open-reading frames (ORFs) in close proximity to the inserts. Two lines yielded putative ORFs—line 284 that contained one insert and line 304 that contained two inserts at separate loci. cDNAs of all three predicted ORFs were recovered. TR-284 has one coding region which encods a predicted 231-amino acid polypeptide. Searches of the GenBank database showed similarity to putative, unknown proteins from Arabidopsis and rice. Northern analysis of wild-type poplars detected expression in roots and leaves, but not in stems. Quantitative Real-Time PCR analysis of stem bases undergoing root formation showed transcript accumulation four days after cutting preparation. Hand sectioning of histochemically stained line 284 cutting bases also showed GUS protein accumulation in day 4, primarily in the cortical regions surrounding the emerging root primordium and in the new root cap. One ORF in line 304 (ORF-304-A) is predicted to encode a 285-amino acid polypeptide, with one exon containing a conserved domain (DUF296). The putative domain is found in proteins that contain AT-hook motifs, suggesting a DNA-binding function for the protein. ORF-304-A mRNA was detected in roots, but not in stems or leaves, using northern analysis. Quantitative Real-Time PCR of stem cutting bases showed transcript accumulation beginning four days after cutting preparation. ORF-304-B is predicted to code for a transcript with four exons. Three different transcripts were recovered by RACE, apparently the result of differential splicing in the third and fourth exons. The conserved region of the ORF was expressed in roots, leaves and stems and constantly from Day 0 through Day 6 in stem bases, with no apparent association with root formation. Hand sectioning of histochemically stained line 304 revealed GUS activity beginning in Day 2 in the stem cortex and in Day 4 in the organized root meristem and the cortex surrounding it. In the emerging root primordium and elongated root, GUS activity was mainly observed in the root cortex, with some staining in the root cap and the distal portion of the root meristem. In situ hybridization and transgenic experiments with these genes and further study of other lines are underway to further identify the role of gene expression during adventitious root formation and development.
Date: 2004-06-29
Degree: MS
Discipline: Forestry

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