Characterization of PME promoters and transposon Tag1 in tomato (Lycopersicon esculentum)
Abstract
Availability of fruit specific promoters is a serious limitation for genetic modification of fruits, including coffee where there is interest to modify attributes of economical value. With the goal to increase the number of fruit specific promoters, 5′-upstream regions from two tomato pectin methyl-esterase (PME) genes were characterized in homologous and heterologus species. To determine spatial and temporal expression of the LePME genes, chimeric constructs containing 2.7 kb and 0.4 kb DNA flanking LePME, and 0.8 kb DNA flanking LePME3 and GUS as reporter gene were made and used to create transgenic tomato and tobacco plants. Transient expression of GUS after particle bombardment of these chimeric genes showed that both PME promoters are capable of expression in tomato pericarp. GUS expression analysis from independent transgenic plants of tomato and tobacco demonstrated that both LePME promoters expressed GUS in both species. However, the 0.4 kb LePME2 5′-flanking region showed higher expression than the 2.7 kb 5′-flanking region in tomato, indicating presence of upstream negative regulatory element in this promoter. The LePME3-0.8 kb flanking region showed expression patterns similar to the LePME2-0.4 kb. In tobacco, expression patterns under these promoters were similar to that observed in tomato with much higher expression in fruits. In general, expression of PME promoters tested was higher than CaMV 35S promoter. Although detectable expression of LePME3 was not observed by Northern or RT-PCR analysis, my results clearly demonstrate ectopic expression under this promoter. Behavior of Tag1 in tomato was examined to isolate desirable fruit specific genes. Tag1-insertion mutants were created and progenies of 500 independent transgenic plants were characterized under the field conditions. Molecular characterization of segregating progenies shows that in Micro-Tom Tag1 excises at rate 8% but reinserts at the rate 2% in subsequent generations. However, genetic analysis of three Tag1 induced mutations, with alteration in plant size, fruit color and cold tolerance, failed to establish correlation between the presence of Tag1 and observed phenotype. Taken together these results suggest that Tag1 has a potential to induce mutations but more independent tagged plants are needed to isolate Tag1-insertion mutations.
Degree
Ph.D.
Advisors
Handa, Purdue University.
Subject Area
Genetics|Botany|Agronomy
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