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- Author or Editor: A. Callahan x
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During the past several years we have been involved in identifying seasonally regulated proteins and genes from peach bark. In the present study, we describe the cloning of a protease inhibitor from a cDNA library made from winter bark tissues. A partial clone obtained from the library was extended to full length by 5' RACE. The full-length cDNA clone (final3b) is 613 bp in length, not including the poly A+ tail. The open reading frame of 237 bp codes for a 79 amino acid protease inhibitor related to the defensin family of proteins. This family of small, cysteine-rich, extracellular proteins play a role in the plantís defense response through their antifungal properties. Sequence comparison of the encoded protein using BLAST analysis revealed significant homology to protease inhibitors from Glycine max, Arabidopsis thaliana, and a defensin protein from bell pepper (Capsicum annuum). Similar to these other cysteine-rich proteins, the peach defensin contains a consensus cys arrangement and is predicted to have an amino terminal signal peptide, presumably targeting it for extracellular transport. RNA-blot analysis indicated that the gene is seasonally expressed in bark tissues of 1-year-old shoots. Transcript abundance of final3b increased in the fall, reached a peak in midwinter and then decreased. The gene was also expressed during early stages of fruit development. RNA-blot analysis of the gene in other tissues, and in response to environmental stress and wounding, is in progress.
Flow cytometric analysis, of leaf nuclei from three cultivars, was used to estimate the DNA content of peach (∼0.61 pg or ∼0.59 × 109 bp/diploid nucleus; 2x=16), and ndicated that the peach genome is only slightly larger than that of Arabidopsis. This value was indirectly confirmed by measurements of nuclei from haploid, triploid and “tetraploid” (cytochimera) peach accessions. cDNA and genomic clones have been used to determine the level of polymorphism among various peach cultivars and related species. Overall, ∼33% of the clones detected polymorphic loci. As expected, the highest level of polymorphism was found in interspecific hybrids (∼50%); whereas in intraspecific populations, only 1 in 5 genomic clones, and 1 in 3 cDNA clones were able to detect polymorphisms (RFLPs). These clones, as well as RAPD primers, are being used to construct a genetic linkage map by analyzing their segregation in 3 intraspecific peach populations (an Fl from France and two F2s from the U.S.). Taken together, these populations are segregating for 12 Mendelian traits and a number of quantitative traits. Our results have enabled us to identify a number of linkage groups, some composed of both molecular and phenotypic markers. The current structure of the peach map is reported.
We are interested in identifying and isolating genes which affect the rate of softening in peach fruit. It may be possible through the engineering of these genes to delay or extend the softening. This could ultimately allow for the harvest and transport of more mature, higher quality fruit. The clone, pch313, was isolated from a ripe peach fruit cDNA library. RNA homologous to this clone is detected at a low abundance in fruit until softening when a >100 fold increase in abundance of the RNA occurs. Pch313 RNA is also detected 30 min after wounding leaf or fruit tissue and peaks in accumulation within 2-8 hours. Wound ethylene was measured from the same tissue and its rate of evolution paralleled the accumulation of the RNA. The cDNA was sequenced and found to have 78% sequence identity with pTom13, a tomato gene that is expressed during fruit ripening and wounding (Holdsworth et al., NAR 15:731-739, 1987). To determine the universality of pch313 related gene expression, RNA accumulation was measured in other fruits during softening, and in leaf tissue upon wounding.
We are interested in identifying and isolating genes which affect the rate of softening in peach fruit. It may be possible through the engineering of these genes to delay or extend the softening. This could ultimately allow for the harvest and transport of more mature, higher quality fruit. The clone, pch313, was isolated from a ripe peach fruit cDNA library. RNA homologous to this clone is detected at a low abundance in fruit until softening when a >100 fold increase in abundance of the RNA occurs. Pch313 RNA is also detected 30 min after wounding leaf or fruit tissue and peaks in accumulation within 2-8 hours. Wound ethylene was measured from the same tissue and its rate of evolution paralleled the accumulation of the RNA. The cDNA was sequenced and found to have 78% sequence identity with pTom13, a tomato gene that is expressed during fruit ripening and wounding (Holdsworth et al., NAR 15:731-739, 1987). To determine the universality of pch313 related gene expression, RNA accumulation was measured in other fruits during softening, and in leaf tissue upon wounding.
Peach [Prunus persica (L.) Batsch.] is considered the best genetically characterized species of the genus Prunus. We therefore used it as a model in our study of the genome organization in Prunus by means of restriction fragment length polymorphisms (RPLPs). Initial results indicated that 60% of cloned DNA sequences examined occur at low copy number within the peach genome. After selecting and examining these sequences, polymorphisms sufficient for RPLP mapping were found. We determined that ≫33% of our cDNA clones and 20% of our genomic clones detected RPLPs among peach cultivars. Analysis of RPLP segregation in two families, both of which segregate for known morphological characters, revealed segregation in 12 RFLP markers for one family and 16 for the other. Although we have not detected linkage between RFLP and morphological markers, preliminary analyses indicate possible linkage between two RPLP markers.
Plants transgenic for potyvirus coat protein (cp) genes have been shown to be resistant to viruses homologous and heterologous to the cp source virus. We have produced plum plants transgenic for the papaya ringspot virus (PRSV) cp gene. PRSV is a potyvirus related to plum pox virus (PPV). PRSVcp transgenic plants have been inoculated with PPV under containment conditions at the USDA Foreign Diseases-Weed Science Research Facility, Frederick, MD, and evaluated for two years. At least one plant is apparently resistant or tolerant to PPV based on symptomology, ELISA and RT-PCR assays. This suggests the potential utility of cp-mediated virus protection in tree fruits. To further test this potential, both short and long-term studies are in progress to evaluate resistance and cp expression in various organs, throughout the year and over the commercial life of individual trees. Plum plants have also been transformed with the PPVcp gene. Studies are underway to evaluate the protection derived from this cp gene.