| American chestnut (Castanea dentata) once dominated the forests of the eastern United States until introduction of Cryphonectria parasitica. This fungus enters through wounds on stems and invades the nearby vascular cambium to form lethal cankers. In order to genetically engineer this species so that transcription of introduced resistance genes is limited to the stems, promoters of stem-specific genes were cloned from American chestnut. As a first step, genes expressed predominantly in the stems were identified. Three cDNA libraries were created and analyzed. From two of these libraries, namely, a leaf and seed subtracted library and an unsubtracted stem cDNA library, 71 cDNA clones were sequenced and submitted to GenBank as ESTs (expressed sequence tags). Of twenty clones analyzed via RNA dot blot, thirteen were expressed predominantly in the stem and leaf tissues, while the other seven were constitutively expressed. Using reverse transcription-PCR transcripts of three selected genes (2/700, A9/ 800, 10A/700) were found in the stem and leaf tissues, and expression in the seed was more variable. Upstream regions of A9/800 (promoter ACS9A), 10A/ 700 (promoter ACS10A), and 2/700 (promoter ACS2) were amplified and subcloned upstream of a promoterless gus gene. Transformation of Arabidopsis thaliana with the recombinant plastids produced eight lines carrying promoter ACS2, three lines with promoter ACS9A, and six lines with promoter ACS10A. Vascular-predominant activity of promoter ACS2 was observed in all eight transgenic Arabidopsis lines, while the activities of the other two promoters were not as consistent. Additionally, a preliminary characterization of EST A19/700 (CASde:Pic1 ), a putative cystatin, was completed. Genomic fragments of CASde:Pic1 were obtained for Chinese and American chestnuts, and comparison of these sequences revealed significant differences within a putative intron. Restriction site analysis was performed using the enzyme MseI, and variations in the banding pattern were observed. Similar analysis using the amplified CASde:Pic1 from F1 and F2 hybrids of C. dentata × C. mollisima cross did not produce this polymorphic banding pattern. Using RT-PCR, cystatin transcript was found in healthy stem, leaf and seed tissues, as well as diseased tissues. The results presented here represent the first significant exploration into gene expression in Castanea dentata, and these data suggest that there are important biotechnological applications for these sequences in forest tree improvement. |
