This report identifies S-RNases of sweet cherry (Prunus avium L.) and presents information about cDNA sequences encoding the S-RNases, which leads to the development of a molecular typing system for S-alleles in this fruit tree species. Stylar proteins of sweet cherry were surveyed by two dimensional polyaclylamide gel electrophoresis (2D-PAGE) to identify S-proteins associated with gametophytic self-incompatibility. Glycoprotein spots linked to S-alleles were found in a group of proteins which had Mr and pI similar to those of other rosaceous S-RNases. These glycoproteins were present at highest concentration in the upper segment of the mature style and shared immunological characteristics and N-terminal sequences with those of S-RNases of other plant species. cDNAs encoding these glycoproteins were cloned based on the N-terminal sequences. Genomic DNA and RNA blot analyses and deduced amino acid sequences indicated that the cDNAs encode S-RNases; thus the S-proteins identified by 2D-PAGE are S-RNases. Although S1 to S6 -alleles of sweet cherry cultivars could be distinguished from each other with the genomic DNA blot analysis, a much simpler method of PCR-based typing system was developed for the six S-alleles based on the DNA sequence data obtained from the cDNAs encoding S-RNases.
Ryutaro Tao, Hisayo Yamane, Akira Sugiura, Hideki Murayama, Hidenori Sassa, and Hitoshi Mori
M. Wisniewski, T. Artlip, R. Webb, C. Bassett, and A. Callahan
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.
Tomomi Tsuda, Masami Yamaguchi, Chikako Honda, and Takaya Moriguchi
We used RNA blot analysis to examine the expression of six genes of the anthocyanin biosynthesis pathway in the flowers and fruit skins at three developmental stages of white and red peaches and a deep-red nectarine [Prunus persica (L.) Batch]. In the red peach `Akatsuki' and the deep-red nectarine `Flavortop', expression levels of anthocyanin biosynthesis genes were related to anthocyanin accumulation in the fruit skin; expression of all six genes dramatically increased at Stage III of fruit development, and anthocyanin concentration also increased at this stage. In the white peach `Mochizuki', however, expression of the chalcone synthase gene (CHS) and the dihydroflavonol 4-reductase gene (DFR) was undetectable in Stage III, although the chalcone isomerase gene (CHI), the flavanone 3-hydroxylase gene (F3H), the anthocyanidin synthase gene (ANS), and the UDP-glucose-flavonoid 3-O-glucosyltransferase gene (UFGT) were expressed. We occasionally found red pigment in the skin of `Mochizuki' peach. In these red skin areas, both CHS and DFR were clearly expressed in Stage III. These results suggest that CHS and DFR are the key regulatory genes in the process of anthocyanin biosynthesis in mature red peach and nectarine.
Hassan Salehi, Zahra Seddighi, Alexandra N. Kravchenko, and Mariam B. Sticklen
Bermudagrass (Cynodon L.C. Rich.) is grown on more than 4 million ha in the southern United States. The black cutworm (Agrotis ipsilon Hufnagel) is the most commonly encountered pest of bermudagrass, especially on golf course greens. Developing insect-resistant cultivars is a very desirable substitute, both environmentally and economically, to using current synthetic pesticides. Here we report, for the first time, Agrobacterium-mediated transformation of `Arizona Common' common bermudagrass [Cynodon dactylon (L.) Pers.] with the Bacillus thuringiensis Berliner cry1Ac gene encoding an endotoxin active against black cutworm. Mature seeds were used for producing embryogenic callus, and calli were transformed with a plasmid containing a synthetic cry1Ac and the kanamycin resistance (nptII) genes. Putative transgenic calli and plantlets were selected on media containing 100 and 50 mg·L-1 G418, respectively. RNA-blot analysis of PCR-positive lines revealed the expression of the cry1Ac transgene in three out of five putative transgenic lines. The larvae fed on transgenic plant leaves experienced highly significant (over 80%) mortality.
Xiyan Yu, Xiufeng Wang, Jide Fan, Hongmei Tian, and Chengchao Zheng
detected in the leaves, stems, and mature fruit, but cannot be detected at all in roots and flowers ( Fig. 2 ), suggesting that CmSPS1 may play a role in leaf, stem, and fruit development in muskmelon. Fig. 2. RNA blot analysis of CmSPS1 spatial
Hisayo Yamane, Yukinobu Kashiwa, Tomomi Ooka, Ryutaro Tao, and Keizo Yonemori
. In total, 15 to 18 buds, located in the top portions of the branch parts, were counted. RNA blot analysis. The seasonal expression pattern of the clone obtained from the SSH/MOS library was investigated by RNA blot analysis. Total RNA was
Elysia K. Krieger, Edwards Allen, Larry A. Gilbertson, James K. Roberts, William Hiatt, and Rick A. Sanders
ripe transgenic (4523, 3951, 3949) tomatoes and not in null (4193, 4131, 3975) sibling tomatoes. ( B ) RNA blot analysis of 21- to 24-nt siRNAs associated with PG and NptII coding regions. 5S rRNA/tRNA bands and miR159 are shown as loading controls
Satoru Kondo, Hiroko Yamada, and Sutthiwal Setha
cyclopropane-1-carboxylic acid (MACC) concentrations, ACC oxidase activity, and ethylene production from ‘La France’ pear fruit skin at the preclimacteric stage. For RNA blot analysis, total RNAs (20 μg) were hybridized with ACS1 , ACS3 , ACS4 , and ACO1
He Lisi, Su Jiale, Liu Xiaoqing, Li Chang, and Chen Shangping
officinalis ) stored in air ( Hurst et al., 1997 ). Two broccoli ( Brassica oleracea var. italica ) acid invertase cDNAs were cloned and RNA blot analysis showed that their transcripts accumulate during senescence of broccoli florets ( Coupe et al., 2003