Plant reglatory sequences for selective control of gene expression
A technology of transgenic plants and plants, applied in the fields of plant peptides, plant products, genetic engineering, etc., can solve problems such as uselessness
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Embodiment 1
[0135] Maize embryos 13 days after pollination (13-DAP) (SATMON033 library) or corn germs 21 days after pollination (21-DAP) (SATMON017 library) were used as embryo-13-DAP or embryo-21-DAP cDNA libraries, respectively. source material. These libraries were generated from maize (DK604, Dekalb Genetics, Dekalb, Illinois U.S.A.). Seeds were planted to a depth of approximately 3 cm in soil in 2"-3" pots containing Metro 200 growth medium and transplanted 2-3 weeks later into larger 10" pots containing the same soil. After transplanting Peters 15-16-17 fertilizer was used 3 times a week at 150ppm for a total of 2-3 times during the life of the plant from transplant to flowering. A total of approximately 900mg iron was added to each pot for a total of 2-3 times during the life of the plant from transplant to flowering 2 to 3 additions. Corn plants were grown in the greenhouse on a 15 hr day / 9 hr night cycle. Day temperature was about 80°F and night temperature was about 70°F. Suppl...
Embodiment 2
[0143]Promoters were identified from a database of EST sequences derived from cDNA libraries prepared from various maize tissues including embryonic and embryogenic tissue-enhanced or callus-enhanced mRNA. This sequence was also used as a query sequence to GenBank containing the previously identified and annotated sequences and the BLAST program was used to search for regions of homology. The choice of expressed sequence tags (ESTs) for subsequent promoter isolation reflects the presence of one or more sequences in representative ESTs randomly sampled from an individual cDNA library or collection of cDNA libraries. To identify regulatory sequences that regulate the expression of transcribed sequences in maize embryos, a subsetting function was run requiring that all ESTs were found in the target embryo library and that other non-target EST libraries in the database were absent or present in low abundance. The resulting candidate ESTs were subjected to an electronic northern fu...
Embodiment 3
[0163] From maize DNA (obtained from maize hybrid Fr27xFrMo 17) isolated using the CsCl purification protocol according to Ausubel et al., 1992, or by the CTAB purification method (Rogers and Bendich, Plant Mol. Biol., 5:69 (1985) Genomic library preparation. Reagents are available (eg, Sigma Chemical Co., St.Louis, MO). Libraries are prepared according to the manufacturer's instructions (GENOME WALKER, a trademark of CLONTECH Laboratories, Inc, Palo Alto, CA). In the reaction, the DNA was subjected to restriction enzyme digestion overnight at 37°C with the following blunt endonucleases: EcoRV, Scal, Dral, PvuII, or StuI (CLONThCH Laboratories, Inc. Palo Alto, CA). With phenol: The reaction mixture was extracted with chloroform, precipitated with ethanol, and resuspended in Tris-EDTA buffer. The purified blunt-ended genomic fragments were then ligated into GenomeWalker TM Ligation of adapters, ligation of the resulting DNA fragments to the adapters was accomplished according t...
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