Functional plant, promoter used for producing the functional plant and its application method
A technology for promoters and plants, applied in the fields of botanical equipment and methods, angiosperms/flowering plants, biochemical equipment and methods, etc., can solve problems such as difficulty in cultivating transgenic plants
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Embodiment 1
[0149] (Example 1: Identification of a specific promoter using a cDNA library)
[0150] (RNA extraction from rice tissue)
[0151] RNA was extracted from the following rice tissues: callus treated with benzyl adenine, callus treated with gibberellin, callus treated with heat shock, callus during propagation, young roots, young green leaves, young Yellow leaves, spikes at flowering stage. A simple approach is to protein-modify ground plant tissue in the presence of guanidinium isothiocyanate and phenol and recover total RNA along with the aqueous layer. Since the 3-terminus of eukaryotes has a polyA sequence, it can be adsorbed to a carrier immobilized with oligo dT and eluted to allow specific separation.
[0152] (Construction of cDNA library from extracted RNA)
[0153] A complementary DNA strand (1st strand) was synthesized from the extracted mRNA using reverse transcriptase, RNaseH and DNA polymerase were allowed to act to synthesize the 2nd strand, and a cDNA library w...
Embodiment 2
[0162] (Example 2: Confirmation of the specificity of the selected promoter)
[0163] Through this example, it was confirmed whether each clone selected in Example 1 had target specificity. The specific steps are as follows.
[0164] (determine the base sequence of the selected clone)
[0165] First confirm whether the selected clone is identical to the expected sequence. When determining the sequence, it can be carried out by the dideoxy dye termination method known to those skilled in the art. Briefly, the DNA strand to be sequenced is made into a single-stranded state by heat denaturation, and a primer is attached at a position upstream from the position to be sequenced at this time. DNA synthesis reaction is performed by DNA polymerase in the presence of dideoxynucleotide triphosphates and deoxynucleotide triphosphates to which different fluorescent dyes are attached to each base. The elongation of DNA synthesis stops when the dideoxynucleotide triphosphate is incorpor...
Embodiment 3
[0167] (Example 3: Acquisition of a promoter region)
[0168] In this example, a clone confirmed to have the target sequence was used to obtain the promoter region. It can be simply expressed as the following steps.
[0169] First, primers were designed based on the data registered in DDBJ, and the promoter region of the target gene was obtained by using a commercially available kit (Genomewalker; Clontech, USA) for isolating the upstream of known sequences using basic PCR techniques. Its program such as figure 2 shown.
[0170] These fragments have a length of around 0.6kb-2.5kb.
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