Pollen-specific expression of dcp2 promoter in a flowering plant
A plant pollen, pollen-specific technology, applied in the field of genetic engineering, can solve laborious, not completely reliable, time-consuming and other problems
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Embodiment 1
[0030] Based on the existing public DNA sequence and the inventor's previous research on the Arabidopsis DCP2 gene, the inventor designed specific primers, cloned and obtained the DCP2 promoter by PCR method, and sequenced it. This example briefly introduces the PCR process of the pollen-specifically expressed promoter DCP2 as follows.
[0031] (1) Extract Arabidopsis genomic DNA
[0032] Refer to the instructions of the plant DNA isolation kit to extract wild-type Arabidopsis genomic DNA; the extracted genomic DNA is subpackaged and stored at -20°C for future use.
[0033] (2) Design specific amplification primers and perform PCR amplification
[0034] To facilitate directional cloning, AgeI and SpeI restriction endonuclease sites were added to the 5' ends of the primers respectively, and the specific primer sequences were designed as follows:
[0035] DCP2-F: 5'-AACCGGTCTTGGAACAATTTGGCTC-3',
[0036] DCP2-R: 5'-AACTAGTAGTTCTTGCCTTTAAAACCCT-3';
[0037] Using the Arabidop...
Embodiment 2
[0048] Since the construction of related vectors is the basis for the research of related genes and the cultivation of transgenic plants, this example briefly introduces the construction process of the pollen-specific expression vector pDCP2-GUS as follows.
[0049] (1) Connect the DCP2 promoter to the pMD18-T vector
[0050]Referring to the instructions of the 2X ligation kit, ligate the specific amplification product of about 1300 bp recovered in Example 1 to the pMD18-T vector, and the 10 μL ligation system is designed as follows:
[0051] Ligation Mix, 5 μL;
[0052] pMD18-T, 1 μL;
[0053] 2.5 μL (about 200 ng) of the recovered PCR product in step (1);
[0054] wxya 2 O, 1.5 μL;
[0055] Ligation overnight at 16°C.
[0056] (2) Transform and screen
[0057] Take 5 μL of the ligation product in step (1), transform Escherichia coli competent cells by heat shock, spread on solid LB medium containing ampicillin (50 mg / L) for recombinant screening, and pick positive clon...
Embodiment 3
[0068] Using the recombinant plasmid pDCP2-GUS constructed in Example 2, plants can be further transformed and used to construct transgenic plants. This example briefly introduces the relevant experiments as follows.
[0069] (1) Transform Agrobacterium and prepare transfection solution
[0070] Take 100 μL of Agrobacterium GV3101 competent cells, add 1 μL of the pDCP2-GUS plasmid prepared in Example 2, and transform using liquid nitrogen freeze-thaw method;
[0071] After transformation, culture at 28°C on YEB culture plates containing kanamycin, rifampin and streptomycin (50mg / L, 25mg / L and 50mg / L respectively), pick positive clones, and perform PCR verification , PCR verification primers were designed as follows:
[0072] F: 5'-GGTCTGCACCATCGTCAACCACTACA-3',
[0073] R: 5'-GGCAGGCTGAAGTCCAGCTGCCAGAA-3';
[0074] The verified correct transformed strains were preserved for later use.
[0075] (2) Transformation of Arabidopsis
[0076] Take the correct transformed strain ...
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