Calcium-dependent protein kinase gene in rice, and its application
A protein and rice technology, applied in the fields of application, genetic engineering, plant gene improvement, etc., can solve problems affecting rice quality and reducing rice yield, etc.
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Embodiment 1
[0052] Example 1. Screening and Cloning of Rice CDPK Gene Related to Disease Resistance Defense Response
[0053] 1. Preparation of Magnaporthe grisea inducer
[0054] The preparation method of blast fungus induced crude extract is as follows:
[0055] (1) The strains of Magnaporthe grisea are preserved in the form of filter paper sheets covered with fungal hyphae. When inoculating, the filter paper sheets are placed on the tomato-oat solid medium, and 3-4 filter paper sheets are placed on each plate.
[0056] (2) Place the plate in an incubator at 30°C for 4-5 days. At this time, a large number of hyphae will grow centered on the paper sheet.
[0057] (3) Add 4-5mL of sterile water to each plate, and use a coating stick to spread back and forth on the plate to scrape the hyphae.
[0058] (4) Wash the mycelium with sterile water, add it to the tomato-oat liquid medium, and culture it with shaking at 28°C and 200rpm in the dark for 4-5 days.
[0059] (5) Collect the culture,...
Embodiment 2
[0096] Embodiment 2, preparation and phenotypic analysis of transgenic rice
[0097] 1. Cloning of OsCPK10 and OsCPK20 genes
[0098] (1) The primers for the OsCPK10 gene were designed as follows:
[0099] OsCPK10-sense pair: 5'-ATGGGGAACACGTGCGTC-3'
[0100]OsCPK10-antisense pair: 5'-TCAAGACATCCTCAAGGCCTC-3'
[0101] The primers for the OsCPK20 gene were designed as follows:
[0102] OsCPK20-sense pair: 5'-ATGGGCAACTGCTGCGTGAC-3'
[0103] OsCPK20-antisense pair: 5'-CTATTGGGTAGTTGTTAACTGCAATG-3'
[0104] (2) Extract the RNA of the Nipponbare rice callus treated with the blast fungus inducer for 1 h in Example 1, obtain cDNA after reverse transcription, use the cDNA as a template, and perform PCR amplification with the primer pairs of OsCPK10 and OsCPK20 genes respectively, The OsCPK10 and OsCPK20 genes were obtained.
[0105] The PCR reaction system is as follows:
[0106]
[0107] (3) Agarose gel electrophoresis to detect the size of the PCR amplified fragment, the ...
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