Disease-resistant correlated wheat MYB albumen, coding gene and application thereof
A technology for encoding genes and proteins, which is applied in the fields of application, genetic engineering, plant gene improvement, etc., can solve the problems of MYB protein gene cloning, expression characteristics and function research that have not been reported yet
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Embodiment 1
[0047] Example 1. Obtaining of TaPIMP1 and its coding gene TaPIMP1 related to plant disease resistance
[0048] 1. Amplification of the 5'end sequence
[0049] The Gibberella-resistant wheat variety Sumai No. 3 (purchased from the Germplasm Bank of Jiangsu Academy of Agricultural Sciences) that grew to the two-leaf stage was treated with Gibberella, and the specific treatment method was: Fusarium graminearum (Jiangsu Agricultural) (Institute of Biotechnology of the Chinese Academy of Sciences) Mycelium blocks were inoculated by friction inoculation to the leaves of Sumai No. 3 wheat that grew to the two-leaf stage, and the Gibberella hyphae blocks were placed in the leaf sheaths. After 48 hours, the wheat treated with Gibberella hyphae was cut out. The leaves, treated with liquid nitrogen, were operated in accordance with the instructions of the Invitrogen TRIZOL Reagent total RNA extraction reagent manual to extract the total RNA from the above-mentioned wheat leaves. According t...
Embodiment 2
[0060] Example 2. Induced expression analysis of TaPIMP1 gene
[0061] Scab mycelium blocks were used to rub inoculate the leaves of Sumai No. 3 wheat cultivated in flowerpots for 10 days, and the blocks of scab mycelium were placed in the leaf sheaths of wheat; Rhizoctonia cerealis (Jiangsu Academy of Agricultural Sciences) The seedlings of Rhizoctonia solani (National Germplasm Bank of China) resistant to sheath blight (China National Germplasm Bank) seedlings were cultured in flowerpots for 15 days (two-leaf stage) with mycelium blocks by friction inoculation of mycelium blocks, and the blocks of sheath blight mycelium were placed in the leaf sheaths. After 48 hours, the wheat leaves with the above-mentioned different treatments were taken, and stored in an ultra-low temperature refrigerator at -80°C after quick freezing in liquid nitrogen.
[0062] The RNA from the wheat leaves with different treatments was extracted (approximately 5 μg total RNA for each sample), and reverse ...
Embodiment 3
[0064] Example 3 Analysis of disease resistance of transgenic tobacco with TaPIMP1 gene
[0065] 1. Obtainment of transgenic tobacco with TaPIMP1 gene
[0066] Use the plasmid pT-TaPIMP1 containing the full-length ORF of TaPIMP1 gene as template, and MBD-F: 5-CA with XbaI and BmaHI restriction sites TCTAGA ATGGACATGGACAAGG-3 and
[0067] MBD-R: 5-GT GGATCC TTCATCGTCAGCAGTAA-3 is a primer to amplify the ORF of TaPIMP1 by PCR. The 982bp PCR product was digested with XbaI and BmaHI, and then ligated to the large fragment obtained by digesting vector pBI121 (Beijing Baierdi Company) with XbaI and BmaHI. The ligation product was transformed into E. coli TOP10 competent cells. Recombinants were screened from the kanamycin-resistant colonies, and sequenced analysis was performed. After sequencing, the recombinant plasmid containing the ORF of TaPIMP1 gene was named pBI121-TaPIMP1, and the recombinant plasmid was controlled by the 35S promoter. Transform pBI121-TaPIMP1 into Agrobacterium...
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