Cloning and application of R gene TN2 for regulating and controlling powdery mildew resistance of plants
A powdery mildew, plant technology, applied in the direction of plant genetic improvement, application, plant peptides, etc., can solve problems such as infection and economic losses in agricultural production
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[0051] Example 1. TN2 mutation inhibits exo70B1 mutant powdery mildew resistance
[0052] 1. Obtaining mutant exo70B1tn2
[0053] Through EMS mutagenesis, a mutant library of exo70B1 was obtained. In the M2 generation, a mutant exo70B1tn2 that can inhibit exo70B1 mediated resistance to powdery mildew was screened.
[0054] The mutant exo70B1tn2 can also be prepared as follows: the mutant exo70B1 (N717829) is crossed with the mutant tn2 to obtain the hybrid F1 generation, and the homozygous double mutant exo70B1tn2 can be obtained by screening the F2 generation obtained by selfing of the F1 generation.
[0055] Wild-type Arabidopsis Col-0 (hereinafter referred to as wild-type Arabidopsis), recorded in the following documents: Catherine A. Frye, Dingzhong Tang, and Roger W. Innes (2001). Negative regulation of defense responses in plants by a conserved MAPKK kinase.PNAS 98(1):373-378; the public can obtain it from the Institute of Genetics and Developmental Biology, Chinese Academy of ...
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[0069] Example 2. TN2 mutation inhibits the expression of PR1 gene (AY064023, submitted on November 19, 2001) in exo70B1 mutant
[0070] In order to verify whether the TN2 gene is involved in plant disease resistance, real-time quantitative PCR was used to detect the PR1 gene expression patterns of Col-0, mutant exo70B1 and double mutant exo70B1tn2 before and after inoculation with powdery mildew. The instrument used is the German Eppendorf Mastercyclereprealplex 4S, as follows:
[0071] After 4 weeks of growing Arabidopsis wild-type plants Col-0 (WT), mutant exo70B1 and double mutant exo70B1tn2 were inoculated in large quantities with powdery mildew for 3 days, the leaves of the uninoculated and inoculated plants for 3 days were taken and put into the liquid Quickly freeze in nitrogen, then store at -70°C, and extract RNA for later use.
[0072] Extract the total RNA from the leaves with Trizol (Invitrogen) reagent, digest the contaminated DNA with DNase I (Promega) for half an hou...
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[0075] Example 3. TN2 mutation inhibits the accumulation of free salicylic acid in exo70B1 mutant
[0076] Salicylic acid is the central molecule that regulates plant disease resistance. Signal transduction that depends on salicylic acid is essential for the establishment of local and systemic resistance in plants. The content of free salicylic acid in Col-0, mutant exo70B1 and double mutant exo70B1tn2 before and after inoculation with powdery mildew was detected.
[0077] The seedlings of Arabidopsis wild-type plant Col-0 (WT), mutant exo70B1 and double mutant exo70B1tn2 were inoculated with powdery mildew in large quantities, and after normal growth for 4 weeks under the conditions of 22℃, 9 hours of light / 15 hours of darkness, they were taken separately The leaves of each material are quickly frozen in liquid nitrogen, then stored at -70°C, and salicylic acid is extracted for later use.
[0078] The content of salicylic acid was determined by high performance liquid chromatograp...
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