Application of gene TaPT16 in improving resistance of plants to powdery mildew

[0023] Example 1

[0024] Substation and low phosphorus treatment

[0025] Transplanting the three-hectic wheat to the fine sand, meteorite and pearl rock, 1: 1: 1 ratio of moxili and pearl rock, respectively, 200. Spore / 15g. In the light period of 16 h / 8h, the temperature of 18 ° C / 15 ° C is grown, all inoculation and control plants are poured twice a week of 1/2 Hoagland nutrient solution, 5μmkh 2 PO 4 Hypophosphorus treatment, 500μmkh 2 PO 4 High phosphorus treatment, 6W, take wheat roots, clean water, harvest the root sample, and store the liquid nitrogen-80 ° C to preserve the spare.

[0026] Treatment of bacterigen and hypophyscented samples and high-phosphorus were extracted with total RNA by Trizol Reagent (Invitrogen).

[0027] Templates were made with 1 μg of RNA, and the first chain cDNA was synthesized using a cDNA synthesis kit and the extracted RNA reverse transcribed into cDNA.

[0028] A real-time quantitative PCR reaction is performed by the CDNA of the above different treatment materials. Use relative quantification 2 -ΔΔCt The method was relatively quantitatively analyzed to the expression amount, and the Actin (Gene Bank login number KC775780) gene was used as a reference gene.

[0029] Tapht-myc is known as a known branty-specific wheat phosphorus transporter, which is hardly expressed when not vaccinating the bacteria (+ p-m), and the expression of the bacteria is significantly increased. Similar to TAPHT-MyC, the expression of TAPT16 and TAPHT-Myc gene was inhibited under low phosphorus, TAPT16 and TAPHT-Myc gene expression was inhibited, and Mosa Chanteidus was seeded under different phosphorus concentrations (low phosphorus and high phosphorus concentration). Surface Ballicle Casic Nippythrosis is significantly increased, such as figure 1 As shown, the TAPT16 gene increased from about 9-16 times after the increase of the bacteria, and therefore, TAPT16 may be a colostrum-induced wheat phosphorus transfer protein gene.

[0030] Example 2 Overgramps TAPT16 in wheat to resistance analysis of wheat white powder disease

[0031] In order to further analyze the function of TAPT16, the full length of TAPT16 was constructed to the plant carrier IPK003 in the maize Ubiquitin promoter, and the recombinant carrier IPK003-TAPT16 was obtained, and the recombinant carrier and the empty vector were converted by the thermostat. Agrobacterium. GV3101, transformed from the Agrobacterium-mediated conversion method to the wrist wheat 26, passed through the screening of 5 single-copy germical transgenic strains, simultaneous inoculation on the three-leaf blade of wild-type week wheat 26 and 5 transgenic plants Fresh wheat white powder spores, placed on a 1% agar plate addition of 85 μm benzimidazole, placed in a climate incubator of 20 ° C in a climate incubator for 4 h, and then treated with a suitable density (about 250 spores) / cm 2 Emperor shaking the fresh spores of the white powder inoculation into the blade, respectively, inoculated with whitefin, and 7d samples, respectively. After the TAPT16 transgenic plants and control plants were cultured in a climate incubator at 22 ° C for 60 h, the leaves were decolored (ethanol / acetic acid = 1: 1 (v / v)), followed by Caumass, Blue R250. Liquid (0.6%, w / v, dissolved in methanol) was stained with a few times in a BX61 microscope after being rolled by deionized water (spores, which were greater than 1000 per plant blade). At the same time, the photo of the blade of the vaccination BGT7DPI was observed with Canon EOS 600D.

[0032] After the blade of IPK003-TAPT16 and the control plants were inoculated with PB, the microscope was observed that the number of microphylfacieta on the white powder on the vane blade of IPK003-TAPT16 was significantly less than that of the control plants, such as figure 2 Middle A shows the statistical analysis of the microchid index, TAPT16 overexpressed plants is significantly lower than the control plants, such as figure 2 In Central C; consistent with the microscopic observation, the phenotypic analysis of the vaccination of PBH 7D indicates that the white powder filament on the plant blade is less than the control plants, such as figure 2 Down B.

[0033] The five single-copy pure transgenic strains of TAPT16 expressed TAPT16 were planted in large fields, further analyzed the resistance of the transgenic plants on white powder. The following four May, respectively, in the control and TAPT16 overturned transgenic strain vaccination, 7-10 days after the analysis of phenotypic results showed the same as the seedling period, compared with the control, TAPT16 Overgraph of plants, stalks, stems and small spikes and other plants are significantly less than the control plants, such as image 3 The A and B shown in Central A and B; at the same time, the control and TAPT16 hypotencetes the biomass of the whitefasteaceous fungi on the plant leaf, and the DNA of the vaccination of Popper bacteria is extracted, and the reflection gene of wheat white powder is selected from the EF-Lα gene of BGT ( HM538432), each template in each quantitative experiment, three synchronous amplification and detection, and three biological repeats, the primer sequence is as follows:

[0034] BGT-EF1A-F: 5'-gtcggatttaaccccaaggt-3 ',

[0035] BGT-EF1A-R: 5'-TTTATCGTAGGGCGACTTG-3 '.

[0036] QRT-PCR results show that the amount of white powder in TAPT16 transgenic plants is significantly lower than that of the control plants, such as image 3 Moine C. The above results indicate that TAPT16 is the positive regulatory factor of wheat white powder.

[0037] Example 3 Overexpression of TAPT16 in wheat TAPT16 on the resistance analysis of wheat stem rot

[0038] In order to further analyze the function of TAPT16 in the homogeneity of wheat and other pathogens, the pathogenic analysis of wheat stem-based corruption in TAPT16 is performed. First, inoculation of the potato-80 ° C-80 ° C-80 ° C-80 ° C-80 ° C-Fikiferi pacine, cultivate 3-5 days in 25 ° C incubator, choose a good holiday Hegu 4-6 pieces of 5-6 pieces of CMC camospore medium (carboxymethylcellulose 15g / L, ammonium nitrate 1 g / L, potassium phosphate 1 g / L, magnesium sulfate 0.5 g / L , Yeast extract 1 g / L, 121 ° C high pressure sterilization 20min), 25 ° C, 180 rpm shaker after 5-6 days; in the ultra-net gauze, remove the fungus, pour 50 ml of centrifuge tube 5000 rpm Centrifuge 15 min , Discard the supernatant, sterile water was cleaned twice, resuspended with 2 mL of sterile water, calculated the number of spores with the blood ball count plate, and diluted the suspension to 1 × 10 5 , Add 4 ° C refrigerator standby or inoculated. The seeds of the 5 transgenic plants of the control plants and TAPT16 were respectively bred to 1 cm, so soaking it into 1 × 10 5 1min in spore fluid, after washing seed surfaces with water, and the amount of watering was controlled during the period, and it was conducive to the onset, and the morbidity of plants was observed after 18-20. As a result, it was found that over-expression plants were comparable to the degree of brown rot, no significant difference, indicating that TAPT16 had failed to enhance the resistance of wheat to stem rotism.