Protein for improving plants fastness and accelerating vegetation and encoding gene thereof
A technology to promote plant growth and plant resistance, applied in plant genetic improvement, genetic engineering, botanical equipment and methods, etc., can solve the problem of undiscovered Alternaria protein and nucleotide sequence, etc.
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Embodiment 1
[0007] Example 1 Isolation and purification of PEAt1 protein
[0008] Cultivate Alternaria with PDA liquid medium, at 28°C, 180r.min -1 , constant temperature shaking culture for 2 days, the mycelium obtained by suction filtration, take the mycelium block and grind it into a fine powder with liquid nitrogen in a mortar, quickly add phosphoric acid extraction buffer, boil in a boiling water bath for 20 minutes, then put it in an ice-water mixture to cool down , 12000r.min at 4°C -1 Centrifuge for 15 minutes, and filter the supernatant with a microporous filter (Ф=0.22 μm) to obtain an extract. Add ammonium sulfate directly to the protein extract to a final concentration of 85%, continue to stir with a magnetic stirrer for 30 minutes, then centrifuge at 5000 rpm for 30 minutes to take the precipitate, and re-dissolve the precipitate with the extraction buffer, according to the volume ratio of the sample to ethanol is 4:1 Add -20°C pre-cooled absolute ethanol, precipitate at -2...
Embodiment 2
[0009] Example 2 The promoting effect of PEAt1 protein on plant root growth
[0010] Soak wheat seeds separately in purified protein and dilute to 3μg.ml -1 , 2μg.ml -1 , 1μg.ml -1 The seeds were soaked in distilled water as a negative control. After soaking the seeds for 8 hours, spread the seeds on a petri dish and culture them under light in a light incubator at 28°C, and add appropriate amount of distilled water to maintain humidity. After 7 days of cultivation, the main root length of each treatment wheat was randomly measured, and 30 parallel measurements were taken for each treatment and the average value was taken.
[0011] It can be seen from Table 1 that when the protein concentration is 1 μg / ml, it can still promote the growth of wheat roots. Roots grow at an activin concentration of 3 μg.ml -1 , 2μg.ml -1 , 1μg.ml -1 Compared with the control group, the root length of wheat increased by 12.29%, 11.7%, and 8.06%, respectively.
[0012] Table 1 Changes of pro...
Embodiment 3
[0014] Example 3 Inhibitory effect of PEAt1 protein on tobacco mosaic virus
[0015] Shanxi tobacco is cultivated in an insect-proof greenhouse (20-27°C). When the plant grows to the 8-leaf stage, use the half-leaf method and use PEAt1 protein 2μg.ml -1 Leaves were soaked for 10 minutes, and water was used as a control, with 6 leaves per treatment, and each treatment was repeated 3 times. The treated leaves were placed in a petri dish with a diameter of 15 cm, kept moist at 25° C. for 2 days, and then inoculated with the virus.
[0016] The conventional juice friction inoculation method was used to inoculate, and the diseased leaves of the poisonous tobacco source were added with 0.01mol / L phosphate buffer (pH7.2) for grinding (the concentration of the virus juice was 50mL of 0.01moL / L phosphate buffer per gram of diseased leaves). The treated leaves were inoculated with TMV virus in an equal amount using the half-leaf method, and the number of dead spots was recorded after 3...
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