Separation,Purification And Biological Activity Research Of Activator Protein From Pestalotiopsis Maculans (Cda.) Nag Raj

Activator protein is a kind of new type of protein with elicitor function, which is separated from plant pathogenic fungi, which can induce many kinds of plants to gain disease resistance, and enhance the cultivation of resilience of plants, and promote plant growth. At the same time, Activator protein can significantly enhance the system broad-spectrum resistance of plants for many kinds of important plant pathogens, and can induce strongly. At present, the research of intracellular soluble active protein in mycelium of Pestalotiopsis maculans (Cda.) Nag Raj has not yet been reported. In this study, we use Pestalotiopsis maculans (Cda.) Nag Raj as the experimental materials, by ammonium sulfate fractional precipitation combined with gel filtration chromatography, and have purified a 17.7 kDa activation bioactive protein from the mycelium of Pestalotiopsis maculans (Cda.) Nag Raj. Biological activity determination showed that the protein can significantly improve the corn, cabbage, cucumber, pea seed germination rate and root growth, and can promote the growth of pea, corn seedling plants, and improve the peas, corn seedling leaf chlorophyll content and improve the root-shoot ratio and root activity of pea, corn seedling. This study obtained the following results:1. By using filtrate and protein crude extract processing corn, cabbage, cucumber, pea seed, we found that Pestalotiopsis maculans (Cda.) Nag Raj can produce something that can promote seed germination and root growth, and the active ingredients is found in protein crude extract. The experiment that using ammonium sulfate classification precipitation to salting out protein crude extract, then the ammonium sulfate precipitation supernatant and precipitation on the test, test results show that there is no active in supernatant fluid, precipitation with activity, that prove the existence of active substances in precipitation. That active substances may be as a part of the macromolecular protein. May be the mycelium component of intracellular soluble protein of Pestalotiopsis maculans (Cda.) Nag Raj, so as to lay a foundation for further purification.2. The experiment proved that activated protein exists in fungus mycelium of Pestalotiopsis maculans (Cda.) Nag Raj, for as much as possible in the shortest possible time to obtain the most hyphae. This study from inoculation quantity,incubation time, temperature, dissolved oxygen, shaker rate, pH six factors of shake cultivation to found the optimized conditions of production of mycelium of Pestalotiopsis maculans (Cda.) Nag Raj in shake cultivation conditions. That the above six culture condition, respectively, to achieve the optimal combination is the best oscillation culture conditions. Such training program that temperature 25 ℃, pH of 6, three bacteria cake,200 mL medium, cultivating 11 days is the best and the most economic.3. The experiments that using ammonium sulfate classification piecewise precipitation to salting out Pestalotiopsis maculans (Cda.) Nag Raj protein crude extract, have proved that the optimal degree of salting out saturation is 60%. The crude extract by Sephadex G-75 thin gel column chromatography has got two peaks, peak 2 was the activity peak, then the peak 2 collected by Sephadex G-75 thick gel column chromatography has got seven peaks, peak 2-7 was the activity peak, when SDS-PAGE electrophoresis detected, there was a single stripe, with relative mobility (mR) calculate the protein molecular weight of 17.7 kDa.4. The experiments study on biological activity of the above purified protein, to study that the peas, corn seeds after processing, the change of germination rate, the change of root growth, the change of plant growth, the change of chlorophyll content and root cap ratio, the change of root activity. After the purification activator protein samples from Pestalotiopsis maculans (Cda.) Nag Raj processing pea, corn seeds, pea seed germination rate increased by 8.84%, corn seed germination rate increased by 8.17%, pea root length increased by 33.20%, corn root length increased by 39.75%, pea seedling plant height increased by 18.06%, corn plant height of seedling increased by 26.33%, peas chlorophyll content increased by 21.22%, corn chlorophyll content increased by 18.21%, pea root cap ratio increased by 84.62%, corn root cap ratio increased by 21.74%, pea root activity increased by 28.57%, corn root activity increased by 66.67%, the growth of the plant is promoted.