阅读说明：本技术 用于烟草花叶病毒病生物防治的超敏蛋白的制备及应用 (Preparation and application of hypersensitive protein for biological prevention and treatment of tobacco mosaic virus disease ) 是由 张家韬 崔传斌 安德荣 董绘阳 陈超 耿伟 李立丹 王安然 于 2019-10-17 设计创作，主要内容包括：本发明涉及用于烟草花叶病毒病生物防治的超敏蛋白的制备及应用,以枯草芽孢杆菌为生产菌株,通过对枯草芽孢杆菌发酵粉的温水溶解、离心、膜分离、喷雾干燥处理制备超敏蛋白成品,采用两个连续梯度密度离心,再经过分级超滤膜分离,最大限度去除菌体和杂蛋白,得到活性较高的超敏蛋白浓缩液,再利用了超敏蛋白的耐热性,通过低温喷雾干燥技术,使超敏蛋白快速干燥,提高了超敏蛋白制备效率,且麦芽粉作为辅料具有溶解性好、稳定性高、含有丰富微量元素和矿物质等优点。本发明制得超敏蛋白对烟草花叶病毒病的防治具有明显的防治效果,防效可达82.66％；本发明分离纯化超敏蛋白活性高,成本低廉,工艺简单,后期处理损耗较低。(The invention relates to preparation and application of hypersensitive protein for biological control of tobacco mosaic virus diseases, bacillus subtilis is taken as a production strain, a hypersensitive protein finished product is prepared by warm water dissolution, centrifugation, membrane separation and spray drying treatment of bacillus subtilis fermentation powder, two continuous gradient density centrifuges are adopted, and then a graded ultrafiltration membrane is used for separation, so that thalli and foreign protein are removed to the maximum extent, a hypersensitive protein concentrated solution with higher activity is obtained, the heat resistance of the hypersensitive protein is utilized, and the hypersensitive protein is rapidly dried by a low-temperature spray drying technology, so that the preparation efficiency of the hypersensitive protein is improved, and the malt powder has the advantages of good solubility, high stability, rich trace elements and minerals and the like as an auxiliary material. The hypersensitive protein prepared by the invention has obvious prevention and treatment effects on tobacco mosaic virus, and the prevention and treatment effects can reach 82.66%; the invention has the advantages of high activity of the separated and purified hypersensitive protein, low cost, simple process and lower post-treatment loss.)

1. The preparation method of the hypersensitivity protein for biological prevention and treatment of the tobacco mosaic virus disease is characterized in that the extraction process of the hypersensitivity protein comprises the steps of warm water dissolution, density gradient centrifugation, fractional ultrafiltration membrane separation, pH value adjustment and low-temperature spray drying which are sequentially carried out.

2. The method for preparing hypersensitive protein for biocontrol of tobacco mosaic virus disease according to claim 1, wherein said warm water dissolution step is performed according to the ratio of bacillus subtilis fermentation powder to water 1: 100 portions are added into warm water with the temperature of 36 ℃.

3. The method for preparing the hypersensitive protein for biological control of tobacco mosaic virus disease according to claim 1, wherein in the step of separating by the graded ultrafiltration membrane, the supernatant after centrifugation is graded and filtered, the filtering temperature is 36 ℃, the pressure drop of an inlet and an outlet of a ceramic membrane device is 0.2-0.25 MPa, and the average flux is 0.3m³The bacteria and foreign protein removing rate reaches 98.99-99.99%, and the bacteriostatic titer of the hypersensitive protein in the supernatant after membrane separation is 12000 units/mg.

4. The method for preparing a hypersensitive protein for biological control of tobacco mosaic virus disease according to claim 3, wherein the fractionating filtration comprises a tertiary filtration; the first stage of filtration adopts 35 ten thousand molecular weight ceramic membranes, the second stage of filtration adopts 15 ten thousand molecular weight ceramic membranes, and the third stage of filtration adopts 5 ten thousand molecular weight ceramic membranes.

5. The method for preparing the hypersensitive protein for biological control of tobacco mosaic virus disease according to claim 1, wherein the specific adjusting method in the step of adjusting the pH value is as follows: collecting the clear liquid separated by the graded ultrafiltration membrane, adjusting the pH value to 4.5, standing for 36h, filtering the supernatant to obtain a concentrated solution containing the precipitate, and repeating the filtering operation until the volume of the concentrated solution is 40% of that of the supernatant before the pH value is adjusted; the regulator for regulating the pH value is hydrochloric acid.

6. The method for preparing the hypersensitivity protein for biological control of tobacco mosaic virus disease according to claim 5, characterized in that 30% malt flour is added into the concentrated solution as an auxiliary agent, and after being uniformly stirred, the hypersensitivity protein dry powder is obtained by low-temperature spray drying according to the treatment capacity of 40L/h.

7. The preparation method of the hypersensitivity protein for biological control of tobacco mosaic virus disease according to claim 6, wherein the air inlet temperature of the low-temperature spray drying is 30-45 ℃ and the air outlet temperature is 30-40 ℃.

8. The method for preparing hypersensitive protein for biological control of tobacco mosaic virus disease according to claim 1, wherein the bacillus subtilis fermentation product is solid fermentation powder, which is preserved in China general microbiological culture Collection center (CGMCC) with the preservation number of CGMCC No. 18089.

9. The application of the hypersensitivity protein prepared by the preparation method of the hypersensitivity protein for biological control of tobacco mosaic virus disease according to claim 1 is characterized in that the application mode of the hypersensitivity protein is leaf surface spraying, the spraying is carried out at the tobacco seedling leaf stage at the optimal time, the hypersensitivity protein is diluted by water by 300 times, and the using amount is 1g per square meter.

Technical Field

The invention relates to the technical field of biological extraction processes, in particular to preparation and application of a hypersensitive protein for biological control of tobacco mosaic virus diseases.

Background

The tobacco mosaic virus is a single-stranded RNA virus, has strong infectivity and pathogenicity to plants, has a wide host range, can infect more than 350 kinds of plants, wherein the damage is the most serious in solanaceae, cruciferae and compositae, and the serious loss and even field damage are often caused after the crops and the plants are infected with diseases. Tobacco is an important economic crop, is very easy to infect TMV, can be infected by tobacco mosaic virus when being operated by various farming, particularly has large spread in the current industrialized centralized seedling culture, has small twisted leaves, generates various necrotic spots, is dwarfed and dead, and loses the utilization value of tobacco leaves. Tobacco virus diseases are therefore the most important plant diseases in tobacco production and other susceptible crops.

Prevention and control of mosaic disease, which is difficult to control and has poor chemical control effect and large residue, has been a major problem in the production of tobacco and other solanaceous plants. At present, a great deal of research is carried out at home and abroad on the prevention and treatment of the tobacco mosaic disease, but the effect of various pesticides for preventing and treating the tobacco mosaic disease popularized in the market at present is not ideal. Moreover, the common chemical pesticide has low effect and high toxicity and causes serious problems of environmental pollution and the like, and the problems of residue of a large amount of pesticides, environmental pollution, improvement of drug resistance of viruses and the like are easily caused by long-term continuous high-dose application of single antiviral chemical pesticide.

Disclosure of Invention

The invention aims to solve the problems of low effect, high toxicity and environmental pollution of common chemical pesticides in the prior art, and provides preparation and application of a hypersensitive protein for biological control of tobacco mosaic virus diseases.

In order to achieve the purpose, the invention adopts the following technical scheme: the preparation method of the hypersensitive protein for biological prevention and treatment of the tobacco mosaic virus disease comprises the steps of warm water dissolution, density gradient centrifugation, fractional ultrafiltration membrane separation, pH value adjustment and low-temperature spray drying which are sequentially carried out.

In the above preparation method of the hypersensitive protein for biological control of tobacco mosaic virus disease, the warm water dissolving step is carried out according to the ratio of bacillus subtilis fermentation powder to water 1: 100 portions are added into warm water with the temperature of 36 ℃.

In the preparation method of the hypersensitive protein for biological control of the tobacco mosaic virus, the supernatant after centrifugation is subjected to graded filtration in the step of separating by using a graded ultrafiltration membrane, the filtration temperature is 36 ℃, the pressure drop of an inlet and an outlet of a ceramic membrane device is 0.2-0.25 MPa, and the average flux is 0.3m³The bacteria and foreign protein removing rate reaches 98.99-99.99%, and the bacteriostatic titer of the hypersensitive protein in the supernatant after membrane separation is 12000 units/mg.

In the above preparation method of the hypersensitive protein for biological control of tobacco mosaic virus disease, the fractionating filtration comprises three-stage filtration; the first stage of filtration adopts 35 ten thousand molecular weight ceramic membranes, the second stage of filtration adopts 15 ten thousand molecular weight ceramic membranes, and the third stage of filtration adopts 5 ten thousand molecular weight ceramic membranes.

In the above preparation method of the hypersensitive protein for biological control of tobacco mosaic virus disease, the specific adjusting method in the step of adjusting the pH value is as follows: collecting the clear liquid separated by the graded ultrafiltration membrane, adjusting the pH value to 4.5, standing for 36h, filtering the supernatant to obtain a concentrated solution containing the precipitate, and repeating the filtering operation until the volume of the concentrated solution is 40% of that of the supernatant before the pH value is adjusted; the regulator for regulating the pH value is hydrochloric acid.

In the preparation method of the hypersensitive protein for biological control of tobacco mosaic virus diseases, 30% of malt flour is added into the concentrated solution as an auxiliary agent, the mixture is uniformly stirred, and low-temperature spray drying is carried out according to the treatment capacity of 40L/h to obtain the hypersensitive protein dry powder.

In the preparation method of the hypersensitive protein for biological control of the tobacco mosaic virus, the air inlet temperature of the low-temperature spray drying is 30-45 ℃, and the air outlet temperature is 30-40 ℃.

In the preparation method of the hypersensitive protein for biological control of tobacco mosaic virus diseases, the adopted bacillus subtilis fermentation product is solid fermentation powder, the bacillus subtilis is preserved in the common microorganism center of China Committee for culture Collection of microorganisms with the preservation number of CGMCC No. 18089.

In the application of the hypersensitive protein prepared by the preparation method of the hypersensitive protein for biological control of tobacco mosaic virus diseases, the application mode of the hypersensitive protein is leaf surface spraying, spraying is carried out at the tobacco seedling stage at the optimal spraying time, water is used for diluting 300 times, and the using amount is 1 g/square meter.

Compared with the prior art, the invention has the advantages that:

1. the method comprises the steps of centrifuging a warm water extracting solution through two continuous density gradient centrifuges, and separating the warm water extracting solution through a grading ultrafiltration membrane, so that thalli and foreign proteins can be removed to the maximum extent, and the hypersensitive protein with high activity is obtained;

2. when the hypersensitive protein is processed into dry powder, the heat resistance of the hypersensitive protein is utilized, the hypersensitive protein is quickly dried by a low-temperature spray drying technology, the preparation efficiency of the hypersensitive protein is improved, and the malt flour serving as an auxiliary material has the advantages of good solubility, high stability, rich trace elements and mineral substances and the like;

3. the invention is a green environment-friendly pollution-free biocontrol microbial inoculum obtained by extracting bacillus subtilis fermentation powder, is a green resistance protein, can improve the antiviral property of plants, and is harmless to human and livestock and free of environmental pollution;

4. the invention has high-efficiency pertinence to tobacco mosaic virus, belongs to a green prevention and control technology of plant diseases, and has the advantages of high activity of separated and purified hypersensitive protein, low cost, simple process and lower post-treatment loss.

Detailed Description

The following examples are for illustrative purposes only and are not intended to limit the scope of the present invention.