摘要
棉花是我国重要经济作物，而棉花黄萎病是当前影响我国棉花产量与品质的主要病害之一，是由大丽轮枝菌(Vertillium dahliae Kleb．)引起的一种危害性极大的维管束系统病害。国内外在棉花黄萎病防治及抗病育种方面虽然取得了一些进展，但进展缓慢。这是因为棉花黄萎病是一种土传病害，利用物理化学手段进行黄萎病的防治效果甚微。培育棉花抗黄萎病品种是解决黄萎病危害的最为经济有效的手段，然而我国陆地棉缺乏免疫抗源，使传统育种困难重重；利用基因工程手段培育棉花抗黄萎病品种已成为防治棉花黄萎病的新途径。本研究主要目的是，通过分离提纯棉花病区土壤微菌核区系拮抗微生物的外分泌抗菌蛋白，研究其抑菌机理，为棉花抗黄萎病基因工程打下基础。
通过挑选在棉花病区土壤微菌核区系分离得到的拮抗微生物，采用硫酸铵沉淀的方法得到其发酵液的拮抗蛋白，检测了抑菌活性，并对粗蛋白的蛋白酶稳定性和温度稳定性进行了研究比较。其中BDT-25的抑菌效果最为明显，而且对蛋白酶K、胰蛋白酶、胃蛋白酶和水浴加热处理敏感，避免了环形肽的可能，是转基因棉花的供体基因理想来源。
经过确定硫酸铵分级沉淀在40—60％范围内，对其蛋白沉淀采用了CM Sepharose Fast Flow弱阳离子和DEAE Sepharose Fast Flow弱阴离子两种1ml预装小柱进行了预试验，确定了使用DEAE弱阴离子交换柱，采用pH6.0的MES样品缓冲液，用0～1mol／L的NaCl线性洗脱。收集各个蛋白峰的蛋白质并检测活性，对有抗菌活性的蛋白质再采用Sephacryl S-100凝胶的分子筛层析方法进行分离，经SDS-PAGE电泳检测结果显示获得了一个纯的蛋白组份。
对纯品抗菌蛋白的蛋白酶和水浴加热处理，同样发现纯品抗菌蛋白对二者很敏感，酸碱处理说明蛋白质在pH3.0～pH10的条件下比较稳定。通过喷淋菌落试验、孢子萌发试验和抗菌蛋白对菌丝的作用试验，揭示了BDT-25的抗菌蛋白的抑菌机理为3个方面：其一为造成孢子细胞畸形，继而胞壁破裂；其二为对菌丝的细胞壁的破坏，菌丝断裂，继而消融。其三是对微菌核的抑制萌发作用。
迄今，国内外尚未见从棉花病区土壤微菌核区系拮抗微生物外泌物中分离纯
摘要
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化出单一抗菌活性组分的报道，本试验对棉花黄萎病结抗微生物的抗菌蛋白组分
分离纯化方法进行了摸索，并分离到一种较纯的抗菌蛋白，对于这种抗菌蛋白的
理化及生化等相关性质进行了初步研究。研究结果为棉花病区土壤微菌核区系拮
抗微生物抗菌蛋白的抑菌机理以及棉花抗黄萎病基因工程育种提供一些理论及
实践方面的基础工作。
Cotton is one of the most important economic crop in our country. However, Ver -ticillium wilt of cotton, which is one of the most dangerous fascicular disease caused by Vertidllium dahliae Kleb., is the major diseases which seriously affect the yield and quality of cotton in our country. Although much progress has been made in prevention and control of Vertidllium wilt of cotton, the progess is slow. Because Vertidllium dahliae is soil-borned fungal pathogen, the effect of prevention and control is very little using the physical and chemical method.It is strongly consided that developing resistant and tolerant cultivar is the most economic and effective ways to resolve this problem. But the basic research about the pathogenic mechanism of Vertidllium dahliae shows that developing resistant and tolerant cultivar by conventional breeding is far from enough because lack of resistant resources. Objectives of this study were to isolate and purify the antifungal protein excreted by antifungal microbe in Microscle
rotiniasphere of cotton field infected by Vertidllium dahliae to learn the mechanism of the antifungal protein, and to do the basic work on the wilt resistant gene engineering of cotton.
The antifungal microbe separated from cotton field infected by Vertidllium dahliae were cultivated and the rough proteins of them were isolated by ammonium sulfate from 40% to 60%. The condition of the purification of the rough proteins were determined by HiTrap CM Sepharose Fast Flow column (1ml) and DEAE Sepharose Fast Flow( 1ml). DEAE week anion-exchange chromatogram is used and the best con dition is MES buffer at pH6.0, and 0-lmol/L NaCL is used to wash the column. The proteins were collected at the peak curve and the antifungal activities were tested.Eac
Abstract
-h peak with antiftmgal activities was collected and then molecular sievechromatogra -phy were used. A purified protein was gained,it was test by SDS-PAGE which shows that it has only one ingredient.
Through tests of stabilization of the rough protein done by protein enzyme and by high temperature,we can see that the purified proteins were sensitived to them. The test of stablization of the purified antifungal protein at different pH show that it was steady from pHS.O to pH 10.0. The shower test of fungal lawn, the burgeon of the spo -re and the effect to hypha of Verticillium dahliae showed that the mechanism of anti -fungal proteins had 3 facets: first, the spore become abnormality and then dissolved; second,the cellular wall was split and dissolved; third, the burgeon of Microsclerotinia were resisted.Until now, not a report was seen about the isolation and purification of antifungal protein from antifungal microbe in the Microsclerotiniasphere of cotton fie -Id. This test quested for the method of purification of the antifungal protein, and purif -ied one ingredient with its character was analysed. This research may use for referenc -e on the mechanism of antifungal proteins of the antifungal protein
s of the antifungal mirobe in the Microsclerotiniasphere of cotton field and on the the wilt resistant gene engineering of cotton.
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