麦麸中小麦矮腥黑穗病菌孢子的提取方法

METHOD OF EXTRACTING SPORES OF Tilletia controversa FROM BRAN

对麦麸中小麦矮腥黑穗病菌孢子的提取方法进行了研究。通过建立的“过筛—α-淀粉酶降解”、“过筛—密度梯度离心”和“过筛— α-淀粉酶降解—密度梯度离心”等方法能从麦麸中提取 TCK孢子。提取的 TCK孢子数量与网筛、α-淀粉酶和密度梯度离心的应用情况有关 ,而提取的 TCK孢子纯度则主要与密度梯度离心有关。选用双层纱布和 5种不同规格的网筛或网筛组合进行过筛能弃除83.88%～ 94 .0 9%的麦麸 ,应用“过筛— α-淀粉酶降解”方法能弃除 95 .0 1%～ 99.0 0 %的麦麸 ,并能提取 19.2 %～ 5 1.7%的 TCK孢子。在“过筛—密度梯度离心”实验中 ,只有 60目 +2 0 0目 +30 0目 +30μm+11μm该网筛组合处理能获取 7.2 %的纯孢子 ,密度梯度离心对孢子的网嵴高度、自发荧光和萌发没有影响 ;在“过筛— α-淀粉酶降解—密度梯度离心”实验中 ,60目 +2 0 0目 +30 0目和 60目 +2 0 0目 +30 0目 +30μm+11μm这 2组网筛处理可分别得到 18.8%和 12 .2 %的纯孢子 。

Methods of extracting spores of Tilletia controversa from bran were studied. The methods “sieving—α amylase degradation”,“sieving—density gradient centrifugation” and “sieving—α amylase degradation—density gradient centrifugation” could be used to extract the spores of Tilletia controversa (TCK) from bran. The number of TCK spores extracted depended on the usage of sieving, α amylase and density gradient centrifugation while the purity mainly on the density gradient centrifugation. 83.88%-94.09% of bran could be removed by the method of double layer gauze and 5 different combinations of sieving treatments. 95.01%- 99.00% of bran could be removed and 19.2%-51.7% of TCK spores could be extracted by the method of “sieving—α amylase degradation”. As for the method of “sieving—density gradient centrifugation”, only the treatment of “60 meshes + 200 meshes+300 meshes+30 μm+11 μm” could work so that 7.2% pure spores were obtained after further density gradient centrifugation which didn't influence the reticulum height of spores, autofluorescence and germination of spores. Likewise, as to the method of “sieving—α amylase degradation—density gradient centrifugation”, the treatment of “60 meshes+200 meshes+300 meshes” and “60 meshes+200 meshes+300 meshes+30 μm+11 μm” could work so that 18.8% and 12.2% of pure spores were obtained after further density gradient centrifugation which didn't influence the reticulum height of spores. The most effective condition for α amylase to degrade insoluble starches is at 71℃ and 350 r/min for 3 mins.These methods have been extensively applied in the routine quarantine at the port.