HT-毒素特异性组分的分离及其在Ht1基因玉米质膜存在结合蛋白的证据
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摘要
本研究对玉米大斑病菌1号小种毒素的特异性组分进行分离,建立了高效的特异性毒素分离纯化方法,并对其进行了紫外波谱分析;用荧光素双醋酸酯(FDA)染色法,测定了毒素特异性组分对原生质体死亡率的影响;进而用1-苯胺基-8-萘磺酸(ANS)荧光探针标记法和SDS-PAGE电泳法对特异性毒素组分与Htl基因玉米细胞互作过程中的质膜蛋白进行了研究,旨在证明Htl基因玉米细胞质膜上存在特异性毒素组分结合蛋白(TBP),为从细胞和分子生物学角度阐明毒素的致病机理提供证据。主要结果如下:
1.对玉米大斑病菌的1号小种菌株(99-2)的粗毒素进行硅胶薄层层析(TLC),分离到Ⅰ(Rf0.06)、Ⅱ(Rf0.21)、Ⅲ(Rf0.45)、Ⅳ(Rf0.60)、Ⅴ(Rf0.75)5个条带,分别对此5个条带进行生物测定,发现条带Ⅱ对带Htl基因的玉米具有较强的特异性。
2.在检测波长220nm、流速1.0mL/min的HPLC分析条件下,对条带Ⅱ的洗脱剂中甲醇与水的比例进行摸索,最后确定60%甲醇为最佳洗脱条件。经对条带Ⅱ进行高效液相色谱(HPLC)的进一步纯化,得到3种组分Ⅱ-1、Ⅱ-2、Ⅱ-3,其中只有Ⅱ-3具有特异致病活性。对Ⅱ-1、Ⅱ-2、Ⅱ-3进行紫外-可见光谱扫描。发现只有Ⅱ-3在300nm处有吸收峰,Ⅱ-1和Ⅱ-2的峰形相似,可能为类似物。
3.从HT-毒素Ⅱ-3组分处理玉米幼苗的叶片中提取原生质体,在0-1.5h内其死亡率与对照(蒸馏水处理幼苗)无明显差别,但1.5h后Ⅱ-3处理一直明显高于对照,说明Ⅱ-3加速了叶片细胞的死亡;在6h左右处理有显著上升,而对照变化不大。说明Ⅱ-3与叶片细胞作用后并不直接伤害细胞,而需经过一定时间,这一过程很可能是Ⅱ-3与受体结合后信号不断放大,最终使原生质体失去活力的过程。
4.以玉米叶片原生质体为研究对象,以ANS(1-苯胺基-8-萘磺酸)作荧光探针,用荧光法研究了Ⅱ-3与质膜蛋白的互作。在被ANS标记后的原生质体悬浮液中加入不同浓度HT-毒素Ⅱ-3组分后,ANS荧光强度明显增加,呈现出了剂量—效应关系,表明Ⅱ-3与质膜上ANS某些受体发生了互作。本试验采用两种试剂,从两个角度表明了ANS受体和质膜上存在的Ⅱ-3受体具有蛋白特性。推测HT-毒素特异性组分的原始作用位点在质膜外侧,且特异性毒素组分与其受体互作具明显的饱和性。
5.利用SDS-PAGE电泳技术分析了HT-毒素Ⅱ-3组分对玉米叶片中可溶性蛋白和质膜蛋白的影响。结果表明,Ⅱ-3对叶片中可溶性蛋白没有影响,但在叶片质膜蛋白中找到了3个与Ⅱ-3致病相关的蛋白,经分析其分子量分别为31.622KD、63.125KD和93.536KD,其中分子量为31.622KD和63.125KD的蛋白为叶片本身所特有。
In this dissertation, firstly, the conditions of isolation and purification of specific toxin fractions produced by Exserohilum turcicum has been studied, and a high-efficient method has been established;secondly, the toxins were analysed by UV-Vis spectrophotometry;then the effect of specific toxin on death rate of corn leaves protoplast was studied by FDA as a tinct reagent;finally, using ANS as a probe and SDS-PAGE to study the membrane protein of the corn leaves with Htl gene. In a word, the aim of the research is to search some existent evidences of the toxin binding site on protoplasmic membrane, and provide proofs for nosogenesis of specific toxin in terms of cytology and the molecular biology. The main results are as follows:1. Five fractions, I (Rf0.06)、 II (Rf0.21)、 III (Rf0.45)、 IV (Rf0.60)、 V (Rf0.75) have been obtained after HT-toxin from race 1 of Exserohilum turcicum 99-2 isolated by TLC. In all of these fractions, only fractions II had specific toxicity to the corn leaves with Htl gene.2. Under the analysis conditions that inspect wavelength 220nm and velocity of flow1.0mL/min, 60% methanol in flowed phase was the optimum conditions. Under the above analysis conditions, fractions II-1、 II-2、 II-3 were isolated from fractions II by HPLC purification, and the bioassay result showed only fraction II-3 was toxigentic to corn leaves with Htl gene but non-toxigentic to corn leaves without Htl gene. Fractions II-1、II -2 and II -3 were scanned by UV-Vis spectrophotometer. It was shown that the fractions II-1 and II-2 had analogous spectrum, and especially the fraction II-3 had a special peak at 300nm.3. The death rate of protoplast from corn leaves treated by toxin was consistent with control (corn treated by distilled water instead of toxin) before 1.5h, but it's higher than control after 1.5h, which demonstrated the toxin had accelerated the death of corn cells. And moreover, the treated had a obvious ascend, which suggested that the toxin play a greatest role in 6h from side to side. So it is possible that the toxin does not injure cells
directly, but needs a certain time. And this course will be probably that the signal is amplified constantly after the toxin combines with its receptor, which make protoplasts lose vigor finally.4. The effect of the toxin on protoplasmic membrane protein was studied by the method of fluorescence, using 8-anilino-1 -naphthalene-sulfonic acid (ANS) as probe and taking the mesophyll protoplasts from maize leaves as the research system. After the liquid of protoplasts labeled ANS was injected with different toxin cone, dosage-effect relationship existed because of the ANS fluorescence intensity increasing obviously, which indicated the toxin had some kinds of effects on receptor of ANS. Two types of reagents were employed to prove the proteic characteristic of ANS receptor, the result of which demonstrated the proteic characteristic of the toxin. It was concluded that the first action site of the specific toxin might be on the outside of protoplasmic membrane, moreover, the toxin receptor has the proteic characteristic and the combination between toxin and its receptor has saturation activity.5. The effects of HT-toxin on soluble protein and membrane protein extracted from corn leaves treated by toxin were studied by the means of SDS-PAGE. The electrophoretic results showed that soluble protein bands had no change, but three membrane proteins(the molecular weight is 31.622KD, 63.125KD and 93.536KD) related to the toxin nosogenesis were found, and protein 31.622KD and protein 63.125KD are inherence of Htl gene corn leaves.
1.对玉米大斑病菌的1号小种菌株(99-2)的粗毒素进行硅胶薄层层析(TLC),分离到Ⅰ(Rf0.06)、Ⅱ(Rf0.21)、Ⅲ(Rf0.45)、Ⅳ(Rf0.60)、Ⅴ(Rf0.75)5个条带,分别对此5个条带进行生物测定,发现条带Ⅱ对带Htl基因的玉米具有较强的特异性。
2.在检测波长220nm、流速1.0mL/min的HPLC分析条件下,对条带Ⅱ的洗脱剂中甲醇与水的比例进行摸索,最后确定60%甲醇为最佳洗脱条件。经对条带Ⅱ进行高效液相色谱(HPLC)的进一步纯化,得到3种组分Ⅱ-1、Ⅱ-2、Ⅱ-3,其中只有Ⅱ-3具有特异致病活性。对Ⅱ-1、Ⅱ-2、Ⅱ-3进行紫外-可见光谱扫描。发现只有Ⅱ-3在300nm处有吸收峰,Ⅱ-1和Ⅱ-2的峰形相似,可能为类似物。
3.从HT-毒素Ⅱ-3组分处理玉米幼苗的叶片中提取原生质体,在0-1.5h内其死亡率与对照(蒸馏水处理幼苗)无明显差别,但1.5h后Ⅱ-3处理一直明显高于对照,说明Ⅱ-3加速了叶片细胞的死亡;在6h左右处理有显著上升,而对照变化不大。说明Ⅱ-3与叶片细胞作用后并不直接伤害细胞,而需经过一定时间,这一过程很可能是Ⅱ-3与受体结合后信号不断放大,最终使原生质体失去活力的过程。
4.以玉米叶片原生质体为研究对象,以ANS(1-苯胺基-8-萘磺酸)作荧光探针,用荧光法研究了Ⅱ-3与质膜蛋白的互作。在被ANS标记后的原生质体悬浮液中加入不同浓度HT-毒素Ⅱ-3组分后,ANS荧光强度明显增加,呈现出了剂量—效应关系,表明Ⅱ-3与质膜上ANS某些受体发生了互作。本试验采用两种试剂,从两个角度表明了ANS受体和质膜上存在的Ⅱ-3受体具有蛋白特性。推测HT-毒素特异性组分的原始作用位点在质膜外侧,且特异性毒素组分与其受体互作具明显的饱和性。
5.利用SDS-PAGE电泳技术分析了HT-毒素Ⅱ-3组分对玉米叶片中可溶性蛋白和质膜蛋白的影响。结果表明,Ⅱ-3对叶片中可溶性蛋白没有影响,但在叶片质膜蛋白中找到了3个与Ⅱ-3致病相关的蛋白,经分析其分子量分别为31.622KD、63.125KD和93.536KD,其中分子量为31.622KD和63.125KD的蛋白为叶片本身所特有。
In this dissertation, firstly, the conditions of isolation and purification of specific toxin fractions produced by Exserohilum turcicum has been studied, and a high-efficient method has been established;secondly, the toxins were analysed by UV-Vis spectrophotometry;then the effect of specific toxin on death rate of corn leaves protoplast was studied by FDA as a tinct reagent;finally, using ANS as a probe and SDS-PAGE to study the membrane protein of the corn leaves with Htl gene. In a word, the aim of the research is to search some existent evidences of the toxin binding site on protoplasmic membrane, and provide proofs for nosogenesis of specific toxin in terms of cytology and the molecular biology. The main results are as follows:1. Five fractions, I (Rf0.06)、 II (Rf0.21)、 III (Rf0.45)、 IV (Rf0.60)、 V (Rf0.75) have been obtained after HT-toxin from race 1 of Exserohilum turcicum 99-2 isolated by TLC. In all of these fractions, only fractions II had specific toxicity to the corn leaves with Htl gene.2. Under the analysis conditions that inspect wavelength 220nm and velocity of flow1.0mL/min, 60% methanol in flowed phase was the optimum conditions. Under the above analysis conditions, fractions II-1、 II-2、 II-3 were isolated from fractions II by HPLC purification, and the bioassay result showed only fraction II-3 was toxigentic to corn leaves with Htl gene but non-toxigentic to corn leaves without Htl gene. Fractions II-1、II -2 and II -3 were scanned by UV-Vis spectrophotometer. It was shown that the fractions II-1 and II-2 had analogous spectrum, and especially the fraction II-3 had a special peak at 300nm.3. The death rate of protoplast from corn leaves treated by toxin was consistent with control (corn treated by distilled water instead of toxin) before 1.5h, but it's higher than control after 1.5h, which demonstrated the toxin had accelerated the death of corn cells. And moreover, the treated had a obvious ascend, which suggested that the toxin play a greatest role in 6h from side to side. So it is possible that the toxin does not injure cells
directly, but needs a certain time. And this course will be probably that the signal is amplified constantly after the toxin combines with its receptor, which make protoplasts lose vigor finally.4. The effect of the toxin on protoplasmic membrane protein was studied by the method of fluorescence, using 8-anilino-1 -naphthalene-sulfonic acid (ANS) as probe and taking the mesophyll protoplasts from maize leaves as the research system. After the liquid of protoplasts labeled ANS was injected with different toxin cone, dosage-effect relationship existed because of the ANS fluorescence intensity increasing obviously, which indicated the toxin had some kinds of effects on receptor of ANS. Two types of reagents were employed to prove the proteic characteristic of ANS receptor, the result of which demonstrated the proteic characteristic of the toxin. It was concluded that the first action site of the specific toxin might be on the outside of protoplasmic membrane, moreover, the toxin receptor has the proteic characteristic and the combination between toxin and its receptor has saturation activity.5. The effects of HT-toxin on soluble protein and membrane protein extracted from corn leaves treated by toxin were studied by the means of SDS-PAGE. The electrophoretic results showed that soluble protein bands had no change, but three membrane proteins(the molecular weight is 31.622KD, 63.125KD and 93.536KD) related to the toxin nosogenesis were found, and protein 31.622KD and protein 63.125KD are inherence of Htl gene corn leaves.
引文
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