| 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 cellsdirectly, 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. |
PDF Full Text Request