【作者】 张华；

【导师】 徐朗莱；

【作者基本信息】 南京农业大学 ， 生物化学与分子生物学， 2005， 博士

【摘要】 一氧化氮（Nitric oxide,NO）供体硝普钠（Sodium nitroprusside,SNP）能明显促进渗透胁迫下小麦种子的吸胀、萌发和胚芽胚根的生长,加速贮藏物淀粉降解为还原糖及可溶性小分子糖类;胁迫解除后,仍能维持种子较高的活力从而有利于幼苗的生长。进一步研究发现,SNP还能明显诱导胁迫下种子淀粉酶活性的上升,轻微提高蛋白水解酶活性,加速淀粉胚乳的液化或溶解,而SNP对酯酶影响不大。在探察NO对渗透胁迫下小麦种子萌发过程中抗氧化代谢时发现,一氧化氮（NO）供体硝普钠（SNP）能显著诱导渗透胁迫下CAT（catalase）、APX（acorbate peroxidase）活力的上升和脯氨酸含量积累,抑制LOX（lipoxygenase）活性,降低H₂O₂与MDA（malondialdehyde）的含量,从而提高渗透胁迫下小麦种子萌发过程中抗氧化能力,为种子的正常萌发提供一个良好的细胞内环境。 有意思的是,在小麦种子正常萌发早期,SNP处理使可以显著诱导内源葡萄糖、果糖和蔗糖含量的上升;进一步采用各种糖和SNP,并结合NO清除剂处理小麦种子,发现糖信号可能也介导了萌发早期小麦种子中淀粉酶的激活机制,且与NO之间存在着对话（Cross-talk）现象。而且在正常萌发条件下NO诱导切胚半粒小麦种子萌发早期（6h）的淀粉酶活力上升可能与GA₃无直接相关。并对NO诱导小麦种子淀粉酶活力的可能信号作用机理及其与GA、糖信号通路的关系进行了探讨。 进一步研究外源一氧化氮（NO）与赤霉素（GA）对小麦种子萌发早期（12h）淀粉酶调控的信号机制发现,在萌发早期（12h内）,α-淀粉酶和β-淀粉酶都不受GA所调控;而β-淀粉酶对NO存在着快速应答现象,但是,α-淀粉酶却没有这种应答关系。NO诱导β-淀粉酶活性的迅速上升可能与游离态β-淀粉酶聚合体的解聚以及结合态β-淀粉酶的直接释放有关;而NO促进的蛋白水解酶活性的轻微上升,对β-淀粉酶的激活或释放没有贡献;而且NO对β-淀粉酶单体也没有激活效应。此外,我们的研究结果还表明,β-淀粉酶对NO的快速应答现象同样存在于其它物种,如大麦、大豆、水稻、西瓜、抑南芥等种子的萌发早期,暗示了β-淀粉酶对NO的快速应答可能是种子萌发早期的一个普遍机制。更多还原

【Abstract】 Effects of nitric oxide （NO） donor, sodium nitroprusside （SNP） on the germination and activities of hydrolytic enzymes in wheat （Triticum aestivum L.） seeds were researched. Treatment with SNP dramatically promoted imbibition and germination of wheat seeds, increased the elongation of roots and shoots, accelerated the degradation of reserve starch and the liquefaction of endosperms under osmotic stress. After osmotic stress removed, the wheat seed treated with SNP could also retain higher viability. Further studies showed that SNP could induce amylase activities apparently, enhance protease activities slightly, while exhibit no roles in esterase. The effects of nitric oxide （NO） donor, sodium nitroprusside （SNP） on the antioxidant metabolism in germinating wheat seeds were also investigated. Treatment with SNP dramatically promoted the activities of CAT, APX and the contents of proline simultaneously, inhibited activities of LOX, decreased the contents of H₂O₂ and MDA. All of these functions were beneficial for improving the antioxidant capacity during germination of wheat seed under osmotic stress.Moreover, SNP could strongly increase the content of sugars, such as fructose, glucose and sucrose in wheat seeds during early germination （12 h） under normal conditions. When the seeds treated with SNP with or without PTIO, an NO scavenger, and different exogenous sugars, including fructose, glucose, sucrose and sorbitol, mannitol as controls, it was found that there might be existing a cross-talking between NO and sugars signaling in the activation of amylase in the early stage of germination, while GA acted no such actions in the same period.The effects of nitric oxide （NO） and gibberellic acid （GA₃） on the responses of amylases in wheat seeds were further investigated during the first 12 h of germination. GA3 had no effects on the activities of α-amylase （EC 3.2.1.1） or p-amylase （EC 3.2.1.2）, either in intact seeds or embryoless halves within 12 h. In contrast, addition of sodiumnitroprusside （SNP）, an NO donor, was able to induce a rapid increase in P-amylase activity without affecting a-amylase. Furthermore, the rapid response of p-amylase to SNP in wheat seeds could be attributed to NO and was approximately dose-dependent. Some other aspects of SNP induction of amylase isozymes were also characterized. Further investigations showed that SNP might play an interesting role in the dissociation of free p-amylase from small homopolymers or heteropolymers. Furthermore, SNP also directly induced the release of bound P-amylase from glutenin and its crude enzyme preparation. However, the slight increase in protease induced by SNP might not be responsible for this action. Interestingly, based on the fact that the rapid response of p-amylase to NO also existed in seeds of other species, such as barley, soybean, rice and watermelon, it might be a universal event in early seed germination.Activities of P-amylase in seeds of three wheat species and their germination index were investigated at the same time. It showed that there was a positive correlationship between activities of p-amylase and the index of germination in wheat seeds. Experiment of p-amylase inhibitors also showed us P-amylase played a key role in wheat seeds germination.In this paper, characterization of beta-amylase isozymes induced by NO was further investigated. The pI of the SNP-induced beta-amylases, Type II, as determined by IEF, was in the range 6.0-7.0, and that of the intrinsically active ones, Type I, in the range 4.8-6.0. The optimum pH of these amylases was found to be 4.5-6.5, and they were stable in the pH range 3.0-9.0. The optimum temperature of these enzymes was 40°C, but they active even up to 55 °C. Further investigations using protein synthesis inhibitors, such as actidione D and chloromycetin, showed that beta-amylase isozymes induced by NO were not synthesized de novo.更多还原