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1、分子生物学论文外源一氧化氮促进小麦种子萌发及其信号作用机制研究3目 录目 录摘 要IABSTRACTIII缩略语词汇表V引言1文献综述3 种子萌发机理的研究进展3 谷物种子中淀粉的降解及其水解酶16 植物一氧化氮（NO）研究进展20参考文献25第一部分 NO供体对渗透胁迫下小麦种子萌发与抗氧化代谢的影响33第一章 NO供体对渗透胁迫下小麦种子吸胀萌发和水解酶活性的影响331 材料与方法352 结果与分析373 讨论44第二章 NO供体对渗透胁迫下小麦种子萌发过程中抗氧化代谢的调控501 材料与方法512 结果与分析533 讨论58第二部分 小麦种子萌发早期b-淀粉酶对NO的快速应答及其生化特性
2、研究61第三章 小麦种子萌发早期b-淀粉酶对NO的快速应答611 材料与方法632 结果与分析653 讨论80第四章 小麦种子萌发早期NO诱导的b-淀粉酶同工酶的特性841 材料与方法852 结果与分析863 讨论91全文结论94创新之处95存在的问题与展望96致谢98中文摘要外源一氧化氮促进小麦种子萌发及其信号作用机制研究摘 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。
3、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处理便可
4、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（6 h）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），-淀粉酶和b-淀粉酶都不受GA所调控；而b-淀粉酶对NO存在着快速应答现象，
5、但是，-淀粉酶却没有这种应答关系。NO诱导b-淀粉酶活性的迅速上升可能与游离态b-淀粉酶聚合体的解聚以及结合态b-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b-淀粉酶的激活或释放没有贡献；而且NO对b-淀粉酶单体也没有激活效应。此外，我们的研究结果还表明，b-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b-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。采用-淀粉酶活性大小存在差异的三个小麦品种研究发现，-淀粉酶活性与种子的萌发速率呈正相关，同时也暗示了，在正常条件下，高活性或高含量的-淀粉酶可能是促进种子萌
6、发的诱因之一。同时结合-淀粉酶抑制剂的实验结果也表明，-淀粉酶在小麦种子萌发过程中起着重要作用。研究小麦种子萌发早期NO诱导的b-淀粉酶同工酶的特性发现，萌发早期小麦种子中的b-淀粉酶最适pH为4.5-6.5，在pH3.0-9.0范围内较稳定；最适温度为40，高于55时活力丧失，表现了其对热的不稳定性；自由态的种子中预存的自由态I型-淀粉酶同工酶pI在4.8-6.0范围，中性偏酸；依赖于NO而产生的II型-淀粉酶同工酶的pI在6.0-7.0范围，偏中性。抑制剂实验表明，萌发早期所有的-淀粉酶均含有巯基。进一步采用蛋白质合成抑制剂的实验结果表明，依赖于NO诱导的-淀粉酶同工酶在种子萌发早期（12
7、h）不是从头合成的。关键词： 一氧化氮；种子萌发；渗透胁迫；小麦（Triticum aestivum L.）；赤霉素；-淀粉酶英文摘要SIGNAL MECHANISMS OF EXOGENOUS NITRIC OXIDE ON THE PROMOTION OF WHEAT SEEDS GERMINATIONSTRESSABSTRACTEffects of nitric oxide (NO) donor, sodium nitroprusside (SNP) on the germination and activities of hydrolytic enzymes in wheat (Tri
8、ticum 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 remo
9、ved, 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 ant
10、ioxidant 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 impro
11、ving 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 witho
12、ut 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
13、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 -amylase (EC 3.2.1.1) or -amylase (EC 3.2.1.2), either in
14、 intact seeds or embryoless halves within 12 h. In contrast, addition of sodium nitroprusside (SNP), an NO donor, was able to induce a rapid increase in -amylase activity without affecting -amylase. Furthermore, the rapid response of -amylase to SNP in wheat seeds could be attributed to NO and was a
15、pproximately 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 -amylase from small homopolymers or heteropolymers. Furthermore, SNP also directly induced t
16、he release of bound -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 -amylase to NO also existed in seeds of other species, such as barley
