【作者】 顾甘雨；

【导师】 董汉松；

【作者基本信息】 南京农业大学 ， 植物病理学， 2006， 硕士

【摘要】 核黄素在生物体内形成黄素单核苷酸FMN及黄素腺嘌呤二核苷酸FAD，它们是黄素酶的辅酶。核黄素所形成的辅酶参与机体组织维生素C抗氧化作用的呼吸电子传递及氧化还原过程，参与植物抗氧化及过氧化过程，从而影响氧化性损伤及后续过敏反应过程中活性氧中间体(ROIs)的产生，而ROIs是过敏细胞坏死的重要信号，能调节生长、抗病、抗虫、抗逆，所以核黄素与植物免疫反应有关。外施核黄素可诱导植物抗病性，体内核黄素水平可能对抗病防卫和生长发育过程有影响。因此，如果能够控制核黄素水平，就能相应控制这些重要过程，提高植物抗病防卫的能力并促进植物生长发育。这是研究核黄素参与的植物防卫和生长调控交叉的重要点切入点。核黄素运载蛋白质(RCP)或受体蛋白质(RfBP)，首先在鸡蛋和卵生鸟类的血清中发现。它是一种单体、酸性的磷糖蛋白，对自由态的核黄素有高度的亲和性。最近报道，RCP是排卵卵母细胞和早期胚胎细胞膜一种组份，同时也是啮齿动物和其他的哺乳动物的成熟精液神经元体细胞末梢区域的一种组份。核黄素通过母鸡生蛋传送到鸡蛋的过程，就是被鸡核黄素运载蛋白或结合蛋白RfBP，一种27，000 Da的蛋白所调控的。在本研究中，作者克隆了软体海龟的核黄素受体基因(RfBP)，将其克隆至pGEM-T Easy载体经测序验证无误后分别克隆至pET30a(+)载体，构建了表达融合蛋白His-tagged RfBP的原核表达载体。研究表明，融合蛋白表达菌株BL21(DE3)pLySs：：pRfBP在1 mM IPTG、37℃诱导4 h的条件下都能够很好地表达融合蛋白His-tagged RfBP，且主要以可溶形式存在。继续提高诱导剂IPTG的浓度和延长诱导时间对其表达量影响不大。利用HisTrap HP Kit试剂盒对RfBP进行了纯化。每升诱导培养细胞可收获RfBP蛋白2 mg，浓度达到了0.5～1.5 mg／ml。His-tagged RfBP融合蛋白不能够在烟草上引起过敏反应。分别用提纯的软体海龟RfBP和鸡的RfBP蛋白免疫新西兰大白兔制备了抗血清，抗软体海龟RfBP血清的效价可达1：64，000以上，而抗鸡RfBP血清的效价可达1：256，000以上。在酶联免疫、Western blot分析中，两种RfBPs的多克隆抗体均能够检测含有RfBP蛋白的不同样品。两抗体之间存在一定程度的交叉反应。由于软体海龟和鸡的RfBP基因的推测产物氨基酸序列一致性达71.3％，这是它们之间存在交叉反应的重要原因。为进一步探讨核黄素在植物抗病及生长过程中的作用，本研究使用核黄素受体蛋白(RfBP)喷洒烟草和拟南芥，接种烟草赤星病菌和丁香假单胞番茄致病变种DC3000，并以野生型植株作对照，观察其在植物抗病和生长中的作用。结果显示：喷洒核黄素受体蛋白有助于提高植物的抗病性，但对植物的生长没有明显作用。同时，实验证明了外施核黄素受体蛋白，有助于提高内源核黄素的含量，这也为核黄素通过什么途径介入植物的抗病防卫提供了帮助。更多还原

【Abstract】 Riboflavin （vitamin B₂） participates in many physiological processes, particularlythose characterized by oxidation and reduction. The vitamin can combine with nucleotidesto form flavin adenine dinucleotide （FAD） and flavin mononucleotide （FMN）, which arecoenzyme of flavoproteins, and which play vital roles in many reactions, especially theproduction of reactive oxygen intermediates （ROIs）. In plants, ROIs are essential forhypersensitive cell death, growth, and defense against pathogens and insects, as well asmany aspects of responses to environmental cues. Levels of riboflavin in plants arebelieved to be important to these processes. As such, if riboflavin levels could bemanipulated, effects of the compound on the important processes could be monitored andcontrolled as a consequence. This would result in great improvement of plant defense andproductivity. This also could provide us with a particular insight into studying coordinationof plant defense with growth regulation, which may be affected by levels of riboflavin.Riboflavin carrier protein （RCP） or -binding protein （RfBP）, first identified in chickenegg and the serum of egg-laying birds is a monomeric, acidic, phosphoglycoprotein andbinds the free vitamin with high affinity. It has recently been shown that RfBP is amembrane component of ovulated oocytes, early embryo and of acrosomal region of maturesperms of rodents and other mammals. Riboflavin transport to the egg by the laying hen ismediated by a 27,000 Da protein, chicken riboflavin carrier or binding protein RfBP.The gene encoding RfBP from soft-shelled turtle was amplified and cloned into theexpression vector pET30a（+）, and the prokaryotic expression vector pET30a（+）::RfBP,called pRfBP, was constructed. Plasmid containing RfBP was transformed into competenceEscherichia. coli BL21 （DE3）pLysS. The bacterium was induced byisopropyl-Beta-D-thiogalactopyranoside （IPTG） and its expressed products were loadeddirectly onto sodium dodecyl sulphate polyacrylamide gel electrophoresis （SDS-PAGE）,approximately 35 kD exogenous protein was observed on the SDS-PAGE. His-tagged RfBP fusion protein was expressed well at 37℃for an additional 4 h after 1mM IPTGwas added, and a majority of fusion proteins was soluble. Higher concentration of IPTGand longer induced time did not accelerate the expression of the fusion protein.RfBP was purified using the HisTrap HP Kit. The yield was more than 2 mg purifiedRfBP proteins per liter of culture and the concentration of purified RfBP protein was about1.5-2.0 mg/ml. RfBP could not elicit hypersensitive response on tobacco. The polyclonalantibodies against RfBPs of both chicken and soft-shelled turtle were produced. The titer ofthe antibodies against chicken and soft-shelled turtle RfBP reached 1:64,000 and 1:256,000,respectively. Two antibodies against RfBPs could recognize the samples containing RfBPprotein in the analysis of ELISA and Western blot. It is imaginable that the cross reactionof two antibodies, because the amino acid sequence identity of chicken and soft-shelledturtle RfBPs reaches 71.3%.In conclusion, the exogenous application of RfBP to plants induces pathogen defense,but does not affect plant growth. Defense response is consistent with the increase in theendogenous riboflavin. These results provide a good basis for further study on the role ofRfBP modulating for riboflavin contents in plant growth and development regulation.更多还原