【作者】 李鑫；

【导师】 吴元华；

【作者基本信息】 沈阳农业大学 ， 植物病理学， 2009， 博士

【摘要】 本文以栽培烟草品种K 326（Nicotiana tabacum L.CV.K326）为试材,对矿质元素调控烟草抗PVY^N的作用进行了研究,从营养抗性角度揭示了矿质元素可通过调控烟草自身生理生化特性以提高植株的抗病毒能力,并明确了矿质营养调控烟草对病毒抗病性的分子机制,为矿质营养调控植物抗性利用奠定了重要理论基础。本论文主要研究结果如下:1.明确了镁、铁、铜、硼素可提高烟草抗病毒能力,几种元素处理均使植株病害减轻及病毒含量降低,几种元素调控烟草抗PVY^N的最佳浓度分别为240·L^-1、33.6mg·L^-1、0.8 mg·L^-1和20 mg·L^-1。本试验证实Mg3(240 mg·L^-1)、Fe4(33.6 mg·L^-1)、Cu3(0.8 mg·L^-1)及B3(20 mg·L^-1)处理的烟株病情指数显著低于接种对照,并且该四种元素浓度处理均能降低烟株体内病毒含量,由此可见Mg3、Fe4、Cu3及B3处理对PVY^N有较好的抗性,从表观上看,亦能延迟脉坏死症状的发生;不同元素处理24、72、120、168 h后接种PVY^N植株体内病毒含量差别显著,其中镁、铁、硼三种元素处理后24 h接种效果较好,能明显降低病毒含量,而铜元素处理24、72、120 h后烟株体内的病毒含量相近。因此,本试验选定喷施元素后24 h接种PVY^N为最佳作用时间。2.采用上述试验筛选的最佳铁、铜、镁、硼浓度处理,明确了矿质元素可通过调节烟株生理生化特性以提高抗性。研究表明:元素处理后,提高了烟株体内可溶性蛋白、脯氨酸、可溶性糖、叶绿素含量,促进PAL、POD、CAT等防御酶的活性,提高光合效率,降低植株体内有害物质丙二醛的含量,减少电解质的外渗,提高烟株木质化程度;喷施矿质元素也可在短期内迅速提高活性氧的含量,活性氧的迸发能刺激寄主植株防御反应的应答;而与叶脉褐变有关的PPO、总酚、类黄酮等物质则表现为前期高于对照后期低于对照烟株,以减少这些物质的破坏作用,因为过多的PPO、总酚、类黄酮会加重褐变。由此可见,喷施矿质元素一方面能增强烟株抗病性,同时也可减轻感病植株症状,减轻PVY^N对烟株的伤害。3.采用气相色谱（GC）、高效液相色谱（HPLC）的测定方法,研究发现铜素能调控感染PVY^N的烟株体内抗病信号物质水杨酸积累和乙烯释放。试验结果表明,水杨酸含量在接种后第15 d时达到最大值,为接种对照的1.3倍:而乙烯释放量在3～9 d内均高于接种对照,在第12 d时乙烯释放量显著低于接种对照,分析认为前期Cu素处理后诱导了乙烯释放的增强,进行抗性信号的传递,后期接种对照严重发病是其释放大量乙烯的直接原因。因此,喷施Cu素后可以诱导烟草植株产生抗病性,增强烟草对PVY^N侵染的抵抗力,并且诱导了相关抗性信号的传递。4.明确了铁、铜、镁素具有改变钙离子浓度、调节钙信使系统的功能,使与抗性信号传导相关的钙调素（CaM）、磷脂酶C（PLC）和蛋白激酶C（PKC）活性提高。通过测定不同元素调节钙离子的动态变化发现,元素处理后接种与未接种的烟株体内全钙及可溶性钙含量均有不同程度的增加,同时细胞膜上的Ca²⁺-ATPase和钙离子受体NADKase明显被激活,研究发现细胞内钙离子浓度升高与二者活性增加密切相关;研究发现,铜素处理后可诱导烟株体内CaM、PKC活性不同程度的提高,且均发生在较早阶段,而磷脂酶C（PLC）的活性变化随钙离子浓的变化而变化,二者呈负相关。由此可见,矿质元素可通过对钙离子浓度的调节,进而参与钙信使系统的抗性信号传递。5.采用SYBR GreenⅠ作为荧光染料的Real-time PCR方法,明确了铜、镁素能调控烟株体内蛋白激酶级联（MAPKs）信号转导途径的关键基因WIPK、SIPK及抗病信号通路末端PR1基因的过量表达,进一步揭示了矿质营养调控烟草对病毒抗病性的分子机制。本研究发现铜、镁元素分别处理及对照烟株中均有WIPK、SIPK及PR1基因的表达,但表达量有所差异,元素处理的烟株中表达量要高于对照烟株,接种处理的烟株表达量要高于未接种的烟株。其中,除SIPK最大表达量在96 h外,WIPK和PR1基因最大表达量均出现在168 h。由此可见,铜、镁元素处理能诱导信号通路中关键基因的过量表达,使烟株产生抗性,提高植株对病毒的抵抗力。更多还原

【Abstract】 The regulating action of mineral elements on tobacco plants for resistant to PVY^N was studied by using tobacco variety K326.The mineral elements can improve the tobacco resistant to PVY^N through promoting the physiological and biochemical property.The resistant molecular mechanism of tobacco regulated with mineral elements to PVY^N were revealed in this paper.1.Magnesium,iron,copper and boron can improve the tobacco resistant to PVY^N, which made the disease symptom lightened and the content of virus decreased at the best concentration of Mg 3(240 mg·L^-1),Fe4(33.6 mg·L^-1),Cu3(0.8 mg·L^-1) and B3(20 mg·L^-1). Effect on content of virus in tobacco which treated with different mineral elements at 24,72, 120,168 h showed that,the best induced time to tobacco treated with iron,magnesium and boron was 24 h,and that of copper treatment having no difference between 24,72 and 120 h. In a word,24 h was the best induced time to tobacco treated different elements.2.The Magnesium,iron,copper and boron can improve the tobacco resistant to PVY^N through promoting the physiological and biochemical property.The results showed that the activity of PAL,POD,CAT and the content of proline,soluble sugar,chlorophyll and lignin in tobacco leaves which inoculated with PVY^N after spraying mineral elements were increased,the content of leakage rate and MDA of electrolyte restrained.Those indicated that the induced resistance was promoted and the symptom was lightened.Furthermore,the production rate of activated oxygen raised sharply in a short time after treatment,those indicated that the induce resistance of tobacco was induced.Besides,the activity of PPO and content of total phenol and flavonoid,which closely linked to vein necrosis,were higher than those of the control in 12 days to resistant PVY^N,but much lower than those of the control when the vein necrosis came severity,which showed that the degree of vein browning was related to the PPO activity and the content of total phenol and flavonoid decreasing.In conclusion spraying iron can induce tobacco plant resistance to PVY^N.3.Content of salicylic acid and ethylene in tobacco were increased by spraying mineral elements.The results showed that the content of salicylic acid（SA） increased 1.3 times in 15th after treatment.The release of ethylene（ETH） was much higher than that of control in a short time.At the 12^th day,ETH released of control was sharply increased,which was much higher than that of copper treatment,because the signal pathway in tobacco leaves was activated when treated with copper solution in early time.But the symptom of control was seriously at later time,which is the factor of ETH released rose.In conclusion spraying copper solution can induce tobacco plant resistance to PVY^N and activate the signal pathway.4.The calcium system was regulated by changing of Ca2⁺ in tobacco treated with mineral elements,and the activity of CaM,PLC and PKC which related with signal transduction promoted.The effect of iron,copper and magnesium solution on the Calcium signal pathway had been preliminarily identified.Consequently,there has a close relative between the content of Ca²⁺ and Ca²⁺ -ATPase and NADKase activity in tobacco leaves. Compared with those of control treatments,the content of Ca²⁺ and total calcium was increased in different time,Ca²⁺ -ATPase and NADKase activated simultaneity.Through the method of ELISA,the activity of CaM and PKC in tobacco which treated with mineral elements was promoted in early time.But there has a a negative correlation between PLC and the content of Ca²⁺.So that the calcium signal pathway could adjust by mineral elements through altering the content of Ca²⁺.5.Take SYBR Green I as the intercalating fluorescence dye in Real-time PCR,the overexpression of WIPK and SIPK,which were the key genes of MAPK cascades,and the resistant gene PR1 in tobacco treated with copper and magnesium solution were studied.The results showed that,the expression of WIPK、SIPK and PR1 were appeared in tobacco of all treatment and control.But the amount of expression in tobacco treated with copper and magnesium solution were much higher than that of control.Except the expression levels of SIPK largest in the 96h,WIPK and PR1 expressions were both the largest in the 168h.So the key genes over expression of signal pathway in tobacco were induced by spraying copper and magnesium solution,the system resistance of tobacco to PVY^N appeared.更多还原