【作者】 胡永建；

【导师】 金幼菊；

【作者基本信息】 北京林业大学 ， 植物学， 2010， 博士

【摘要】 马尾松(Pinus massoniana)是我国南部特有乡土树种,面积居全国针叶林首位,在荒山造林及采脂中发挥着重要的生态效益和经济价值。但常遭受马尾松毛虫(Dendrolimus Punctatus Walker)的危害,猖獗年份针叶全部食尽,给林业带来巨大损失。为解释马尾松毛虫周期爆发的机理,发挥森林生态系统自主调节作用,完善害虫综合治理策略,本文从马尾松诱导防御角度进行了以下几个方面的研究。1)用扫描电镜观察了马尾松毛虫危害后马尾松针叶横切面树脂道变化,发现针叶树脂道中有许多紧密排列、且不易挥发的白色颗粒状树小球,推测其为二萜酸类物质,表明马尾松诱导防御反应中物理抗性增强。同时,单萜类具有与其相同的丙酮酸／3-磷酸甘油醛代谢途径,间接说明虫害后起主要防御作用的萜烯类挥发物通过重新合成来发挥其重要生态功能。2)对盆栽马尾松接虫咬食处理后,通过TCT-GC／MS分析了同株中未受害轮枝针叶挥发物成分及相对含量的时序变化。结果表明,与对照相比,挥发物组分相同,但多数挥发物相对含量变化显著,萜烯类化合物如莰烯、4(10)-宁烯、β-蒎烯、p-散花烯、柠檬烯、β-水芹烯、β-石竹烯、α-石竹烯、罗汉柏烯和含氧化合物辛醛、壬醛、癸醛、冰片乙酯、戊二烯二丁酯等的相对含量都有明显的时序变化。另外,邻枝针叶内源茉莉酸含量测定结果显示,茉莉酸含量在2h显著高于对照,与受害枝的内源茉莉酸相比,具有滞后性。表明虫害后马尾松的伤信号可以从受害部位传递至周围邻枝,引起整个植株产生防御反应。茉莉酸在调节挥发物的系统诱导防御中起重要作用。3)通过自制密闭玻璃箱对虫害马尾松邻株针叶挥发物及信号物质进行研究。结果表明,在感受到虫害株释放的伤信号后,健康马尾松针叶茉莉酸含量迅速增加,并在48h达到高峰,72h降至正常水平。脂氧合酶是茉莉酸合成途径中的关键酶,在虫害邻株针叶中,其活性呈先升高后回落的趋势,且在12h达到最高,与内源茉莉酸的变化相一致。水杨酸在处理后所测各点始终高于对照,12h达到峰值。ABA方差分析差异不显著。4)在上述研究的基础上,进一步持续检测了密闭环境中可能的报警信号及邻株健康马尾松挥发物的时序变化。结果表明,箱体中没有检测到茉莉酸甲酯,但却检测到乙烯的存在,在处理的10h内一直维持在5μg／L左右。邻株挥发物的成分与对照相同,但相对含量差异显著。多数挥发物的相对含量在处理后就有所升高,在24h或36h达到峰值。初步证明虫害马尾松与相邻健康马尾松之间存在明显的化学通讯,邻株能够迅速感知虫害株挥发物指纹图的改变,并产生相应的防御功能。邻株马尾松激发了茉莉酸信号途径,且与乙烯、水杨酸表现出协同作用,共同调节挥发物的变化。5)为了深入了解茉莉酸对马尾松防御相关蛋白调节机制,通过外源茉莉酸甲酯喷施处理,利用蛋白质双向电泳及生物质谱技术,首次研究了处理前后蛋白表达的差异。胶图结果显示,蛋白主要集中在pH4-7的区间内,分子量从15-82kD。从中找出56个差异点,处理后9个蛋白点丢失,新合成27个,其余20个差异显著的蛋白点中,11个表达上调,9个下调。生物质谱鉴定出21个差异蛋白,以1,5-二磷酸核酮糖羧化酶为主,在诱导防御中的能量供给上发挥重要作用。同时,通过优化提取试剂,挑选最适pH范围的等电聚焦胶条等,成功建立了适合马尾松针叶蛋白样品的提取方法及电泳条件,为马尾松蛋白质组学研究奠定了良好的基础。6)模拟吸附热解析方式,首次通过ATD-GC/MS建立了常见挥发物的外标定量曲线,并对其条件进行了优化,即直接进样方式、流速50±3ml／min、干吹时间1min、温度50℃,当标准化合物含量在几十pg～几百ng之间时,其线性范围最佳。这一研究弥补了热脱附所测挥发物的绝对定量问题,为今后挥发物定量提供了科学依据。更多还原

【Abstract】 Pinus massoniana is a native endemic species in southern China, holding the biggest areas among coniferous forests. It has enormous ecological and economic value, especially in barren mountain afforestation and resin tapping. However, it often suffers from the feeding of Dendrolimus Punctatus seriously, resulting in huge losses to the forestry. For explaining the pest outbreak mechanism, revealing the self-regulation function of forest ecosystem, improving integrated pest management strategies, several aspects about the rapid induced resistance of P. massoniana have been researched in this dissertation.1) The needle transverse plane of P. massoniana had been observed by scanning electron microscope (SEM). There were many white granular resin acid arranged closely in the resin ducts after punctatus feeding, showing the physical resistance is increased by their re-synthesis. Meanwhile, according monoterpenea and diterpene acids sharing the same pyruvate/3-glyceraldehyde phosphate metabolic synthesis pathway, this proved indirectly that the volatile terpenoids were also re-produced in the resin ducts, which played an important function in plant induced resistance.2) After one branch needles of potted plant P. massioniana was fed by D. Punctatus, the composition and relative contents of volatile organic compounds (VOCs) from the adjacent upper branch needles of insect-damaged branch at different times was analyzed by TCT-GC/MS. The results showed that terpenes were the dominant, while the others such as oxygenated and nitrogenous compounds were less abundant VOCs. Most of them in treated plants had higher contents than those in the control after 1h treatment and remained much high levels at 2h, such as camphene, 4(10)-Thujene,P-pinene, o-Cymene, limonene,β-phellandrene, p-Metha-1.4(8)-dien,β-Caryophyllene, a-Caryophyllene, as well as some oxygenates including octanal, nonanal, decanal, borneol acetate, glutaric acid, dibutyl ester and cyclohexyl isothiocyanate. The content of methyl jasmonic acids (MeJA) in adjacent needles of infected P. massioniana was increased after 1h treatment and nearly doubled that of control plants at 2h. The data indicates that the defensive system of P. massioniana was initiated and leading to the resistance reaction of the neighborhood undamaged branches by inset-pest.3) For investigating whether and how gymnosperm can perceive chemical compound as pre-warning signals from adjacent neighbors, insect-damaged P. massoniana and healthy ones were placed in one transparent closed glass box. After treated for 10h, the dynamic change of endogenous jasmonic acid (JA), abscisic acid (ABA), salicylic acid (SA), lipoxygenase (LOX) and VOCs from healthy needles had been studied. The results showed that the content of JAs increased immediately and reached twice times as control after treatment, then it was keeping in a higher level than control until another peak come again at 48h. LOX is a key enzyme in the synthesis of JA, its activity was increased at first and then trend down, the change trend was consistent with that of JA. SA in measured points was always higher than controls, and reached its peak in 12h. Although in most points ABA higher than the control, the analysis of variance was not significant.4) Furthermore, compared with control, no new compounds produced emitted from adjacent no-feeding P. massoniana needles, but most of their relative percentages like Tricyclene, a-Pinene,β-Phellandrene promoted and reached the peak between 24-36h. In addition, to explore what kinds of chemicals can be the potential elicitor, the blend of gases in the glass cube were collected and analyzed, apart from the common VOCs from P. massoniana, ethylene (ETH) was detected and was keeping stable content about 5μg/L among the treating points. The reported volatile MeJAs was not found. The date indicates that the neighboring plants make evident response to the received information and initiated the rapid resistance reaction, plant-to-plant communication indeed exists between damaged and undamaged P. massoniana. In the inspired signal conduction pathway, JA, SA and ET showed synergy effect in the regulation of volatiles.5) To understand the regulation mechanism of JA on the pinus defense-related proteins better, by using two dimensional electrophoresis (2-DE) and tandem mass spectrum techniques,56 JA-responsive proteins were sieved and analyzed between control and exogenous MeJA spraying treatment in P. massoniana needles. Those protein spots mainly distributed in pH4～7，MW15kD～82kD. Among of them, the expression of 11 proteins increased,9 decreased,9 disappeared, and 27 new synthesized. Twenty-one proteins was analysized by TOF-MS. R ibulose-1,5-bisphosphate carboxylase/oxygenase is the main differential expression proteins. Meanwhile, by optimizing the extraction, selection of the optimum pH range of isoelectric focusing strips, etc, the 2-DE maps was gained with less tailing, clearly and well distributed protein spots, and have high repetition rate. The successful establishment of pine needle protein extraction and electrophoresis conditions has laid a good foundation for future pine proteomics research.6) Simulating adsorption-thermal desorption procedure, external standard quantitative curves of common pinus VOCs were established by atuo-themal-desorber (ATD)-GC/MS. When the flow rate of N2 is 50±3ml/min, dry blow time lmin, temperature 50℃, the results are repeatable well. For the MS, the injection content of standard compounds between 50pg-700ng, the linear range is best. The external standard method could determine the absolute content of VOCs, which supplies a more accurate and reliable quantitative manner for plant VOCs research.更多还原