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Study On The Communication Between Healthy And Pest-fed Pinus Massoniana And Differential Expression Proteins Induced By MeJA

Posted on:2011-12-19Degree:DoctorType:Dissertation
Country:ChinaCandidate:Y J HuFull Text:PDF
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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.
Keywords/Search Tags:Pinus massoniana, volatile organic compounds, endogenous signals, systemic resistance, interplant communication, proteomics, external standard method
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