【作者】 郭笑彤；

【导师】 左元梅；

【作者基本信息】 中国农业大学 ， 植物营养学， 2014， 博士

【摘要】 花生(Arachis hypogaea L.)是我国重要的油料作物,在石灰性土壤上种植容易出现缺铁黄化现象,而与玉米(Zea mays L.)间作可以有效缓解这种现象。玉米/花生田间和盆栽间作试验都证明间作能够改善花生铁营养这一生理现象,并且确定了麦根酸在间作改善花生铁营养过程中起到重要作用,而在分子水平上也对花生中铁吸收和转运基因进行了初步研究。尽管如此,间作体系调控花生铁营养的机制仍不清楚。本文通过研究花生铁吸收和转运基因生物学功能解析玉米/花生间作体系中花生铁吸收和转移的分子生物学机制,进而阐明玉米/花生间作改善花生铁营养的分子生态调控机制。为确定花生铁吸收和转移相关基因的分子生物学功能,对石灰性土壤条件下生长的花生AhIRT1转基因烟草和水稻以及AhNRAMP1、AhFRO1、AhYSL3.1转基因水稻株系进行了分析。结果表明,石灰性土壤条件下转基因植株的生长状况和铁营养状况明显好于非转基因植株,表明诱导表达花生铁吸收转运相关基因在植物中发挥作用能够提高植物耐缺铁能力；同时,在玉米/花生间作体系中应用抑制性差减杂交的方法获得AhOPT1基因,它在花生根系和叶片都受到缺铁诱导上调表达,酵母功能互补试验证明AhOPT1能够吸收铁。玉米/花生间作盆栽试验表明间作体系有效降低花生根际pH,促进根际铁活化,根际有效铁含量较单作显著升高,而有效氮、磷养分显著降低,减少其对铁的螫合作用；而玉米/花生间作田间试验结果也表明间作后花生根际有效铁、锌养分含量在整个生育期呈上升趋势。综上说明间作动态调控了花生根际环境。通过对田间盆栽条件下单间作花生根系铁吸收相关基因表达分析确定间作效应在开花期调控AhNRAMP1、AhFRO1、AhYSL1基因高效表达进行Fe(Ⅱ)和Fe(Ⅲ)的吸收,在结荚期主要调控AhYSL1基因高效表达进行Fe(Ⅲ)的吸收,在饱果期间作效应不再调控花生铁吸收基因表达,而AhIRT1基因在花生全生育期都受到花生铁营养状况调控,由此可以说明,间作体系中花生铁吸收是由间作效应和花生铁营养状况共同调控的。通过对玉米/花生间作田间试验花生地上部铁分配的研究发现,间作后花生不同生育期铁分配发生明显的改变,而这种变化与不同时期花生各部位铁营养需求相一致。而在田间和盆栽试验中通过对单间作花生铁转运基因AhYSL3.1、AhFRDL1以及AhOPT1表达差异分析发现,间作后花生铁转运基因在不同部位不同时期表达发生变化,而这种变化调整了花生铁转移从而满足不同时期各部位花生铁营养需求。更多还原

【Abstract】 Peanut (Arachis hypogaea L.) is an important oil crop in China and it easily shows iron deficiency-induced chlorosis in calcareous soils. This phenomenon can be effectively improved when peanut intercropped with maize (Zea mays L.). The peanut/maize intercropping system can improve iron nutrition of peanut, which has been identified at physiological level via both field and pot experiments, and the DMA has been proved playing an important role in improving peanut iron nutrition in intercropping system. Besides, the preliminary studies of iron absorption and transport genes have been reported. Even so, the mechanism involved in this process remains unclear. In this study, by investigation the biological functions of iron-related genes of peanut the molecular mechanism of iron absorption and translocation in intercropped peanut was elucidated. Moreover, the molecular and ecological mechanism of peanut/maize intercropping improving iron nutrition of peanut was unraveled.To indentify the biological functions of iron-related genes of peanut, the transgenic tobacco and rice plants of induced expression of peanut AhIRT1gene and the transgenic rice plants by induced expression of AhNRAMP1, AhFRO1and AhYSL3.1genes in calcareous soils were investigated. The results showed that the growth and nutrient status of transgenic plants were obviously better than that of non-transgenic plants, suggesting induced expression of peanut iron-related genes in plants conferred enhanced tolerance to iron deprivation. Meanwhile, AhOPT1gene was isolated by suppression subtractive hybridization from the roots of monocropped and intercropped peanut. The mRNA level of AhOPT1was obviously induced by iron deficiency in both roots and leaves. Moreover, yeast complementation assays implied that AhOPT1encoded a functional iron transporter.In the pot experiments, compared with monocropping system, the pH of rhizosphere in intercopping system had been reduced in most peanut growth period while the available iron increased remarkably. Meanwhile, the concentration of soil Olsen-P and total nitrogen significantly decreased in intercopping system, which reduced chelation of iron and thus improved the availability of iron in rhizosphere. And the results from the field experiments also indicated that the concentration of available iron and zinc of rhizosphere tended to increase in intercopping system during the whole growth stages. In conclusion, peanut/maize intercropping dynamically regulated the rhizosphere environment. The mRNA levels of peanut iron absorption-related genes in intercropping and monocorpping were analyzed. The results showed AhNRAMP1, AhFRO1and AhYSL1genes were highly expressed in intercropped peanut during anthesis, which may result in effectively uptake of Fe(Ⅱ) and Fe(Ⅲ). Only AhYSLl gene was still highly expressed in intercropped peanut for absorption of Fe(Ⅲ) during pod-setting stage. No gene was regulated by intercropping system in pod filling stage. Besides, AhIRT1gene was regulated by peanut iron status in the whole growth period. Therefore, we concluded that the uptake of iron in intercropping peanut was affected by both intercropping system and the iron status of peanut.In field experiment, the iron distribution of peanut shoots in monocropping and intercropping system was investigated and it demonstrated that the iron distribution in intercropped peanut was remarkably changed during different growth stages, which is consistent with the demand of iron in peanut. The expression levels of peanut iron transport genes AhYSL3.1, AhFRDLl and AhOPT1from both field and pot experiments indicated that intercropping regulated the expression of these genes in different parts of peanut during different growth stages, which modified the translocation of iron and thus meet the iron demands of peanut in various growth periods.更多还原