【作者】 陈松；

【导师】 张国平；

【作者基本信息】 浙江大学 ， 作物学， 2010， 博士

【摘要】 改善施肥技术和挖掘超高产水稻的氮素利用潜力,从而提高水稻氮素利用效率已成为当前研究的热点。本研究以超高产水稻品种为材料,利用大田试验,探索了超高产水稻养分利用的品种特性及其在不同耕作、播种方法、氮肥运筹处理下的产量形成和养分吸收特性,利用重组自交系进行养分利用效率的QTL分析,并结合双向电泳与质谱分析技术,研究超高产水稻养分利用效率的分子遗传机理。主要结果如下：1、超高产水稻品种的产量显著受年份和地点的影响,供试的两个超高产水稻品种的高产表现归因为有较高的库容,如较多的单位面积穗数和每穗粒数。茎鞘和叶片的干物质积累主要集中在分蘖期到齐穗期,在灌浆期减少。光合特性,如净光合速率(Pn)、叶面积指数(LAI)、比叶面积(SLA)、叶绿素荧光(Fv／Fm)、SPAD值和养分吸收和利用等指标,均存在着显著的环境效应,而部分生理性状受环境影响较小,如分蘖期的SLA、齐穗期的Fv／Fm和LAI、齐穗期和收获期的茎鞘干物质积累量和叶片氮含量,启示出这些性状可用于育种程序。另外,相关分析表明,齐穗期净光合速率等可作为超高产水稻高产的衡量指标。2、不同耕作方式对产量的影响因地区而异。与翻耕相比,免耕水稻齐穗前干物质积累能力较弱,而灌浆期则相反。免耕的净光合速率显著高于翻耕,而Fv／Fm则无显著差异。耕作方式对蒸腾速率和SPAD值的影响因地点和年份而异。免耕的茎鞘和穗部氮浓度要低于翻耕。磷浓度在齐穗前不同耕作方式之间没有显著差异,但是在收获期,免耕稻穗部磷含量显著高于翻耕。免耕稻茎鞘和叶片的钾浓度显著高于翻耕。在收获期,耕作方式对养分积累的影响因部位不同而有所差异。与翻耕相比,免耕稻在生育后期茎鞘和叶片养分积累量略高,而在生育前期其差异又因地点和年份而异。免耕条件下,氮收获指数(NHI)和钾收获指数(KHI)都显著较高,而磷收获系数(PHI)受环境影响较小。与翻耕稻相比,免耕稻的养分利用效率较低。3、不同播种方式(直播、移栽)对超高产水稻品种产量及其构成、干物质积累、叶片光合特性、稻米品质以及养分利用的影响表现为：直播稻在粒重、分蘖前期每株分蘖数、叶绿素荧光参数Fv/Fm以及蒸腾速率上具有优势,而在产量、单位面积穗数、结实率、穗重、净光合速率和气孔导度上则刚好相反。此外,在每穗粒数、成熟期茎鞘、叶片干重等指标上,播种方式之间无显著差异。播种方式对植株生长和产量的影响存在着显著的品种差异。供试品种中,相对高产的春优58和相对低产的甬优6号具有不同的生理特性及对处理的反应。春优58的单位面积穗数、每穗粒数和穗部干物质含量较高。春优58在移栽条件下,净光合速率较高,而甬优6号则以直播中较高。大部分稻米品质性状和养分利用特性在不同播种方式下存在着显著的环境效应,但移栽稻的整精米率、糙米率以及粒宽在不同环境下均显著高于直播稻,而恶白度和胶稠度则刚好相反。相关分析表明,稻米垩白度、透明度、胶稠度、直链淀粉含量以及蛋白质含量与氮、磷、钾的吸收利用呈显著相关。4、不同肥料运筹对超高产水稻品种产量、高产特性以及养分利用的影响表现为,同对照(F1)相比,减量后重处理(F4)与减量(F2)的产量略低但差异不显著,后重处理(F3)与对照基本持平或略高。单位面积穗数和每穗粒数,减量处理(F2或F4)显著降低,而后重处理(F3)变化不大；减量和后重处理(F2、F3、F4)均不同程度地提高结实率,且对粒重有显著影响。这表明,库容大小受氮肥用量的影响,后期增施有一定的补偿作用。氮肥对前期干物质的影响,主要集中在茎鞘上,表现为分蘖期F4显著低于对照,始穗和齐穗期则无显著差异,而收获期和分蘖期相似。这表明,前期减少氮肥的水稻有较大的灌浆期茎鞘干物质转运能力。SPAD值和净光合速率在生育前期处理间无显著差异,而在齐穗期,后重处理(F3)要略高于对照。茎鞘NPK的吸收和积累前期以F4最低,其他处理无显著差异。减肥、后重(F2、F3、F4)均能不同程度提高氮利用效率(NUE),但磷利用效率(PUE)、钾利用效率(KUE)、NHI、PHI和KHI则受环境和品种的影响,且它们的表现各不相同。5、利用超级杂交稻汕优10号(珍汕97×密阳46)重组自交系247个株系,分别于2007年和2008年种植在中国水稻研究所(浙江、富阳),成熟后测定植株氮磷钾含量,计算养分利用效率和养分收获指数。应用有207个RFLP和SSLP标记构建的遗传连锁图来确定养分利用性状的QTL位置和效应。结果表明：6个养分利用性状在2个环境中均连续分布,且都存在一定数量上的双向超亲遗传类型。共有7个主效QTL基因,6对上位性QTL基因在2年试验中都检测到,分布在除4、7、9外的其他染色体上。其中NUE、PUE都检测到1个主效QTL；NHI、PHI和KHI分别检测到2、2和1个主效QTL,单个QTL对性状的贡献率为3.36%-7.20%,其中控制磷素利用效率的QTL座位(RM1-RM151)与控制氮素收获指数的QTL座位(RM151-RG532)相邻,说明控制N、P利用的QTL可能是连锁的。6、研究了不同氮素利用效率的两个超高产水稻品种在氮胁迫条件下的蛋白质表达谱,以明确水稻氮素利用效率的分子机理。在水稻4叶期进行氮胁迫处理,处理后12小时、3天、7天取最大展开叶分析。叶片总蛋白提取后,双向电泳以分离蛋白。双向电泳监测到1000多个点,其中只有一小部分具有显著差异。在春优58和甬优6号中分别找到了8和16个上调蛋白,2和11个下调蛋白。结果显示,与NUE相关的生化途径可能相对简单,说明NUE易于人为调控。基于质谱分析结果,找到了37个蛋白点,其中6个与胁迫诱导相关,包括DegP2蛋白、harpin蛋白、热激蛋白、谷胱甘肽巯基转移酶(GSTF14)、类Fibrilin蛋白和3-磷酸甘油醛脱氢酶(Glyceraldehyde-3-phosphate dehydrogenase)。除了与胁迫相关的蛋白外,其他蛋白点都与叶片功能相关,如光合作用Rubisco激酶和Rubisco大亚基等。本研究还发现了2种与氮胁迫相关的新蛋白：harpin结合蛋白和oryzainsγ前体。更多还原

【Abstract】 Recently, the investigation on increasing the rice nutrient utilization efficiency by upgrading the cultivation system and exploring the genotypic potential has been emphasized. We studied yield performance and its physiological characters, as well as nutrient utilization traits of high yielding rice varieties through different treatments, including cultivation (conversional tillage and no tillage), seeding (transplanting and direct seeding) and fertilizer application strategies. Moreover, the QTL analysis of nutrient utilization efficiency was analyzed using a RIL population. In addition, proteomic analysis of the high yielding rice exposed to the different nitrogen levels was also conducted. The main results are as follows:1. There was a large variation in grain yield for the two super-rice varieties among locations and years. The realization of high yield potential for the two varieties was closely related to the improved sink size, i.e. more panicles per m2 or grains per panicle. Stem and leaf biomass was mainly accumulated during tillering to heading stages, and showed slow decline at grain-filling stage. Meanwhile, some photosynthetic characters, including net photosynthesis rate (Pn), leaf area index (LAI), specific leaf area (SLA), fluorescence parameter (maximum quantum yield of PSII, Fv/Fm), chlorophyll content (expressed as SPAD value), as well as nutrient (N, P, K) uptake were also measured to determine their variations over genotypes and environments and their relationships with grain yield. Although there were significant differences between years or locations for most measurements, SLA at tillering and heading stages, Fv/Fm and LAI at heading stage, stem biomass at heading and maturity stages, and leaf nitrogen concentration at tillering and heading stages remained little changed, indicating their possible applications as selectable characters in breeding programs. It was also found that stem nitrogen accumulation at tillering stage is one of the most important and stable traits for high yield formation.2. The influence of no tillage (NT) on leaf photosynthesis nutrient uptake, remobilization and partitioning in rice plants was determined. Te experiments with treatments NT and conventional tillage/plough follow (CT) were carried out at two locations (Jiaxing and Hangzhou) for two years (2005 and 2006). Grain yield was constantly lower in Jiaxing, but slightly higher in Hangzhou for NT cultivation than for conventional one in the both two years. In comparison with the conventional cultivation, no-tillage cultivation had less biomass accumulation before heading and higher capacity of matter production during grain filling. A significantly higher leaf net photosynthetic rate was observed for the plants under NT than for those under CT. A fluorescence parameter, Fv/Fm did not show any difference between the two cultivations. The effect of cultivation methods on transpiration rate (Tr) and SPAD value was dependent on the location and year. N concentrations in stem and panicle in no tillage (NT) were lower than those in conventional tillage (CT). However, leaf N concentration was relatively higher in NT plants after heading stage. No significant difference was found in P concentration between two tillage treatments before heading stage. However, at mature stage, NT had higher P concentration in panicle. NT plants had higher K concentrations in both leaf and stem than CT ones before heading stage. At maturity stages, the difference about the nutrient accumulation between the two tillage treatments varied with plant organs. NT plants had significantly higher stem and leaf nutrient accumulations than CT ones at late stages, while at early stage, the difference between the two tillage treatments varied with location and year. Both N and K harvest index (NHI and KHI) was significantly higher in NT plants than in CT plants, but the difference between the two tillage treatments in P harvest index (PHI) was changeable over year and location. NT plants had lower nutrient utilization efficiency in comparison with CT plants.3. The responses of six super-rice cultivars to two cultivation methods, transplanting (TP) and direct-sowing was investigated. It was shown that grain weight and tillers per plant at early growth stage, maximum quantum yield of PSII (Fv/Fm) and transpiration rate (Tr) was higher in DS than in TP, while grain yield, panicles per m2, grain-setting percentage, panicle weight, net photosynthetic rate (Pn) and stomatal conductance (Cond) were just opposite. Meanwhile, little difference was found in grains per panicle, stem (shoot) and leaf weight between the two planting methods. The responses of plant growth and physiological traits to planting method differed greatly among the 6 cultivars. It was found that the higher yield of Chunyou 58 was associated with more panicles per m2 and grains per panicle in both planting methods than the other cultivars. Chouyou 58 and Yongyou 6 had the highest and lowest panicle biomass in the both planting methods, while Chunyou 58 had the highest Pn value in TP and Yongyou 6 had the lowest Pn value in DS. Significant genotypic difference were found in the rice mill, appearance and cooking qualities. However, Effects of planting method on the nutritional harvest index, nutritional efficiency and grain cooking qualities (excluding Gel consistency) differed between the different environments, as the three way interactions were significant. For both nutrient harvest index and utilization efficiency, large genotypic differences could be also found. Rice chalkiness, transparency, gel consistency, amylase and protein contents were all significantly correlated with N, P and K nutrient traits.4. Field experiments were conducted in Jiaxing (2006), Xiaoshan (2007), Hangzhou (2008), using Xiuyou 5 and Xiuyou 5, and Yongyou 6 and Chunyou 58, respectively, in order to investigate the effect of different fertilizer applications (Fl: control; F2:-10% amount of fertilizer; F3:+10% panicle topdressing fertilizer; F4:-10% amount of fertilizer with +10% panicle topdressing fertilizer) on yield, physiological response and nutritional utilization of high yielding rice. The result showed that yield were slightly decreased or kept consistent in F2 and F4, and increased in F3, compared with F1. Panicles per m2 and grains per panicle in F2 and F4 were smaller than that in F1, while little difference was found between F1 and F3. Grain filling percentage was improved in F2, F3 and F4 for some extent, while grain weight was found no difference among the treatments. It is suggested that the sink capacity was weaken by the decreased early fertilizer and compensated by the increased topdressing fertilizer. Early stem biomass accumulation were related to the fertilizer directly, with F4 was significantly smaller than F1. For PI and HS, no significant difference was found among the treatments suggesting that there is higher biomass turnover efficiency in F4 than in F1. Differences in SPAD value and net photosynthetic rate were only found at heading stage, with F3 being significantly higher than F1. Lower early stem nutrient concentration and accumulation were found in F4, but little difference in leaf, compared with F1. The treatments of F2, F3 and F4 were significantly increased in NUE, but for NHI, PUE, PHI, KUE and KHI, the influence of the treatments varied with environment and genotype.5. QTL mapping for nutrient utilization traits, including NUE, PUE, KUE, NHI, PHI and KHI were conducted in two year (2007 and 2008) using a population of 247 RILs derived from a super hybrid rice cross of "ZS97 X MY46". The result showed that the tremendous transgressive segregation for nutrient utilization traits was observed in the population and the frequencies of these traits were approximately normally distributed. A total of 7 major QTLs and 6 pairs epistatic effect QTL distributed on all the chromosomes, except 4,7 and 9, were detected in both years. Only QTLs (qNUE 5-23 and qPUE1-5) were found for NUE and PUE, respectively. Two, two and one QTLs for NHI, PHI and KHI were also detected, respectively. Phenotypic variance explained by a single QTL ranging from 3.36% to 7.27%. QTLs controlling PUE and NHI were closely located, indicating the linkage of the nitrogen and phosphors utilization.6. In order to understand molecular mechanisms of the difference in NUE of rice, a systematic proteomic study was carried out to investigate N stress-responsive proteins in two high yielding rice cultivars differing in NUE. Four leaf-old seedlings were treated with N-free solution for 12 h,3 d and 7 d. Total proteins of leaves were extracted and separated by two-dimensional gel electrophoresis. Although more than 1000 protein spots were reproducibly detected, only a very small proportion of spots showed differential expression, including 10 and 24 up-regulated,2 and 12 down-regulated in the two cultivars Chunyou 58 and Yongyou 6, respectively. It was indicated that relatively simply biochemical pathways maybe involved with NUE, thus it should be efficiently manipulated. Mass spectrometry based peptide mass fingerprinting (PMF) procedure identified 31 protein spots. Six stress-induced proteins were found, including DegP2, harpin binding proteins, Heat shock-related proteins, glutathione S-transferase GSTF14, Fibrillin-like protein and Glyceraldehyde-3-phosphate dehydrogenase. Apart from the stress related proteins, the other differential proteins identified were mainly these involved in the regulation of the main leaf biological function, photosynthesis metabolism, such as Rubisco activase, RuBisCo large subunit, etc. The study also detected two novel proteins, harpin binding protein and oryzains gamma precursor. The current study gives new insights into N stress response and theoretical bases for improving NUE of rice.更多还原