【作者】 贾彦博；

【导师】 杨肖娥；

【作者基本信息】 浙江大学 ， 农业资源利用， 2008， 博士

【摘要】 钾是植物生长三要素之一。随着农产品产量和品质的不断提高,作物对钾素的需要量也不断增加。然而,我国土壤普遍缺钾,尤其是在水稻生产体系中,约有70%以上的稻田处于钾素亏缺状态,土壤低钾胁迫已成为水稻高产优质的主要限制因素。水稻适应低钾胁迫的能力存在显著基因型差异,探讨水稻适应低钾的根际土壤化学、根系形态学、生理学和分子生物学机制,提出钾高效水稻基因型筛选和鉴定的性状指标,对于拓展植物营养学的研究领域,挖掘具有特异性能的水稻种质资源具有重要的理论价值和实践意义。本研究以大田试验筛选出的钾效率不同的水稻基因型为试验材料,研究了水稻适应低钾胁迫的大田响应以及根系的形态学及生理学特征;根系分泌物的组成特性以及对矿物钾的活化机制;通过研究水稻T-DNA插入突变体库中钾敏感和钝感突变体株系的差异,为今后水稻钾高效分子生物学研究打下基础。取得的主要研究结果如下:1.通过田间试验研究了不同供钾水平对8个水稻品种钾素吸收利用和稻谷产量的影响。研究结果表明,两种供钾水平下,水稻的稻谷产量、钾利用效率和各生育期地上部钾积累都存在显著的基因型差异。低钾胁迫显著降低水稻的稻谷产量和各生育期地上部钾积累量,显著提高水稻的钾利用效率。相关性分析表明,低钾胁迫下水稻生育前期（秧苗期+分蘖期）地上部钾积累量以及生育中期（抽穗期）地上部钾积累量与稻谷产量呈显著正相关;正常供钾条件下水稻生育前期地上部钾积累量与稻谷产量呈显著正相关。因此筛选和培育具有较高钾利用效率和在生育前期具有较强钾素积累特性的水稻基因型可能是缓解南方水稻土钾素严重缺乏的有效途径之一。2.在低钾条件下,水稻根重和根体积显著降低,但钾高效基因型的降幅小于钾低效基因型;适度低钾条件下,水稻根重和根体积呈增大趋势,且钾高效基因型的增幅大于钾低效基因型。但钾高效基因型根系总表面积、细根数大于钾低效基因型。钾高效水稻基因型根系吸收钾的K_m值、C_min值和β值都小于钾低效基因型,而I_max值在不同钾效率基因型间无规律性变化,说明水稻对低钾胁迫的适应性与根系对K⁺的亲和力、生长介质中临界K⁺浓度有关,而与水稻吸钾的最大速率无关。3.通过比较水稻钾低效基因型铜梁火种（TLHZ）和钾高效基因型HA-881043（HA）缺钾条件下的叶绿体结构和叶绿素荧光参数的差异,研究结果表明:净光合速率（Pn）和气孔导度（Gs）在低钾胁迫下降低,其中钾低效基因型比钾高效基因型降低的幅度要大,但胞间CO₂浓度（Ci）在两个基因型之间却是相反的趋势;这说明钾低效基因型的光合能力降低主要是由于抑制了光化学活性。叶绿素荧光测定结果表明:缺钾条件下,两水稻基因型的初始荧光（Fo）、可变荧光与最大荧光比（Fv/Fm）以及非光化学猝灭系数（NPQ）与对照相比均无显著性差异;两水稻基因型的可变荧光与最大荧光比（F_v/F_m）、光化学猝灭系数（qP）、实际光化学效率（φPSⅡ）以及光合电子传递速率（ETR）与对照相比均有所下降,其中钾低效基因型（TLHZ）的降幅更大。说明缺钾对水稻叶绿素荧光参数的影响与其钾敏感性密切相关,F_v/F_m、qP、φPSⅡ和ETR四个叶绿素荧光参数可作为耐缺钾水稻品种快速筛选的参考指标。TEM分析结果表明:在缺钾处理条件下,钾低效基因型（HA-881043）比钾高效基因型（TLHZ）叶绿体结构受到更严重的破坏。4.采用营养液培养试验研究了低钾（-K）、正常供钾（CK）条件下钾高效基因型HA-881043和钾低效基因型铜梁火种的根系K⁺吸收动力学参数,抗氧化酶活性,根系显微结构及对钾的吸收、积累差异。试验结果表明,2个基因型在耐低钾胁迫能力方面存在显著性差异。相对于对照（正常供钾）,在低钾（-K）时,高效基因型HA-881043植株根系生物量极显著高于低效基因型铜梁火种,其根系MDA、Pro、SOD活性,及根系活力也明显高于低效基因型铜梁火种,这一规律也在根系显微结构中体现出来。表明根系MDA、Pro、SOD等抗氧化酶活性及根系活力可以作为比较水稻品种在耐低钾胁迫能力方面差异的评价指标之一。5.通过水培试验,研究了两种不同钾效率水稻基因型在不同的供钾水平下根系分泌物的组成特征以及对矿物钾的释放能力差异。通过大田试验研究了不同的供钾条件下,两种水稻种植前后根际土壤中不同形态钾的亏缺程度。结果表明,根系分泌物对含钾矿物中的钾具有明显的释放作用,其中钾素利用高效型水稻根系分泌物的释钾能力高于低效型,且根系分泌物中有机酸含量与释钾量呈显著正相关。不同基因型有机酸的分泌能力大小是造成土壤钾吸收利用差异的重要机理之一。6.从以粳稻日本晴为受体构建的经农杆菌介导的T-DNA插入水稻突变体库中获得了2个突变体株系,通过缺钾（5mg/kg）营养液培养筛选发现,两个水稻突变体株系对钾的利用效率不同,表型上与亲本有显著差异。通过PCR检测和潮霉素抗性分析表明:两个株系及其后代钾高效和低效的T3代不同单株与T2代都缺失分子标记Hyg,证明其性状与T-DNA插入突变无关。分析不同突变体株系在低钾条件下的根系形态和生理学机制,表明高效突变体株系相对于野生型有更强的钾积累能力,具有更发达的根系和完整的显微结构;而低效突变体株系则不具有这些特征。更多还原

【Abstract】 Potassium（K）plays a role in a wide range of functions in plants:photosynthesis,enzyme activation,protein synthesis,osmotic potential,and as a counterion to inorganic ions and organic biopolymers.Potassium deficiency in soils is widespread in China,especially in the paddy soils, where low K has become the major limiting factor for obtaining high yield.China is short of K fertilizer resources,so it is importance to understand the mechanisms of plant adaptation to low K. There existed large genotypic differences in K efficiency in lowland rice（Oryza sativa L.）.The major objectives of this study were to investigate the characteristics of root morphology,K absorption kinetics,and root cell membrane tolerance to low K stress,to find out the plant trait for screening and identifying K-efficient rice genotypes,and understand the characteristics of rhizosphere processes of K in the paddy soils.The major results obtained were summarized as follows:1.A field trial was carried out to study the effect of different K supply levels on the grain yield, K uptake and utilization of eight rice cultivars.Results showed that there were significantly genotypic variations in rice grain yield,K utilization efficiency and shoot K accumulation at different growth stage under two different K supply.Compared with normal K supply,rice grain yield and shoot K accumulation were decreased,and K utilization efficiency was increased under low K supply.Correlation analysis showed that under low K supply rice grain yield was positively correlated with K utilization efficiency,shoot K accumulation at both early growth and mid-growth stages（p＜0.05）,and under normal K supply rice grain yield was positively correlated with K utilization efficiency and shoot K accumulation at early growth stage（p＜0.05）.Therefore,screening and breeding rice genotypes with higher K utilization efficiency and higher ability to accumulate K at grow th early stage would probably be one of alternatives to alleviate the K deficiency of paddy soil in south of China.2.Differential effects of K deficiency on chloroplast ultrastructure and chlorophyll fluorescence parameters were observed between the K-efficient（HA）and the K-inefficient（TLHZ） rice（Oryza sativa L.）genotypes.The net photosynthetic rate（Pn）and stomatal conductance（Gs） decreased at low K,but to a greater extent in TLHZ than in HA.Chlorophyll fluorescence measurements showed that PSⅡ-available photochemical efficiency（F_v/F_m）,electron transport rate of PSⅡ（ETR）,and photochemical quenching（qP）declined more significantly,whereas the non-photochemical quenching（NPQ）increased more in TLHZ than in HA-881043 under low K, indicating that the K-efficient genotype has greater ability to keep PS reaction center less damaged and maintain stronger photosynthetic ability with high resistance to photoinhibition under K deficiency.In addition,better preserved chloroplast ultrastructure was noted in HA-881043 than in TLHZ under K deficiency.The high efficient photochemical capacity in K -efficient genotypes may be a key factor for high K efficiency in rice.3.Disparity in the root morphology of six rice（Oryza sativa L.）genotypes varying in potassium（K）efficiency was studied with three K levels:5 mg L^-1（low）,10 mg L^-1（moderate） and 40 mg L^-1（adequate）in hydroponic culture.Morphological parameters included root length, surface-area,volume,and count of lateral roots,as well as fine（＜0.2 mm）and thick（＞0.2 mm） roots.The results indicated that root growth of all genotypes was reduced under low K,but moderate K deficiency increased root length of the efficient genotypes.At deficient and moderate K levels,all the efficient rice genotypes developed more fine roots（＜0.2 mm）than the inefficient ones.Both fine root and root surface area were found to be the best parameters to portray K stress in rice.In accordance with root morphology,higher relative K concentration was noted in shoots of the efficient genotypes when grown at moderate and deficient K levels,indicating that root morphology parameters are involved in root uptake for K and translocation it up to shoots. Potassium deficiency affected not only the root morphology,but also the root ultra-structure.The K_m,C_min,andβvalues of K uptake by root of the efficient genotypes were both lower than those of the inefficient ones,whereas minimal differences were observed for the I_maxvalues among different rice genotypes.The results indicated the K efficiency was relative to the root affinity for K⁺,the minimal K⁺ concentration in the culture medium(C_min),but not to the maximum K⁺ uptake rate.The roots of high-efficient genotypes had stronger tolerance to K deficient stress for root membrane damage,and could maintain developed root architecture to adapt the low K in growth medium.And the results of field experiment were similar with hydroponic study.4.Differential changes of K fractions in the rhizosphere in field experiment and root exudates constitutes were investigated between the K-efficient and K-inefficient rice cultivars under low and adequate soil K supply.The results showed that considerable depletion of readily-available K and exchangeable K in rice rhizosphere occurred at both low and adequate K levels.At low K,the efficient cultivar（HA-881043）reduced more dramatically the rhizospheric concentrations of both readily-available K and exchangeable K than the inefficient cultivar（TLHZ）,but the opposite trends were noted at adequate K supply.Both cultivars could deplete readily-released K in the rhizosphere.And similar difference was noted for pH of root exudates.Moreover,the root exudates of the K-efficient cultivars grown at low K contained higher organic acids and showed more effective in extracting K from philogopite.These results indicated the K efficiency in lowland rice resulted from more effective use of slowly-released K and mineral K in soil by means of induced root exudation of acids and/or organic acids at low K.5.We chose 200 isolated rice（Oryza sativa L.subsp.Japonica,cv.Ribenqing）mutants as material from the mutant system derived from gene transformation mediated via Agrobacterium tumefaciens.By different K treatment,we obtained the K-sensitive/tolerant mutant rice（STOW）. Our results indicated that K accumulation of the K-tolerant mutant were higher than that of the wild type,so the capacity of transporting K to shoots might be greater in the mutant.Then the high/Iow -accumulation K-tolerant mutant was added a generation to analyze the amplification patterns of Homomycin gene in T3 plants of K-tolerant mutant.The results indicated that there was not the Hyg in T2 and T3 lines,so we concluded that different inheritable character of accumulating K in the mutant was not caused by T-DNA inserting.Our studies suggested that tolerant mutants had stronger root morphology to adapt to low K,but sensitive mutants had no these characters.更多还原