【作者】 卜媛媛；

【导师】 柳参奎；

【作者基本信息】 东北林业大学 ， 生物化学与分子生物学， 2012， 博士

【摘要】 氮素是水稻生长发育所必须的营养元素,也是限制其产量的重要因素之一。在农业自然生态系统中,氮素主要以三种不同的形式存在：铵态氮(NH4+-N)、硝态氮(NO3+-N)及铵硝混合(NH4+-NO3-的形态。目前,世界上70%的水稻栽培于水田或旱田的土壤中,由于长期的自然选择及进化,水稻为典型的喜铵(NH4+)作物,而陆稻则为典型的喜硝(NO3-)作物。并且高浓度的铵态氮常常会抑制陆稻品种主根的伸长,为了阐明陆稻主根的伸长受高NH4+抑制的作用机理,我们利用耐NH4+较强的水稻品种Akihikari与对NH4+较敏感的陆稻品种IRAT109构建重组自交系群体(BC1F11),开展了数量性状定位(QTL)与代谢组学的研究,主要研究结果如下：1.两亲本Akihikari与IRAT109分别在含有不同浓度的(NH4)2SO4、NH4N03与Ca(NO3)2为唯一氮源的水培液中培养9天,随着处理浓度的升高,在含有NH4+的条件下对两亲本的根长都有明显的抑制作用,而Ca(N03)2则对两亲本主根的伸长没有影响,即使在较高的浓度条件下。2.在含有500μM KNO3、(NH4)2SO4或NH,NO3为唯一氮源的水培液处理下,三种氮素对亲本Akihikari主根的仲长无明显的影响,然而IRAT109在KN03处理液中主根最长,其次是NH4N03,在(NH4)2S04处理下主根最短。3.应用QTL定位方法分析水稻与陆稻主根的伸长对三种不同形态氮素(NO3--N, NH4-N, NO3--NH,4-)的遗传效应,共检测到两个QTLs,其中一个QTL是在NO3-条件下检测到的,Q7-1位于第7号染色体的RM3859-RM5405分子标记之间,促进主根仲长的等位基因来自于IRAT109(陆稻)；另外一个QTL是在NH4+条件下检测到的,Q2-1位于第2号染色体的RM3421-RM5472分子标记之间,促进主根伸长的等位基因来自于Akihikari(水稻)。4.两亲本Akihikari与IRAT109在不同浓度的NO3-与NH4+处理下,Akihikari与IRAT109主根的伸长不受低浓度或高浓度NO3-的影响,表现稳定的生长趋势。而且低浓度与高浓度的NH4+对Akihikari主根的仲长也无影响,然而随着NH4+处理浓度的升高IRAT109主根的伸长明显受到了抑制作用。此外,通过对伴随离子(SO42-)试验研究表明,陆稻在高浓度铵条件下,NH4+为主要的决定性因子抑制主根的伸长。5.利用QTL Cartgrapher2.0软件对水稻Akihikari与陆稻IRAT109的重组自交系群体(BC1F11)133株系进行QTL分析显示,在20μM与2mM的NH4+处理下共检测到6个基因组区域控制主根的伸长效应,分别位于第1、2、6(2个QTLs)、9和11号染色体上。其中,IRAT109等位基因促进主根伸长的QTL有4个,包括qSRLl-1, qSRL6-1, qSRL6-2与qSRL9-1,这个结果与IRAT109实际具有比Akihikari较长的根系统相一致。比较在低浓度与高浓度处理下检测到的QTL,20μM NH4’处理下没有特异的QTL存在,然而在2mM NH4处理下存在两个特异的QTLs, qSRL2-1与qSRL6-2,其中,只有qSRL2-1在高NH4+条件下参与IRAT109主根伸长的抑制作用。6.利用毛细管质谱分析法(CE-MS)分析两亲本Akihikari与IRAT109根部的29种氨基酸含量。在低浓度NH4+处理下,Akihikari与IRAT109根部氨基酸组成非常相似,含量最高的是谷氨酰胺(Gln),其次是天冬酰胺(Asn)、丙氨酸(Ala)、谷氨酰胺(Glu)、丝氨酸(Ser)及天冬氨酸(Asp)。在高浓度NH4+处理下,这6种氨基酸在Akihikari与IRAT109根部的含量明显不同,谷氨酰胺、谷氨酸与天冬氨酸在Akihikari根部的积累量明显升高,而丝氨酸主要在IRAT109根部积累。丙氨酸与天冬酰胺含量在两亲本问无明显差别。7.利用重组自交系群体(BC1F11)133株系在20μM与2mM的NH4+处理下的每种氨基酸含量进行QTL遗传分析,结果检测到2个与根长相关的丝氨酸含量的QTL,分别位于2和11号染色体上,低浓度NH4+(2号染色体)与高浓度NH4+(11号染色体)促进丝氨酸含量的等位基因都来自于IRAT109。而且这两个丝氨酸含量的QTL与IRAT109等位基因抑制根长的QTL (qSRL2-1与qSRL11-1相重叠。暗示了在高NH4+条件下丝氨酸在IRAT109根部的积累可能参与根长的抑制作用。另外,向培养基中分别添加0.1mM或0.5mM浓度的谷氨酰胺、谷氨酸、天冬酰胺、丙氨酸及丝氨酸对两亲本主根伸长的影响结果显示：添加丝氨酸严重抑制了Akihikari与IRAT109的根长,而添加的谷氨酰胺、谷氨酸、天冬酰胺或丙氨酸都对Akihikari与IRAT109的根长无任何的影响,我们得出的结论是,丝氨酸与主根的伸长相关,至少在高NH4+条件下参与主根的抑制作用。更多还原

【Abstract】 Nitrogen is one of the necessary nutrition for plant growth and development, and also one of the limited factors for yield. In the agricultural natural ecological system, the nitrogen mainly exist by three different forms:ammonium (NH4+-N), nitrate (N03-N) and ammonium nitrate (NH4+-NO3-). At present,70%of the world’s rice cultivation in paddy field and upland soil, due to long-term natural selection and evolution, lowland rice prefer ammonium as the major nitrogen source, and upland rice utilize nitrate for growth. In generally, high concentration of NH4+often inhibits seminal root elongation in upland cultivars. To elucidate mechanism underlying this inhibition by NH4+on root elongation, we carried out quantitative trait locus (QTL) and metabolome analyses using a mapping population derived from the cross between "Akihikari"(NH4+-tolerant lowland cultivar) and "IRAT109"(NH4+-susceptible upland cultivar), the main research results are as follows:1. Akihikari and IRAT109were hydroponically for9days in different concentration of three nitrogen forms, containing (NH4)2SO4、NH4NO3and Ca(N03)2, respectively. With increasing the concentrations of (NH4)2SO4and NH4NO3, Akihikari and IRAT109seminal root elongation inhibited, while Ca(N03)2have no effect on seminal root elongation, even in high concentration conditions.2. Akihikari and IRAT109were grown in500μKN03、(NH4)2SO4, and NH,N03as the sole nitrogen source treatment, all three kinds of nitrogen forms no significant effects on Akihikari seminal root elongation, however IRAT109with the longest seminal root in KNO3solution, followed by NH4NO3, and in (NH4)2SO4, has the shortest root. 3. Perform QTL analysis of genetic effects on lowland and upland rice seminal root elongation which treated in three different nitrogen forms (NO3--N、NH4+-N and NO3--NH4-),wo specific QTL regions were detected, Q7-1was located on chromosome7between the marker RM3859-RM5405, and IRAT109(upland rice) allele promote seminal root elongation in N03condition (upland soil),while Q2-1was located on chromosome2between marker RM3421-RM5472, and Akihikari (lowland rice)allele promote the seminal root elongation in NH4+condition (paddy field).4. When grown hydroponically over the wide ranges of NH4+concentration (20μM,200μM and2mM), Akihikari and IRAT109seedlings showed different growth responses to NH4+. In Akihikari seminal root length was almost uniform over the ranges of NH4-, while seminal root length was progressively reduced with increasing NH4+concentration in IRAT109. Such inhibitory effect on IRAT109roots was never seen in NO3-:seminal root elongation was uniform over the ranges of N03in both Akihikari and IRAT109. High concentration of NH4-was much inhibitorier for IRAT109root than for Akihikari root. In addition, through the accompaning ion test study shows that only NH4-inhibit seminal root elongation.5. Analysis BC1F11population with QTL Cartgrapher2.0revealed that a total of six genomic regions, distributed on chromosomes1,2,6(two QTLs),9and11, affected root elongation. The IRAT109allele promoted elongation on four (qSRL1-1, qSRL6-1, qSRL6-2and qSRL9-1) out of six QTLs, which consisted with the fact that IRAT109has a deeper root system than Akihikari. Comparison of QTLs between low and high NH4+treatments revealed that no QTLs were specific for low (20μM) NH4+treatment while two QTLs (qSRL2-1and qSRL6-2) were specific for high (2mM) NH4+treatment. Of the two, one (qSRL2-1) seemed to be involved in the growth inhibition by high NH4; only at this QTL the IRAT109allele reduced root growth under high concentration of NH4+ 6. Measurement of29amino acids content in Akihikari and IRAT109root were analyzed by capillary electrophoresis-mass spectrometry (CE-MS). At low concentration of NH4, amino acid composition was similar between "Akihikari" and "IRAT109", with glutamine as the major component followed by alanine, glutamate, serine and asparagine. Under high NH4+concentration, however, amino acid contents were fairly different between two cultivars; glutamine and glutamate were notably increased in "Akihikari" while serine was increased only in "IRAT109". Alanine and asparagine were increased similarly between the two cultivars.7. We determined QTLs controlling the content of these amino acids. Of these amino acids, serine seemed to be related to root elongation. We detected two QTLs for serine content on chromosomes2and11where the IRAT109allele increased serine content under low (chromosome11) and high (chromosome2) NH4-, respectively. Interestingly, these two QTL were co-located with the seminal root length QTLs with negative effect from IRAT109allele (qSRL2-1and qSRL11-1). Among them was the QTL on chromosome2(qSRL2-1) where the IRAT109allele reduced root growth under high concentration of NH4-, indicating that accumulation of serine under high concentration of NH4+might inhibit root elongation.Morover, additional of0.1mM or0.5mM of glutamine、glutamate、 asparagine、aspartate and serine to the growth medium to examined the effects of these amino acids on root growth. Addition of serine to growth medium severely inhibited seminal root elongation, while glutamine, glutamate, alanine, and asparagine had little effects on seminal root elongation. From these results, we concluded that inhibitory effect of serine may, at least in part, be involved in the inhibition of rice root elongation by NH4.更多还原