【作者】 张艳；

【导师】 何朝族；

【作者基本信息】 清华大学 ， 生物学， 2011， 博士

【摘要】 水稻是重要的粮食作物。增加水稻单产，培育具有优良农艺性状的新品种是一个重大课题。水稻又是单子叶植物基因组研究的模式植物，对其研究已进入“功能基因组时代”。丙酮酸激酶是糖酵解途径的一个关键酶，其对水稻形态发育的影响报道甚少。本课题对T-DNA插入的水稻矮化突变体ospk1进行鉴定，对突变基因OsPK1进行功能分析，探讨胞质丙酮酸激酶OsPK1对水稻生长发育的影响及机制。突变体ospk1的突变表型主要有:矮秆、鞘包穗、结实率低。侧翼序列分析确定T-DNA插入到基因OsPK1起始密码子上游349bp处。定量RT-PCR检测发现，OsPK1在ospk1的根、叶片、节间和幼穗中的表达均下降约90%，表明T-DNA插入到基因上游的转录调控区域。将OsPK1的全长基因组片段转化到ospk1突变体，获得互补转基因水稻，从穗是否伸出叶鞘、株高、结实率和种子萌发等方面判断，基本恢复到野生型的表型，证实了OsPK1的突变导致ospk1的突变表型。亚细胞定位和酶活性分析揭示OsPK1编码一个胞质的丙酮酸激酶。定量RT-PCR结果显示糖酵解/糖异生途径中的4个酶的表达在ospk1突变体中上调或下调。RT-PCR结果显示突变体中的1个PEP羧化酶Os01g0208700表达增加。表明OsPK1的表达降低引起相关代谢途径的其他酶的表达改变。通过定量RT-PCR、GUS染色和石蜡切片分析了OsPK1的表达模式：在叶的表达水平最高；在幼苗期胚芽鞘、胚根根尖的成熟区、不定根表达很强；在花和乳熟期种子也有表达；在叶肉细胞、茎的韧皮部伴胞细胞有强的表达，还见于根的皮层薄壁组织细胞。OsPK1在不同的功能组织和细胞表达较强，暗示OsPK1参与到不同代谢过程。LC/MS检测发现突变体叶鞘中的GA₄和茎中的GA₁含量降低，而前体大量积累，推断具生物活性的赤霉素在突变体中的合成受到抑制是导致其矮化的原因之一。突变体叶鞘和茎中的ABA含量增加，打破了赤霉素和脱落酸之间原有的平衡。GC/MS检测了野生型水稻和ospk1不同器官中葡萄糖、果糖、半乳糖和蔗糖的含量，推断OsPK1表达降低影响了糖的合成、代谢和运输以及根对外源蔗糖（营养）的吸收。本研究阐明了胞质的丙酮酸激酶OsPK1对水稻重要农艺性状（株高和结实）的影响，拓宽了对株高和结实分子调控机制的理解。拟南芥、玉米和高粱均有与OsPK1序列高度一致的同源基因，因此本研究对这些基因的研究具有参考价值。更多还原

【Abstract】 Rice is an important cereal crop. How to increase rice yield and cultivate newvarieties which has favorable agronomic traits is a major issue. Rice is a modelplant of genome research in monocots. Rice research has entered the "functionalgenomics era." Pyruvate kinase （PK） is a key enzyme of the glycolytic pathway,and its effects on morphological development of rice are rarely reported. In thisstudy, we explored the effects of a cytosolic PK （PKc）, OsPK1, on rice growthand development and the mechanism in them by identification of a rice T-DNAinsertion dwarf mutant ospk1and functional analysis of the gene OsPK1.The main phenotypes of mutant ospk1are dwarf, panicle enclosure andreduced seed set. Analysis of the flanking sequences revealed that a T-DNAintegration occurred at nucleotide position349bp upstream of the ATG startcodon of OsPK1. Quantitative RT-PCR analysis showed that the expression ofOsPK1was decreased by approximately90%in the root, leaf, internode, andpanicle of ospk1, which suggested that the T-DNA insertion site is in thetranscriptional regulatory region of OsPK1. OsPK1full-length genomicfragment was introduced into ospk1mutant to obtain transgenic plants forcomplementation. The phenotype of these transgenic plants totally reverted tothat of WT, which was judged from whether panicle is out of sheath, plantheight, seed set and seed germination. This confirmed that the OsPK1mutationis responsible for the ospk1phenotype. Subcellular localization and enzymeassay revealed that OsPK1encodes a PKc. Quantitative RT-PCR analysisshowed altered expression of four putative enzyme genes ofglycolysis/gluconeogenesis pathway. RT-PCR analysis showed that theexpression of Os01g0208700, a PEP carboxylase, was increased in ospk1. Thesesuggest that the decreased expression of OsPK1leads to altered expression ofcertain enzymes involved in related metabolic pathways. The expression patternof OsPK1was analyzed using quantitative RT-PCR, GUS staining andmicrotome sections. The highest level of OsPK1expression was in leaves. Inyoung seedlings, high levels of OsPK1expression were observed in coleoptile, the maturation zone of the radicle and adventitious roots. OsPK1expression wasalso detected in young floret and milky stage seeds. At the cellular level, OsPK1expression was localized in mesophyll cells in leaves, phloem companion cellsin vascular bundles in stems, and in cortical parenchyma cells in roots. Thus,OsPK1appears to be expressed predominately in these functional tissues andcell types, implying that OsPK1is critical for diverse metabolic processes in rice.Using LC/MS, we found that the contents of GA₄in the young sheath and GA₁in the uppermost internode （UI） were decreased in ospk1, and their precursorsaccumulated abundantly. We infer that the suppressed synthesis of bioactiveGAs in ospk1is likely to be one of the factors resulting in its dwarf phenotype.In addition, ospk1showed increased ABA contents in the two tissues, resultingin the variation in the ABA/GA balance. The contents of glucose, fructose,galactose and sucrose in different organs of WT and ospk1were measured usingGC/MS. These results indicated that the decreased expression of OsPK1haseffects on the biosynthesis, catabolism and transport of sugar, as well asexogenous sucrose （nutrient） uptake by root.This study clarified the effects of a PKc, OsPK1, on the important agronomictraits （plant height and seed set） and provided new insights into molecularmechanisms of plant height and seed set. OsPK1has very close orthologs inArabidopsis thaliana, maize and sorghum. Therefore, our work is a valuablereference to future study on those orthologs.更多还原