【作者】 张所兵；

【导师】 沈振国； 张红生； 王才林；

【作者基本信息】 南京农业大学 ， 植物学， 2009， 博士

【摘要】 水稻株高是与产量、光合效率和抗倒性密切相关的重要农艺性状。赤霉素(GA)对株高建成有重要的调控作用。GA调控植物生长发育是通过GA信号调控与生长发育相关基因表达实现的。从GA合成到GA信号传递的任一步阻断,都能影响植物正常的生长和发育,并从表型上表现出来。因而,通过水稻株高突变体研究,不仅可以鉴定在GA代谢途径和信号转导途径中起作用的新基因,而且可以了解GA调控禾本科植物株高建成机理,后者对理想株型育种具有重要的理论和实践意义。9311HR-T是从半矮秆品系9311HR中自然突变获得的高秆突变体。鉴于株高突变体在研究植物激素代谢、信号转导以及植物激素调控株高建成机理等方面有重要价值,本研究对高秆突变体9311HR-T株高变异机制进行了研究。9311HR-T的株高为210.8±4.9cm,是野生型的1.8倍。与野生型相比,高秆突变体9311HR-T地上部各伸长节间均极显著伸长,第1至第6节间的长度分别是野生型9311HR的1.4倍、1.8倍、1.6倍、2.1倍、2.8倍和4.5倍,这是导致其株高增加的主要原因。解剖学研究发现,9311HR-T节间伸长是细胞伸长所致,暗示该突变体可能与GA有关。用不同浓度外源GA3对野生型和突变体第二叶鞘、幼苗进行处理,并对无胚半粒种子α-淀粉酶进行诱导。结果发现,突变体第二叶鞘、苗高及α-淀粉酶诱导对外源GA3的敏感性没有改变。GA合成抑制剂uniconazole能使突变体第二叶鞘长度和苗高恢复为野生型,且uniconazole对高秆突变体9311HR-T苗高抑制作用能被外源GA3解除,表明高秆突变体株高增加是内源活性GA含量增加所致。内源GA含量测定表明,高秆突变体9311HR-T中内源活性GA1含量是野生型的2.6倍,为内源活性GA含量增加导致9311HR-T高秆表型提供了直接证据。为探究引起9311HR-T内源GA增加的内在原因,利用半定量RT-PCR分析了GA合成途径中关键限速酶基因的表达,结果发现GA20ox2表达明显上调,而GA2ox3表达下调,表明GA代谢途径中这2个关键基因未受活性GA调控。序列分析表明,在9311HR中,GA20ox2CDS第1026个碱基C颠换为G,形成一个终止密码子TAG,使翻译提前终止；而在9311HR-T中,GA20ox2CDS第1026个碱基G又颠换为C,使翻译能够正常进行；而GA2ox3序列未发生改变。上述结果表明,GA20ox2恢复突变和表达上调及GA2ox3表达下调共同导致高秆突变体9311HR-T内源GA含量增加,进而导致高秆表型。02428h系从半矮秆水稻02428体细胞培养后代中发现的隐性高秆突变体,其高秆性状受elongated uppermost internode (eui)控制。之后,我们又在02428h中发现一个半矮秆突变体02428ha,该突变体仍然携有eui基因。本研究对半矮秆突变体02428ha矮化机制进行了研究。02428ha的株高为127.3±2.6cm,比野生型02428h矮29.7cm,差异达极显著水平。节间调查表明,第1节间缩短是导致02428ha株高矮化的主要因素。为研究引起02428ha节间缩短的原因,本研究将野生型和突变体最上节间平均分成5段,进行解剖学观察,结果表明：02428ha最上节间细胞长度较02428h相应的细胞长度缩短；从下至上,02428ha细胞长度较野生型02428h缩短的比率分别为36.4%,37.5%,37.1%,40.3%和29.4%。02428ha最上节间较02428h显著缩短,缩短的比率是24.1%。故细胞长度缩短是导致02428ha最上节间缩短的直接原因。外源GA,处理表明,1×10-8M GA3就能促进野生型02428h第二叶鞘伸长生长,而促进突变体02428ha第二叶鞘伸长生长所需的最小GA,浓度为1×10-6M；而且,1×10-4M GA3饱和处理亦不能使02428ha矮化表型完全恢复为野生型。上述结果表明,02428ha对外源GA3敏感性发生改变。内源GA含量测定表明,突变体02428ha中活性GA1含量为15.96ng.g-1F W,是野生型的6倍,进一步证实02428ha株高矮化是对GA敏感性降低所致。因02428ha对外源GA3敏感性降低,故推测其株高矮化与GA信号途径损坏有关。对GA信号转导途径基因表达分析后发现,GA信号转导途径中正调控因子F-box蛋白基因GTD2表达明显下调,表明该突变体对GA敏感性降低是GID2抑制表达所致。对该基因克隆测序后发现,在GID2基因编码区(coding sequence,CDS)第594和595碱基之间插入了15个碱基。插入位点恰好位于GID2的LSL结构域中,而该结构域对GID2是必须的。故插入突变可能改变其功能。上述结果表明,GID2表达下调导致突变体对GA敏感性发生改变,另一方面插入突变亦可能影响GID2功能导致突变体对GA敏感性改变,进而导致半矮化表型。本文还讨论了02428ha在杂交水稻育种中的潜在应用价值。更多还原

【Abstract】 Rice plant height is one of the important agronomic traits, which is closely related to grain yield, photosynthesis efficiency and lodging resistance. Gibberellin (GA) plays an important role in controlling plant height. GA regulated growth and development by converting the GA signal into alterations in expression of the genes controlling plant growth and development. When GA biosynthesis was blocked or GA signaling was impaired, the plant growth and development could be affected and plant morphology could be altered. Thus, the plant height mutants were not only used to identify novel genes related to GA biosynthesis and GA signaling, but also to understand the mechanism of plant height morphogenesis, which had important significance in plant ideotype breeding.The rice tall mutant,9311HR-T, was obtained from a natural occurrence in a semidwarf strain,9311HR. Because plant height mutants can be used to understand phytohormone metabolism, phytohormone signal transduction and the mechanism of plant height morphogenesis, this study aimed to reveal the mechanism that governed the tall phenotype in9311HR-T. The final height of9311HR-T was210.8±4.9cm from the ground to the top of the panicle, which was1.8times as long as wild type. Compared to wild type, the extra plant height of9311HR-T was mainly caused by an increase in length of each elongated internode. The length of the first, second, third, fourth, fifth, and sixth internodes in9311HR-T was1.4,1.8,1.6,2.1,2.8and4.5times longer than those in9311HR, respectively. Microscopic observation of internode was made to find that the cell length in the mutant enhanced compared to the wild type, which indicated that the mutant was related to GA. To investigate the response of9311HR-T to GA, the role of GA in second leaf sheath elongation and in shoot elongation and agar plate assays for amylases were examined. The sensitivity of mutant to GA3application was found to be similar to that of wild type. To determine whether or not the tall phenotype of9311HR-T was caused by abnormal endogenous GA level, uniconazole, inhibitor for GA biosynthesis, was used to pretreat the plants, and then the response to exogenous GA3was investigated. The wild type phenotype of9311HR-T could be rescued by uniconazole treatment. The dwarf phenotype of9311HR-T retarded by uniconazole treatment can be restored by exogenous GA3. These results suggested that the tall phenotype of9311HR-T may be caused by elevating endogenous bioactive GA levels. To determine the specific cause of the elongating of the internodes length in9311HR-T, we measured endogenous GA1levels in the elongating internodes of both wild type and9311HR-T plants. The results showed that endogenous GA1levels in mutant9311HR-T was2.6times over that in wild type9311HR, which directly supported the conclusion that the mutant9311HR-T displayed a tall phenotype, owing to enhanced endogenous bioactive GA levels. To explore the intrinsic reasons for enhanced endogenous GA levels in9311HR-T, the expression of key GA metabolism genes were analyzed by semi-quantitative RT-PCR. The results show that the expression of two key GA metabolism genes was not regulated by endogenous GA. The expression of GA20ox2was obviously up-regulated, while the expression of GA2ox3was obviously down-regulated. Sequence analysis revealed a C to G transversion at position1026in GA20ox2coding region in9311HR, which led to the production of a stop codon TAG and translation termination. A novel G to C transversion was observed at position1026in GA20ox2coding region in9311HR-T, which restored translation of the full-length gene product. However, the coding sequence of GA2ox3in9311HR-T was identical to that in9311HR. Taken together, up-regulation and restorer mutation of GA20ox2and down-regulation of GA2ox3led to elevated GA levels in9311HR-T, which made9311HR-T exhibit a tall phenotype.The rice strain,02428h derived from a semidwarf material02428in somatic cell culture was a recessive tall mutant, and the recessive tall culm was controlled by elongated uppermost internode (eui).02428ha derived from02428h was a semi-dwarf mutant in eui background. Because semidwarf varieties possess high yield potential, the present study aimed to further characterize the dwarfing mechanism in the mutant. The plant height of02428ha was significantly shorter than that of the wild-type02428h plants, and the final height from the ground to the top of the panicle was127.3±2.6cm, which was29.7cm shorter than that of02428h. The dwarfed plant phenotype of02428ha was mainly caused by a decrease in length of the first internode. To investigate the shorten internode of02428ha was caused by abnormal cell elongation and/or cell division, five equally divided sections of the uppermost internode in wild-type and mutant plants were subjected to microscopic observation. In each section, the cell length of02428ha was shorter than that of the wild type, and the cell length decreased ratios were36.4%,37.5%,37.1%,40.3%and29.4%from bottom to top, respectively. The uppermost internode length decreased ratio was24.1%. Therefore, the shortening of the uppermost internode length in02428ha was due to longitudinally decreased cell lengths. To determine whether or not the dwarf phenotype of02428ha was caused by abnormal endogenous GA level or altered sensitivity to GA, the response to exogenous GA3was investigated. When the second leaf sheaths of wild type and02428ha plants were treated with various concentrations of GA3, the wild type elongated at GA3concentrations of1x10-8M or higher, but the mutant02428ha did at GA3concentrations of1×10-6M or higher. Importantly, the dwarf phenotype of02428ha could be incompletely rescued by1x10-4M GA3treatment. The results suggested that mutant02428ha showed decreased sensitivity to exogenous GA3. Endogenous GA1levels in mutant02428ha were15.96ng.g-1fresh weight, which was6times over that in wild type02428h. The results also supported the conclusion that the mutant altered sensitivity to GA. Because02428ha showed a reduced sensitivity to GA, it was necessary to investigate whether or not the GA signaling genes are affected in02428ha. The expression of the F-box gene GID2was down-regulated in02428ha, which was responsible for the reduced sensitivity to GA in the mutant. Especially, gene clone and sequence analysis revealed that a15base pair insertion between base594and595in its coding region from the translation start site was found in the mutant. The insertion mutation just altered the LSL domain, which was essential to GID2. Thus, the insertion mutation might altered its function. The results firstly suggested that the down-regulation.expression was responsible for the phenotype of the mutant02428ha, and that sequence variation of GID2gene might be related to it. The utilization potential of02428ha in hybrid rice breeding was also discussed.更多还原