【作者】 刘莉；

【导师】 林拥军；

【作者基本信息】 华中农业大学 ， 遗传学， 2008， 博士

【摘要】 水稻是人类最重要的粮食作物之一。虽然目前杂交水稻的推广很大程度上解决了世界人民的粮食问题,但是一些优良品种尤其是一些籼型品种在中后期衰老快,限制了更多有机物的形成和积累,不利于结实率和千粒重等产量相关性状的提高。解决这个问题的关键是了解叶片衰老的机制,创造延缓水稻叶片衰老的途径,进而提高有机物在籽粒中的积累。利用衰老特异性启动子驱动细胞分裂素基因表达、抑制衰老上升基因表达或者过量表达衰老下降基因都有可能实现持绿性水稻的目标。本实验针对这个问题,克隆鉴定了水稻来源的衰老特异性启动子,并用该启动子驱动农杆菌来源的细胞分裂素合成酶基因转化籼稻和粳稻,筛选持绿性家系,并且构建了中花11剑叶的早期衰老上升表达文库,主要结果如下:1.克隆水稻启动子P_SAG39,通过报告基因GUS融合表达载体转化水稻鉴定其时空组织特异性表达谱。经过组织化学鉴定发现其在叶、茎、根、花、颖壳及未成熟种子的种皮、愈伤组织中表达,在成熟种子及胚乳中不表达,而且在叶片衰老晚期表达量达到高峰,揭示该启动子是叶片衰老特异性,而且可以应用于基因工程育种改良研究。2.鉴于启动子序列预测出多种激素响应相关的顺式作用位点,本实验用瞬时表达体系检测激素和冷胁迫对一系列5’端缺失启动子的表达情况,并且研究了缺失启动子的衰老特异性表达谱,确定P_SAG39启动子的衰老相关的顺式作用位点存在的区段。通过凝胶阻滞实验鉴定了可能对衰老上升表达起作用的顺式因子。揭示顺式作用位点HBOXCONSENSUSPVCHS和WRKY71OS与叶片衰老特异表达有关。3.在粳稻中成功转化了P_SAG39:IPT表达系统,繁殖并筛选了单拷贝插入的转基因纯合家系,5个以牡丹江8^#为受体:MT1、MT2、MT3、MT4、MT5:3个以中花11为受体:ZT1、ZT2、ZT3。4.研究转P_SAG39:IPT表达系统的阳性转基因水稻的叶片持绿性变化,发现持绿性增强,且这个表型与外源基因IPT的表达量共分离。5.调查了转化植株表达的细胞分裂素在光照下对幼苗生长及抽穗期产生的影响,P_SAG39:IPT的转基因植株表达细胞分裂素在一定光照条件下（14小时或24小时）影响了种子刚萌发时芽的生长,但是不会影响茎杆的继续伸长。在长日照条件下,转基因中花11植株表达细胞分裂素刺激了OsMADS50的表达,从而提前开花。6.探明结实期间碳氮代谢水平是否发生变化,通过分析纯合家系ZT1可溶性糖含量和全氮含量发现,糖及氮素水平的改变引起叶片的库源转换发生变化从而引发叶片衰老。7.完成纯合家系的随机区组试验,每个小区设置三个重复,调查转基因植株的持绿性和其他农艺性状。转基因阳性株系ZT1、ZT2的生物学产量相对其阴性株系降低约8-10%,千粒重增加约5-9%。8.以绿色叶片为driver,早期衰老叶片为tester,用抑制差减杂交法完成水稻剑叶早期衰老特异基因文库的构建,以driver和tester的总RNA为探针分别进行文库克隆的Macroarray杂交,筛选出815个差异表达的ESTs。通过生物信息学分析（阳性克隆聚类及GO分析）,一共确定了533个单基因,其中183个有GO注释,涉及大分子物质代谢、调控蛋白质合成、能量代谢、调节基因、解毒、病原性和逆境、细胞骨架构成和花发育。121个与已报导过的持绿相关QTLs共线性,其余是功能未知的新基因。9.从文库中随机挑选50个阳性克隆的Northern杂交验证了差减杂交的效率和准确度,达到60%以上。对几个未知功能的基因进行定量PCR实验,发现它们不仅在自然衰老早期上升表达,而且不同程度的受到激素的诱导。这些基因为阐明衰老机制提供了一定基础。更多还原

【Abstract】 Rice is one of the most important food crops in the world.Though the spread abroad of hybrid paddy rice greatly resolved the lacking of food for the world population,most of the wildly-used hybrids（especially indica rice） were early senescence in the late development,which limits the sythesis and accumulation of carbohydrates,unfavorable to seed rating or grain yield improvement.The key to solve the problem is to understanding the mechanism of leaf senescence and then find a pathway to delay senescence,so as to improve grain filling.Utilizing cytokinin synthesis gene under control of senescence-specific promoter,suppressing senescence up-regulated genes or overexpression senescence down-regulated genes all possibly lead to creating stay-green rice.In this study,a rice senescence-specific promoter was isolated and identified,and drived a cytokinin synthesis gene from Agrobacterium in transgenic indica and japonica rice,then the stay-green lines were selected.At the same time,we constructed a flag leaf early senescence SSH library of japonica rice Zhonghua 11.The main results were as follows:1.A rice promoter P_SAG39 was cloned and its time and spatial expression patterns were identified in transgenic plant through the repoter gene gus.Gus staining results showd the promoter expressed in leaf,culm,root,flower,glume;immature seed coat and callus,but not in mature seed.mRNA of the gene accumulated at highest level at late senescence stage.Above all,the promoter is senescence specific and could be used in crop genetic improvement.2.According to PLACE,various cis elements were predicted.We checked expression mode of a series of 5’-truncated promoters in response to hormones and cold treatment in rice calli,and to leaf senescence in 5’ end deletion transgenes.In the analysis of gel retardation assay,we obtained two cis-acting elements HBOXCONSENSUSPVCHS and WRKY71OS for responding to leaf senescence.3.P_SAG39:IPT was transformed to japonica rice successfully.We selected homozygous IPT transgenic lines with single copy insertion,containing 5 lines using MDJ8^# as a recipient named MT1,MT2,MT3,MT4 and MT5,and 3 lines using ZH11 as a recipient named ZT1,ZT2 and ZT3.4.Measuring chlorophyll concentration of flag leaves indicated that stay-green phenotype was coseparation with the leves of ipt in P_SAG39:IPT positive transgenes. 5.We investigated the impact of cytokinin in the transgenes on seedling growth and heading mediated by the lighta and found that in a certain light conditions（14 hours or 24 hours） cytokinin has affected the germination of seeds,the growth of buds,but not the stem elongation.In the long-day conditions,cytokinin stimulates the expression of OsMADS50,thereby leads to early flowering.6.The changes of carbon and nitrogen metabolism were detected in ZT1 at reproductive growth stage.By sugar and nitrogen contents determination,we found sink source transition triggered the onset of leaf senescence.7.The plot-test was performed to evaluate green-stay and other agronomic traits by random block designation in triplicates.In ZT1 and ZT2 the biological yield decreased about 8-10%,and 1000-grain weight increased about 5-9%compared with that of the non-transgenic plants.8.We constructed a flag leaf early senescence library of japonica rice Zhonghua 11 by suppression subtractive hybridization using the green flag leaves as the driver and the early natural senescence flag leaves as the tester.815 differentially expressed ESTs were screen using total RNA of driver and tester as probes through macroarray analyses.A total of 533 unigenes have been confirmed by bioinformatics analysis（ESTClustering and GO annotation）.183 of these unigenes have GO annotations,involved in macromolecule metabolism,protein biosynthesis regulation,energy metabolism,gene expression regulations,detoxification,pathogenicity and stress,cytoskeleton organization and flower development.Another 121 unigenes co-localized with previously reported known stay-green QTLS.Else were functional unkown.9.To confirm the efficiency and accuracy of the library,we performed reverse northern blotting analysis using 50 randomly selected cDNA fragments as probes from the SSH library.The result showd the efficiency was more than 60%.RT-PCR analysis on the other novel genes indicated that they can be up-regulated in natural early senescence and induced by hormone in different levels.These genes provide new insight into the onset of leaf senescence mechanism.更多还原