【作者】 罗增科；

【导师】 何光华；

【作者基本信息】 西南大学 ， 作物遗传育种， 2009， 博士

【摘要】 水稻是世界上种植面积最大、最重要的粮食作物之一,也是研究单子叶植物发育分子生物学的理想模式植物。水稻是我国最重要的粮食作物,研究其生殖器官发育的分子机制具有重要的理论意义和应用价值。在水稻中,花器官发育相关的基因大多是通过同源克隆而来,而大多数同源克隆基因的功能因为与性状缺乏联系而无法最终确定。通过花器官突变体的发现和观察研究、克隆基因,并进行功能分析最终确定基因的功能,是研究花器官发育基因的功能和调控模式的有效方法。同时研究表明,单双子叶植物间可能存在相同的花器官和相同的调控模式,但具体机理尚不清晰。因此,对水稻花器官发育的研究在揭示花发育相关基因的作用及其相互关系、探明植物生殖发育,特别是花发育的奥秘中起了十分重要的作用。本研究通过图位克隆的方法从水稻中分离了一个水稻花器官发育相关基因ELONGATEDLODICULES 1（EL1）,在对突变体进行表型观察和细胞学分析的基础上,并对其表达模式和功能进行了分析,明确了EL1基因的功能和作用模式。主要结果如下:1.el1突变体的表型观察和细胞学分析我们在保持系缙2B中发现了1个性状表现稳定遗传的水稻花器官突变体,其花器官形态表现为第1轮内颖弯曲呈S状;第2轮浆片变形伸长,有些多一颖壳状组织;第3轮雄蕊减少,第4轮子房和柱头均出现增多的现象。该突变体四轮花器官均发生突变,育性并没有发生改变。鉴于该突变体的突变性状未见报道,我们将其命名为el1（elongated lodicules 1,浆片伸长）突变体。2.EL1基因的遗传分析和初步定位利用4个恢复系与el1突变体杂交,F₁代植株表型正常,与野生型表型相同;F₂代出现表型分离,正常型与突变型的比例为3:1,表明el1突变体性状为单隐性基因控制。用水稻SSR标记分析其中一个F₂代分离群体（el1×602）,先用分子标记分析亲本间即el1突变植株与602之间的多态性分析,再用在亲本间表现多态性的引物作连锁分析,将EL1基因定位在第1染色体短臂SSR引物RM1232与RM5389之间,遗传距离分别为7.3cM和4.5cM。3.EL1基因的精细定位和候选基因的筛选构建大于5000株的F₂代大群体,利用新合成的SSR,InDel,STS等标记将EL1基因定位在SSR引物RM128（PS2C8）和RM1152之间,遗传距离分别为0.83cM和0.79cM,两标记间的物理距离为645Kb。在定位区间相应位置上发现一个可能与花器官发育相关的MADS-box基因OsMADS32,OsMADS32初步筛选为EL1的候选基因。4.候选基因的克隆分析和确定OsMADS32的测序分析:对el1突变体与野生型的OsMADS32基因的cDNA和DNA进行了测序分忻,发现el1突变体中,OsMADS32基因的编码区内发生了一个碱基T的缺失,导致翻译的提前终止,从而进一步确定OsMADS32为EL1的候选基因。候选基因的酶切验证:根据测序结果,在碱基缺失位置上恰好存在一个内切酶RsaI的位点,设计引物扩增基因内部一段约200bp的序列（包括突变何点）,酶切检测突变群体。结果显示,此突变何点与突变性状完全连锁,确定了OsMADS32就是EL1的候选基因。5.EL1基因的时空表达分析EL1基因在穗部的特异表达:通过半定量RT-PCR和QPCR的方法检测,EL1基因在根、茎、叶、穗中的表达情况,发现EL1基因只在水稻的穗中特异表达。EL1基因的RNA原位杂交分析:在野生型中,花原基发育初期,EL1的转录信号在整个花原基和整个小花分生组织的顶端被检测到;但是在花原基发育后期,EL1的转录信号在浆片、雄蕊和雌蕊比在颖片中更为强烈。6.候选基因的功能验证EL1基因的RNAi分析:构建RNA干涉表达载体,转化粳稻品种中花11的幼胚愈伤组织,阳性植株表型与突变体表型相同,进一步验证和明晰了EL1基因在水稻花器官发育过程中的功能。EL1基因的过量表达分析:我们构建了以35S启动的EL1基因的过表达载体,获得转基因阳性植株,转基因植株小花内三轮器官没有变化,而内颖变短、变细。7.EL1基因的进化分析对主要的MADS-box基因进行分子进化分析,结果表明EL1和小麦MADS-box基因TaAGL14和TaAGL15构成一个分枝,根据现有的基因序列信息,此分枝中只存在于禾本科中。8.花发育相关基因的表达分析选取11个花器官发育相关基因,这些基因在野生型和突变体表达无差异,此结果初步说明了EL1基因在水稻花器官发育过程中的独特性和独立性。更多还原

【Abstract】 Rice（Oryza sativa L.） is not only one of the most important food crops in the world but also a model plant for study of the molecular developmental biology in monocots.Research on rice reproductive mechanism is significant both in theory and in human agriculture.In rice,most of genes involved in floral development were isolated by screening the cDNA or DNA library with the probes homologous to sequences of dicot plants,and their functions were identified by the altered morphology of floral organs in transgenic plant.The acquisition and research on the rice mutants related to floral development play an important role in understanding the function and interaction of genes in reproductive process,especially in floral development.In this study,we isolated a gene,ELONGATED LODICULES 1（EL1） involved in the flower development in rice,we have completed the investigation of the anatomical structure of floral organs,genetic analysis,fine mapping and further analyzed its functions.Our results may provide some information on the molecular mechanism of the controlling of floral organ development in plants.The results as follows:1.Phenotypic Observation and Cytological Analysis of el1The rice el1 mutant was found from a spontaneous mutation in an indica maintainer line Jin 2B. In the whorl 1 of el1 flower,palea/lemma is in the shape of crook "S".In whorl 2 the lodicules were elongated and two stamens were transformed into lodicule-like organs.In the whorl 3 of el1 flower, stamens number decreased.In the whorl 4 of el1 flower,lodicules and/or pistils number increased. Because the phenotypic traits have not been reported,temporarily designated as el1（elongated lodicules 1） mutant.2.Genetic Analysis and Preliminary Mapping of EL1The genetic analysis was conducted on the F₁ hybrids and F₂ populations obtained from 4 crosses.The F₁ population of four crosses showed wild-type phenotype.In the four F₂ populations, all the segregation rates of wild-type and el1 mutation plants fit the ratio of 3:1.Therefore,the el1 mutant phenotype is controlled by a single recessive gene.We used the SSR marker to analyze one F₂ populations（el1×602） of crosses,and in this cross, the gene EL1 was mapped between RM1232 and RM5389 on chromosome 1 with respective genetic distance 7.3 and 4.5 cM.3.Fine Mapping of EL1 and Filtrating the Candidate GenesThe gene EL1 was mapped between（RM128） PS2C8 and RM1152 with genetic distances 0.83 cM and 0.79 cM（physical distant:645Kb） by new developmental SSR,InDel,STS marker.In this region,a prominent candidate gene,OsMADS32,which is a transcription factor with MADS domain. This domain defined function has been well established in the regulation of flower development in higher plants,was identified.So we filtrated OsMADS32 was the candidate gene of EL1.4.Determining the Candidate Gene of EL1 and Cloning AnalysisSequence Analysis of OsMADS32:We sequenced the cDNA and DNA of OsMADS32 of mutant plant and wild-type plant.We compared the OsMADS32 transcripts and found a single nucleotide T deletion resided at the el1 mutated allele,causing a premature translation stop. Furthermore,the results showed OsMADS32 was the candidate gene of EL1.Digestion Analysis of the Candidate Gene:An original Rsal restriction site was identified and experimentally confirmed in the wild-type allele,while in the mutant allele the Rsal restriction site was abolished due to the T deletion.We amplified a 200bp sequence（including mutant locus） and digested the mutant plants population,but none of them was able to be catalyzed.Taken together, these results indicated that the candidate gene that we isolated was the candidate gene.5.Temporal and Spatial Expression Patterns of EL1EL1 was specifically expressed in panicle:To determine the temporal and spatial expression patterns of EL1 gene,semi-quantitative RT-PCR and quantitative PCR were used to detect the expression of EL1 in root,stem,leaf and panicle,and the results showed that EL1 was only specifically expressed in panicle.The in situ hybridization of EL1:While the floral organ initiation,EL1 transcripts were detected in all flower primordia and spikelet apical meritems.After floral organ initiation,EL1 was abundantly expressed in the lodicules,stamens and pistils stranger than in palea/lemma.6.Functional Analysis of the Candidate GeneRNA Interference of EL1:We constructed the RNA interference vector.Construct was introduced into embryo callus of rice cv.Zhonghuall（Oryza sativa L.ssp.japonica） by Agrobacterium-mediated T-DNA transfer.The phenotype of positive plants were the same as mutants’,furthermore,the results confirmed the function of El1 in rice flower development.Overexpression of EL1:We constructed the over expression vector of El1 by 35S promotor and achieved transgenic positive plants.the palea of the ransgenic positive plants become short and narrow but the inner three whorls organs have not changed.7.Phylogeny Analysis of EL1 geneA phylogenetic tree was constructed using the main MADS-box genes,the results showed that EL1 was clustered with two wheat MADS-box genes（TaAGL14 and TaAGL15,respectively）.Based on the gene sequence,it is a unique branch in grass.8.Expression Analysis of Genes Involved in Flower DevelopmentWe chose 11 genes involved in fllower development,and the expression did not detect significantly changes in all the genes between el1 mutant and wild-type.The result preliminary indicated the uniqueness of EL1 gene in flower development in rice更多还原