【作者】 李生平；

【导师】 吴为人； 段远霖；

【作者基本信息】 福建农林大学 ， 生物化学与分子生物学， 2008， 硕士

【摘要】 营养生长是农作物产量和品质形成的基础,而花器官发育的结果直接影响其产量和品质。目前有关植物花器官发育的分子遗传基础的研究已经很深入,但对植物的营养生长和生殖发育起关键作用的多效基因的研究还不多见。本课题组在水稻育种材料中发现了一个植株显著矮化、叶片短而细、花器官明显变异的突变体ddf1（Dwarf and deformed flower 1）。初步研究表明该突变体是由单基因隐性突变所致,暂将该基因命名为DDF1。推断DDF1是维持水稻正常营养生长与花器官发育,调节植株形态建成与生殖发育的重要多效基因。目前尚未见类似ddf1突变体的报道。利用此突变体进行深入研究,对于揭示植物生长发育调节基因在决定植物的形态建成与花器官发育方面的多重作用具有重要的理论意义。本研究对ddf1突变体的遗传特性进行初步分析,并对DDF1基因进行精细定位,为最终克隆该基因奠定基础。主要研究结果如下:1.DDF1基因的遗传分析。由于ddf1突变体的花器官发生变异而高度不育,因此ddf1突变位点只能由杂合体保持。由ddf1杂合体与Acc8558杂交,建立了一个约4000株的F₂代群体,卡平方检测表明,该群体中野生型植株与突变型植株符合3:1分离。以上结果表明,ddf1突变性状由隐性单基因控制。2.DDF1基因的初步定位。从F₂群体中随机取10株野生型植株和10株突变型植株,分别取其叶片等量混合并提取DNA,构成正常DNA池和突变DNA池。利用已公布的473对水稻SSR引物对ddf1杂合体和Acc8558进行检测,共找到150对在两亲本之间显示多态性的引物,然后用这些引物检测两个DNA池之间的多态性,发现位于第6号染色体上的4对引物在两个DNA池之间表现多态性,说明DDF1基因位于第6号染色体上。进一步用F₂群体中的170个突变体进行验证,结果表明DDF1位于微卫星标记RMS1和RMS2之间,与两标记之间的遗传距离分别为3.8 cM和2.4cM。3.DDF1基因的精细定位。在初步定位的基础上,利用已公布的水稻BAC克隆重叠群和基因组序列,在标记RMS1和RMS2之间设计并合成了25对SSR引物,其中有5对引物在两亲本之间表现出多态性。用这5对引物对ddf1杂合体×Acc8558的F₂群体中的986株突变体进行分析,将DDF1基因定位在约60.6kb的区域内。4.DDF1侯选基因的初步确定。通过查询NCBI网站（http://www.ncbi.nlm.nih.gov/）的水稻基因组注释数据库,可知DDF1基因所在的60.6kb区域内共有10个基因,其中有一个基因可能与组蛋白乙酰化作用有关。目前在双子叶模式植物拟南芥中已发现多个与组蛋白乙酰化作用有关的基因参与植株的营养生长与生殖发育过程的调节。因此,初步将这个“与组蛋白乙酰化作用有关”的基因作为DDF1的侯选基因。进一步的验证工作正在进行中。更多还原

【Abstract】 Vegetative growth is the basis of crop yield and quality,while the development of floral organs directly affects the yield and quality of crops.The molecμlar genetic basis of the development of floral organs has been deeply studied,but few studies have been reported on pleiotropic genes that play key roles in the vegetative growth and reproductive development of plants.ddf1（dwarf and deformed flower 1） is a mutant found in rice breeding materials,which is dwarf with short and thin leaves and deformed floral organs.Preliminary analysis suggested that ddf1 was generated due to the recessive mutation of a single gene temporarily named as DDF1.We deduce that DDF1 is an important pleiotropic gene for maintaining vegetative growth and floral organ development and regμlating the morphogenesis and reproductive development in rice.No similar mutants have been reported.Deep study of this mutant will be meaningfμl for the understanding of the functions of pleiotropic genes in the morphogenesis and floral organ development of plants. This study aimed to analyze the genetic features of DDF1 and finely map the gene,which will facilitate the cloning of the gene.The major resμlts are as follows:1.Genetic analysis of DDF1.Due to ddf1’s exceeding sterility,the mutation was maintained only by ddf1 heterozygotes.A 4000 F₂ popμlation was constructed by ddf1 heterozygote and Acc8558 cross-fertilizing.The mutants was characterized with dwarf plants、thin leaves and deformed floral organs.By chi-square test the segregation ratio of wild type plants to mutant plants in the F₂ popμlation was accoding to 3:1.It is indicating that ddf1 is controlled by a single recessive gene.2.Preliminary mapping of DDF1.DNA was extracted separately from two pools constructed with selecting 10 wild type plants and 10 mutants from F₂ segregate popμlation.By filtrating 473 pairs of published SSR primers in ddf1 heterozygote and Acc8558, we found 150 pairs of SSR markers had polymorphism in two parents.Then four markers on chromosome six of rice had polymorphism in two pools were found from the 150 pairs of markers filtrated above.It is indicated that DDF1 was located on chromosome six.Then we used 170 ddf1 mutants selected from F₂ popμlation to certify the resμlt.As the resμlts show,DDF1 was mapped between RMS1 and RMS2.The genetic distance from DDF1 to RMS1 was 3.8 cM and to RMS2 was 2.4 cM.3.Fine mapping of DDF1.On the basis of initial mapping,we enlarged F₂ popμlation to finely mapped DDF1 gene.From F₂ popμlation described above we gained 986 mutants and from 25 pairs of SSR markers designed according to BAC contigs and genome sequence we filtrated five SSR markers had polymorphism between two parents. With the 986 mutants and the 5 pairs of SSR markers we finely mapped DDF1 in a 60.6kb physical distance in chromosome six of rice.4.Preliminary determination of the candidate gene of DDF1.At present,several genes related to acetylation of histones which regμlate the growth and procreation of plants have been found in Arabidopsis.By searching NCBI databases,we found ten genes were annoted in the 60.6kb region where DDF1 was located and a gene related to acetylation of histones may be the candidate gene of DDF1,and further validation is going on.更多还原