【作者】 胡凤益；

【导师】 黎志康；

【作者基本信息】 中国农业科学院 ， 生物化学与分子生物学， 2010， 博士

【摘要】 一年生的栽培稻品种在坡地种植时会导致土壤侵蚀并降低农民的收成,具有地下茎性状的多年生栽培稻品种能够在生产稻米的同时减少土壤侵蚀进而提高粮食和生态安全。具有地下茎的长雄野生稻是多年生稻作品种遗传改良中多年生性状和地下茎性状的逻辑供体。但是,通过传统杂交育种方法获得具有多年生性状的栽培稻品种的努力一直未取得成功。稻属地下茎的生长特性受到许多基因的控制,而与地下茎起始和生长的分子机制一直以来是不清楚的。为了有助于对地下茎性状分子机制的了解,我们利用基因精细定位和芯片技术,在水稻第三染色体和第四染色体上分别精细定位了以前初步分子定位的地下茎基因Rhz2和Rhz3,同时,对地下茎生长点、地下茎节间、地上茎生长点、地上茎节间和嫩叶进行全基因表达谱分析,以此来完善我们在分子水平对地下茎发育的理解。利用分子标记辅助选择方法(MAS),结合地下茎的有无、花粉育性、花药开裂等性状,从包含有5261个单株的F2群体中筛选了4个F2单株自交形成F3群体后进行地下茎基因Rhz2和Rhz3的精细定位。研究结果证实了以前发现的两个显性互补基因Rhz2和Rhz3控制地下茎表达的遗传规律；地下茎基因Rhz2精细定位在长雄野生稻基因组序列的scaffold2675和scaffold9327,这两个scaffold覆盖了长雄野生稻基因组的178.92kb,并整合到水稻第三号染色体上；地下茎基因Rhz3精细定位在长雄野生稻基因组序列的scaffold26912、scaffold30607, scaffold9358和scaffold6613,这四个scaffold覆盖了长雄野生稻基因组的40.49kb,并整合到水稻第四号染色体上。利用Affymetrix公司的水稻基因组芯片(包含有51,279个探针,分别表示48,564个粳稻、1,260个籼稻的转录本)进行长雄野生稻地下茎全基因组表达谱的研究。研究结果表明5种组织中都有特异的基因群表达；58个基因在地下茎生长点中特异表达,其中几个基因与分蘖的起始和延伸相关；和地上茎生长点的表达水平相比,在地下茎生长点中发现了162个上调基因和261个下调基因,在这两种组织中,与植物激素有关的基因和具有冗余功能的基因家族受到明显的不同调控；几种顺式调控元件,包括CGACG, GCCCORE, GAGAC和一个Myb核心在地下茎生长点和地下茎节间中特异表达基因的上游启动子区富集；与地上茎生长点相比,两个涉及到ABA信号通路的顺式作用元件,RY重复和TAAAG在地下茎生长点中过表达；几个地下茎特异表达基因共同定位在地下茎相关QTLs的染色体区域,这些基因可能是地下茎相关基因克隆的功能候选基因。长雄野生稻全基因组表达谱的研究结果表明地下茎的发育和生长是一个非常复杂的基因调控网络,并且在地下茎和分蘖的建成中可能存在一个重叠的调控机制。植物激素,如生长素(IAA)和赤霉素(GA)都涉及到地下茎发育中相应的信号通路。一些在地下茎中富集的顺式作用元件和地下茎特异基因能够共同定位在地下茎相关QTLs的染色体区域以及地下茎基因Rhz2和Rhz3的精细定位结果将会有利于这两个地下茎基因的克隆,也为进一步剖析控制地下茎生长特性的分子机制奠定了基础。研究结果将会有助于我们在禾本科的进化、多年生作物培育以及控制杂草保护环境等方面的理解和认知。更多还原

【Abstract】 Cultivation of annual rice on steep hillsides causes soil erosion and reduces farm productivity. A perennial, rhizomatous rice cultivar would produce food and reduce erosion, subsequently, increase food and ecosystem security. A logical donor of the perennial and rhizomatous traits is Oryza longistaminata, a perennial wild rice that produces rhizomes. However, traditional breeding efforts to transfer the trait have not yet been successful. The rhizomatous growth habit in rice is controlled by many genes, and the molecular mechanism related to rhizome initiation and elongation is still unknown. Using the strategies of fine mapping and microarray technology, we fine mapped the rhizome genes that are Rhz2 and Rhz3 which were mapped preliminary via molecular marker on rice chromosome 3 and chromosome 4, respectively. Meanwhile, we also investigated the gene expression patterns in rhizome tips, rhizome internodes, aerial shoot tips, aerial shoot internodes, and young leaves to improve our understanding of rhizome development at the molecular level.There are 4 F3 populations, which derived from the self-crossing generation of F2 individual that selected via MAS, rhizome present, pollen fertility and anther dehiscence from a F2 population within 5261 individuals, were employed to fine map the rhizome genes, Rhz2 and Rhz3. The two dominant-complementary genes controlling rhizomatousness was confirmed. The Rhz2 was fine mapped to the scaffold2675 and scaffold9327 which are coved 178.92kb on the physical map of O. longistaminata on rice chromosome 3 and the Rhz4 was located on the scaffold26912, scaffold30607, scaffold9358 and scaffold6613, which are coved 40.49kb on the physical map of O. longistaminata on rice chromosome 4.Microarray experiments were performed using one Affymetrix GeneChip Rice Genome Array (Santa Clara, CA) for the five samples. The array contains 51,279 probe sets representing 48,564 japonica and 1,260 indica transcripts. Different gene sets were determined exclusively expressed in five tissues.58 genes were identified as prevalent sets in the rhizome tip. Of these, several genes were functionally involved in tiller initiation and elongation. We found 162 genes up-regulated and 261 genes down-regulated in rhizome tips compared to the expression level in shoot tips that we examined. Strikingly, the genes related to phytohormone and the gene families with redundancy function were obviously differentially regulated in these two tissues. Several cis-regulatory elements, including CGACG, GCCCORE, GAGAC and a Myb Core, were highly enriched in the rhizome tip or internode, and two cis-elements, RY repeat and TAAAG, which are implicated in the ABA signaling pathway, were found overrepresented in the rhizome tip in comparison with the shoot tip. A few rhizome-specific expressed genes were co-localized on the rhizome-related QTLs regions, indicating these genes may be good functional candidates for the cloning of rhizome-related genes.The whole genome profiling of O. longistaminata indicated that a very complex gene regulatory network underlies rhizome development and growth, and that there might be an overlapping regulatory mechanism in the establishment of rhizomes and tillers. Phytohormones such as IAA and GA are involved in the signaling pathway in determining rhizomes. Several cis-elements enriched in rhizomes and rhizome-specific genes co-localized on the rhizome-related QTL intervals provide a base for further dissection of the molecular mechanism that controls the rhizomatous growth habit. The fine mapping of the Rhz2 and Rhz3 will facilitate cloning of the genes, which may contribute significantly to our understanding of grass evolution, advance opportunities to develop perennial cereals, and offer insights into environmentally benign wee-control strategies.更多还原