水稻着丝粒DNA变异及其影响植株生长发育机制研究

The centromere of eukaryotic chromosomes is essential for the faithful segregation and inheritance of genetic information. It serves at the site for assembly of the proteinaceous kinetochore to which spindle microtubules attach during mitosis and meiosis. The centromere is a complex composed of centromeric proteins and DNA motifs in many higher eukaryotes. Although their functions are relatively conserved, centromeres contain highly divergent DNA sequences from all eukaryotic chromosome. However, functional proteins within centromeres are highly conserved. In particular, a centromere-specific histone 3 variant, referred to as CENH3, is a key centromere-specific protein for centromeric chromatin. However, it is not a continuous distribution in centromeric region. Part of the nucleosomes contain H3. Recent researches shows that active genes exist in centromere. The active genes are located at euchromatin area containing H3. However, the genes located at H3 will be inactive when H3 was replaced by CENH3. Because the highly repetitive nature of the multimegabase satellite sequence arrays span centromeres in most plants and animals, thsoe genes locating in centromeres were rarely studied. In rice, there is no complication of satellite-rich DNA in the Centromere 8 (Cen8). As a result, Cen 8 is an ideal model system to research the features of centromere and gene function in the centromeric region. In preliminary studies, we obtained a rice line containing a 250Kb-DNA deletion in the Cen8 region. Although, it's mitosis are normal , it grows weakly and dies under natural environment. In this proposal, we will reveal the structure characteristics of new centromeric genomic DNA using this material. In addition, the candidate genes controlling plant growth and development in the rice Cen8 region will be discovered using the FISH, RNA-seq, ChIP-seq and RT-PCR technologies. Then, RNAi and complementary test will be emploied to confirm the function of the candidate genes. These results will reveal the neocentromeric characteristics of rice chromosome 8. Furthemore, these results will provide powful evidences to support that there are functional genes controlling plant growth and development in centromeric region in rice. In addtion, we will get a new material and method to study function of these genes located at centromeric region.

着丝粒是真核生物染色体基本功能元件，是DNA和蛋白质的复合体。在着丝粒区域存在特异H3 (CENH3) 和正常H3。最新研究表明着丝粒区域存在的活性基因位于H3，而位于CENH3上的基因是失活的。本实验室在多年的水稻细胞遗传研究过程中，积累了一系列水稻染色体着丝粒变异材料，为研究着丝粒区域DNA特点和基因功能提供了丰富的遗传资源。在前期研究中，我们筛选到一个水稻第8染色体着丝粒区域DNA缺失的遗传材料。该材料植株生长发育异常。本研究以该材料为研究对象，运用FISH、IF、ChIP-seq、RNA-seq等技术明确着丝粒区域DNA序列变异特点，确定该区域控制水稻生长发育的候选基因；在此基础上通过RNAi和功能互补试验验证候选基因，开展水稻着丝粒区域基因的生物学功能及其调控植物生长发育的分子机理分析。本研究将为人工染色体的构建提供新的理论依据，同时为着丝粒区域存在活性功能基因的理论提供新证据。

着丝粒是真核生物染色体基本功能元件，是DNA 和蛋白质的复合体。在着丝粒区域存在特异H3 (CENH3) 和正常H3。最新研究表明着丝粒区域存在的活性基因位于H3，而位于CENH3 上的基因是失活的。通过本项目的实施，运用FISH、ChIP-seq，ChIP-PCR，RNA-Seq等技术明确CENH3所结合的着丝粒DNA区域所丢失的片段大小在200kb左右，确定该突变体材料存在8个缺失基因。明确新着丝粒区域向长臂扩张，H3与CENH3发生互换，新CENH3结合区域的基因改变了其原先组蛋白的结合状态，从而基因转录活性受到影响，与之转录活性相关的组蛋白修饰含量也随之减少，而导致3个活性基因的失活。明确在着丝粒变异材料中发生基因组或转录组变异的11个基因，把其作为水稻着丝粒区域调控植株生长发育的候选基因，并通过CRISPR/Cas9技术、RNAi技术和过表达技术对第8染色体着丝粒区域影响植株生长发育基因进行相关转化研究，并重点对其中的DCL基因功能进行了深入研究，研究表明DCL基因控制水稻的叶色与株高，该基因突变后会影响植株正常生长发育。相关结果发表SCI论文3篇，中文权威1篇；培养博、硕士生6名。项目主持人2016年入选江苏省333工程第三层次培养对象，2014年入选省高校“青蓝工程”优秀青年骨干教师培养对象和省优秀本科论文指导老师。