【作者】 周梁良；

【导师】 宋任涛；

【作者基本信息】 上海大学 ， 生物信息工程， 2011， 博士

【摘要】 大刍草是与玉米亲缘关系最近的野生祖先,和玉米同属于玉蜀黍属,但是在外观上两者却有着巨大的差异,如何在短短一万年内将大刍草驯化成外观上截然不同的栽培玉米,至今仍是未解之谜。本论文选择了2个染色体位点,玉米驯化的重要基因tb1(teosinte branched1),以及典型的簇状分部的22kD醇溶蛋白基因家族,在玉米和大刍草间进行比较基因组学分析从而研究玉米的驯化与进化。tb1基因编码了一个TCP家族的转录因子,它控制了玉米的分蘖数和穗形态。在玉米中,tb1基因在腋分生组织和雄蕊原基中相对大刍草有较高的表达,从而抑制了对应组织的生长发育,造成玉米和大刍草的形态差异。以往研究发现tb1基因的驯化区域位于编码区上游58-69kb处,其中58-64kb控制了穗形态而64-69kb控制了分蘖,但驯化的具体位点并不清楚。本研究结合比较基因组学和关联分析,揭示了tb1基因的驯化位点。首先,我们对玉米、高粱、水稻和二穗短柄草在tb1上游的保守非编码序列(conserved noncoding sequences, CNSs)进行分析,发现大部分CNSs位于tb1上游58-72kb处,暗示该区域可能对tb1调控有重要作用。其次,我们从BAC文库中分离并测序了玉米Yu87-1、W22自交系,以及一年生大刍草和多年生大刍草的tb1区域。通过比较分析发现,一年生大刍草和玉米高度保守,而多年生大刍草和玉米则有较大差异,这些差异主要是由于不同的LTR反转座子插入造成。在上游调控区域(58-69kb),发现了两个主要差异可能与tb1驯化相关,一个为玉米tb1上游58.8kb处的LTR反转座子插入,另一个为玉米tb1上游64.5kb处的MITE类型的DNA转座子插入。随后我们在539个玉米品种和189个大刍草品种中对这两个转座因子进行了检测,发现它们的插入与玉米表型紧密关联。最后我们挑选了玉米和大刍草品种,对整个调控区域进行扩增并测序,经过序列比对排除了其它SNP或InDel作为驯化位点的可能性。由此证明,玉米tb1基因上游58.8kb处的LTR反转座子插入和64.5kb处的MITE插入,分别上调了tb1在雄蕊原基和腋分生组织中的表达,导致了tb1基因的驯化。22kD醇溶蛋白是玉米籽粒中主要的储藏蛋白,由一个基因家族编码,在玉米基因组中的分布为一个基因簇和一个单独的拷贝(fl2),是研究基因家族扩增和进化的优良模型。本实验室前期对玉米、高粱和薏苡的22kD醇溶蛋白基因簇进行了比较分析,并对基因家族的进化历史进行了预测。本研究从一年生大刍草和二倍体多年生大刍草BAC文库中筛选22kD醇溶蛋白基因,分别得到了22和40个阳性单克隆。使用基因簇两侧标记引物对所有克隆进行鉴定,同时结合指纹图谱和DNA杂交图谱,对BAC克隆在基因簇中的覆盖情况进行了预测。结果显示,在一年生大刍草和多年生大刍草中都筛选到了两个22kD醇溶蛋白基因簇,基因簇的长度在200-300kb间,并且基因簇两侧都有直系同源标记CR2和CRa。对二倍体多年生大刍草中的5个BAC克隆进行初步测序,并对fl2区域进行了比较分析。在fl2区域,玉米和多年生大刍草间的同源序列仅占28%,造成差异的主要原因是不同的LTR反转座子插入以及后续的染色体重组,与tb1区域类似。本研究揭示了玉米tb1基因的驯化是由于两个转座因子的插入,说明转座因子在玉米的进化过程中不仅仅扩张了基因组,同时也对基因的表达调控起了至关重要的作用;通过对22kD醇溶蛋白基因家族的筛选鉴定,预测了基因簇的基本情况,为研究22kD醇溶蛋白基因家族在玉蜀黍属中的进化奠定了基础;首次分离测序了大刍草大片段基因组序列,并与玉米进行比较分析,丰富了我们对玉蜀黍属基因组以及基因组进化的认识。更多还原

【Abstract】 Teosinte is the most related wild ancestor of modern maize. But the architectures of maize and teosinte are strikingly different. However, how the ancient agriculturists domesticate maize into teosinte in such a short time was still a mystery. In this study, we choose the major domestication gene, teosinte branched1 and the important storage protein gene family, 22kD prolamin gene family, and carry on comparative analysis to study the evolution and domestication of modern maize.Teosinte branched1 encoded a TCP family transcriptional factor and controls maize basal branching and ear phenotype. In maize, tb1 has a relatively high expression level in axillary meristems and stamens of ear primordial thus suppress the development of corresponding organs. Previous studies revealed the domestication region located 58-69kb upstream of tb1. 58-64kb upstream region controls the ear phenotype while 64-69kb upstream region controls the basal branching. But the exact sequences that alter tb1 expression remain unknown.In this study, we use comparative genomics and association analysis to figure out the causative mutation for tb1. We analyzed the conserved noncoding sequences (CNSs) of tb1 upstream region among maize, sorghum, rice and brachypodium. We found most CNSs located 58-72kb upstream of tb1, suggesting this region might be important for tb1 regulation. Then we isolated and sequenced the tb1 region of Zea mays ssp. parviglumis, Zea diploperennis, maize haplotype W22 and Yu87-1. Comparative analysis revealed that PVG was highly conserved with maize, while Zea diploperennis show much less conservation mostly due to retroelements insertions. We find the major difference between maize and teosinte that might cause tb1 mutation were one LTR-retrotransposon and one MITE insertion in maize tb1 regulatory region (58-69kb upstream of tb1 coding region). We detected the presence of these two transposable elements in 589 maize haplotypes and 189 teosinte accessions. The results revealed the LTR-retrotransposon and MITE were strictly associated in maize. Finally, we sequenced tb1 regulatory region in some other maize haplotypes and teosinte accessions. Sequence alignments exclude all the SNPs or InDels that might be candidate locus for tb1 mutation except for the two TEs. We concluded that the LTR retrotransposon and MITE insertions upregulate tb1 expression in axillary meristems and stamens of ear primordial, thus lead to the domestication of tb1.22kD prolamin was the major storage protein in maize kernel. It was encoded by a gene family and was a good model to study the evolution of gene family. It is composed of one gene cluster and one single gene (fl2) in maize genome. Our lab previously compared the 22kD prolamin gene cluster among maize sorghum and coix. We also predicted the formation and evolution history of the gene cluster. In this study, we isolated 22 BAC clones in Zea mays ssp. parviglumis and 40 clones in diploperennis that containing 22kD prolamin gene. According to the fingerprints, DNA blotting and flanking marker screening, we predicted the possible organization of 22kD prolamin gene cluster in teosinte genome. We found two 22kD prolamin gene cluster with flanking orthologous markers in both Zea mays ssp. parviglumis and Zea diploperennis. The gene cluster extended from 200kb to 300kb. We sequenced 5 BAC clones of Zea diploperennis. We analyzed the fl2 region between maize and Zea diploperennis. They only shared 28% homologous sequences. Like in the tb1 region, LTR retrotransposon insertion and following recombination are the major cause of variation.This study revealed that the domestication of maize tb1 gene was the result of two transposable elements insertion events. Transposable elements not only expanded the maize genome, but also had great impact on gene regulation. Through isolation and identification of 22kD prolamin gene family, we predicted the organization of gene cluster, facilitated the further study of 22kD prolamin gene evolution in the genera Zea. We isolated and sequenced teosinte large genomic sequences and compared with maize for first time. This study enhanced our knowledge of genome composition and evolution of the genera Zea.更多还原