【作者】 关媛；

【导师】 吴爱忠； 蔡润；

【作者基本信息】 上海交通大学 ， 生物医学工程， 2008， 博士

【摘要】 黄瓜植株茎、叶、卷须、花萼、子房表面均覆有短刚毛,果实均有刺,通过组织学观察,发现黄瓜叶片上的刚毛和果实上的果刺形态结构一样,均为多细胞无腺体的表皮毛。果实是黄瓜经济性状最重要的部分,而果刺是果实外观品质重要的性状。黄瓜EST序列数量的迅速增加为开发新的SSR标记提供了宝贵的数据资源。本论文从GenBank上公布的黄瓜EST序列中检索到902条来自果实的序列,利用SSRIT软件分析得到63条包含SSR的EST序列,设计了37对EST-SSR引物,结果显示有9对引物在7份黄瓜自交系上扩增有多态,占所设计引物总数的24.3%,平均等位变异数为2.1个,7份自交系间C8和S06遗传相似系数最小为0.22。有27对引物在3份甜瓜上有扩增产物,其中1对引物扩增有多态。这为今后黄瓜基因组研究提供了新的标记。利用中国华北类型的黄瓜自交系S94构建了一个BAC文库,该文库包含约19200个克隆,平均插入片段为105 kb,大约覆盖黄瓜基因组的5倍。为了使黄瓜的遗传连锁群锚定其染色体,从黄瓜的7个连锁群共选择了22个标记,其中15个SCAR标记和7个SSR标记。利用这些标记用PCR的方法筛选BAC文库的DNA池,15个标记筛选到至少2个克隆,共筛选到60个BAC克隆,最后确定了22个BAC克隆作为连锁群特异克隆,这个BAC文库的构建为今后黄瓜基因组研究奠定了基础。本论文利用欧洲温室类型自交系S06(母本)与黄瓜无毛(果刺)突变体gl(父本)构建了分离群体。通过F2和BC1群体的遗传分析,结果表明有毛(Gl)为显性,无毛(gl)为隐性,并且果实、茎、叶、等表面的表皮毛性状为一对核基因控制。同时控制果实、茎、叶、表皮毛性状的无毛基因(gl)对控制果瘤性状的果瘤基因(Tu)存在隐性上位作用。用SRAP标记结合BSA法进行黄瓜果刺形成基因的定位,找到两个与Gl连锁的SRAP标记,其中包括一个与Gl基因紧密连锁的标记ME4EM3,连锁距离为3.2cM,并将该标记转化为一个SCAR标记。用这个SCAR标记筛选黄瓜BAC文库,进行了BAC末端测序,为进一步精细定位与分离果刺形成基因奠定基础。为了克隆控制黄瓜果刺形成相关的基因,本论文利用棉花和拟南芥TTG1基因进行同源克隆,得到一个CsTTG1基因。半定量RT-PCR分析表明,该基因在重要的分生组织及器官中都有表达。Southern blot实验表明,该基因在黄瓜中为单基因。功能互补实验表明CsTTG1能互补拟南芥ttg1突变体的表型,这个结果表明,黄瓜TTG1-like基因与拟南芥的TTG1基因功能同源。这有助于我们理解多细胞表皮毛形成的分子机制,也有助于认识黄瓜果刺这个外观品质性状。更多还原

【Abstract】 The wild-type cucumber has trichomes on foliage and spines on fruit. The leaf trichomes and fruit spines have similar shape and structure with histology analysis in cucumber, and both structures are multicellular, non-glandular trichomes. Cucumber fruit is most important part of economic character, and breeding objectives for improvement are often focused on fruit yield and quality. The fruit spine is important trait of fruit appearance quality.Simple sequence repeat markers derived from expressed sequence tags (EST-SSR) are potentially valuable tools for plant breeding and germplasm collection conservation, and increasingly, efforts have been made for developing this type of marker. In this study, we have identified 9 polymorphic SSR markers from cucumber fruit EST deposited in public sequence database. The average allele number was 2.1 per locus, ranging from two to three alleles during screening 7 cucumber genotypes. Genetic similarity coefficient ranged from 0.22-1, with an average of 0.66 among 7 cucumber genotypes. Amplification products were also detected by 27 pairs of primer in 3 melon genotypes. These informative EST-SSR markers can be used in cucumber genome research.A bacterial artificial chromosome (BAC) library consisting of 19,200 clones with an average insert size of 105 kb has been constructed from a cucumber (Cucumis sativus L.) inbred line S94, derived from a cultivar in North China. The entire library was equivalent to approximately 5 haploid cucumber genomes. To facilitate chromosome engineering and anchor the cucumber genetic linkage map to its chromosomes, 15 sequence-characterized amplified regions (SCAR) and seven simple sequence repeats (SSR) markers from each linkage group of cucumber were used to screen an ordered array of pooled BAC DNA with polymerase chain reaction (PCR). Fifteen markers gave at least two positive clones. As a result, twenty-two BAC clones representing 7 linkage groups of cucumber were identified, which further validated the genome coverage and utility of the library. This BAC library and linkage group specific clones provide essential resources for future research of the cucumber genome.To mapping gene of fruit spine formation, we was constructed segregate populations with inbred line S06 (Europe greenhouse type) and glabrous (gl) mutant. The trichoms characteristic of foliage surface was controlled by a pair of nuclear genes. The characteristic of trichomes (Gl) was dominant to that of glabrous foliage (gl) with heredity analysis. The gene of foliage trichomes also controlled fruit spines. The Gl gene together with Tu gene decided fruit surface, which showed three phenotypes on fruit: have warts and spines, only have spines, without warts and spines, with its proportion being 9:3:4. This result indicated the epistatic recessiveness of glgl gene to Tu_ gene. Combining the bulked segregant analysis (BSA) and the sequence-related amplified polymorphism (SRAP) technology, we found two markers linking to the Gl/gl locus. Among them, the one closely linked SRAP markers flanking the Gl/gl locus were marker ME4EM3 with 3.2cM, and then this marker transform to SCAR markers. A BAC library of cucumber will be PCR screened by this marker and get positive BAC clones with end sequence. This work is base for further fine mapping and isolated gene of fruit spine formation.To identify genes involved in the molecular control of cucumber fruit spine formation, we isolated one putative homologues of the Arabidopsis trichome associated gene TRANSPARENT TESTA GLABRA1 (TTG1). CsTTG1 genes are expressed in many tissues throughout the plant with RT-PCR analysis, including shoot apices and floral buds. The gene used in turn to probe Southern blots of cucumber genomic DNA restricted with various endonucleases. This result showed that cucumber genome contains single copy of CsTTG1 gene.This cucumber TTG1-like gene was able to restore trichome formation in ttg1 mutant Arabidopsis plants. These results indicate that these cotton genes may be functional homologues of AtTTG1. These results helpful understand molecular mechanism about muticelluar trichome pattern.更多还原