【作者】 刘世强；

【导师】 杨清； 黄三文；

【作者基本信息】 南京农业大学 ， 生物化学与分子生物学， 2008， 博士

【摘要】 性别分化是植物的基本发育过程,由于作物的性别表达类型决定了其育种和栽培的方式,所以控制性别表达具有重要经济意义,黄瓜（Cucumis Sativus L.）具有非常丰富的性别表现类型,是研究植物性别表达的模式植物。黄瓜花有雄花、雌花和两性花三种类型,但幼花蕾最初都是两性花原基,在进一步发育中两性花原基中雄蕊的滞育形成雌花,雌蕊的滞育形成雄花,雄蕊和雌蕊都发育则形成两性花。目前已经明确控制黄瓜性别表达的主效基因位点有F、M和A三个。除遗传因素外,黄瓜性别表达还受环境和激素的调控,长日照、高温、赤霉素促进雄花产生,而短日照、低温、乙烯则促进雌花形成。激素在黄瓜的性别表达中起重要作用。黄瓜性别表达的乙烯控制模型认为：乙烯既促进雌蕊发育,又抑制雄蕊发育。该模型推断,F基因的产物控制乙烯在黄瓜植株分布的部位和浓度,促进雌性的表达；而M基因的产物则控制乙烯信号的识别,在乙烯浓度高于阈值时抑制雄蕊的发育。近年来的研究发现对该模型提供了实验支持：F基因已被克隆,为乙烯合成酶基因家族成员之一,符合其控制乙烯浓度功能的预测；M位点直接介导了乙烯诱导的雄蕊滞育。要进一步验证该模型的真实性,必须要克隆M基因,而M基因的精细定位则是该基因克隆的前提。本研究以近等基因系WI1983G（雌性株,基因型为MMFF）和WI1983H（两性花株,基因型为mmFF）、F1、F2和BC1为材料,通过M基因的精细定位以及转录表达谱分析,为图位克隆M基因、研究其功能奠定基础。而后者又为进一步深入了解乙烯在黄瓜性别表达过程的作用机理,揭示葫芦科作物性别表达的进化史,乃至通过基因工程操纵其它作物的性别表达来加速杂种优势的利用提供理论依据。主要结果如下：1.近等基因系亲本WI1983G全为雌花,WI1983H全为两性花,它们的杂交组合F1代群体单株均开单性雌花（性型表现为雌性株）,单性花对两性花为显性性状。该杂交组合的F2代群体单株的性型分离株数统计表明：638株F2单株当中,其中雌性株473株,两性花株165株,经卡平方测验,F2代雌性株与两性花株的比例符合3：1的分离比（χ2=0.38；p>0.5）；BC1代群体751个单株当中,雌性株373株,两性株378株,符合1：1的分离比（χ2=0.03；p>0.95）,表明两性花是由单基因控制。2.采用高通量AFLP技术,共获得4个与M基因紧密连锁的AFLP分子标记,其中PGGMCCC₄50/453和PGTMCTA 185分别位于M基因的两侧,共跨度约5 cM。离M基因最近的侧翼标记EACAMCAT₂02/203和EATGMCAA₈0与M基因的遗传距离分别是0.9和1.6 cM,最终将M基因定位在2.5 cM以内。3.利用M基因的侧翼标记PGGMCCC₄50/453和PGTMCTA₁85,从1984株F2和751个BC1分离群体中筛选出重组单株,然后通过896对AFLP引物组合、2000对SSR引物（来自于中国农业科学院蔬菜花卉研究所功能基因组实验室黄瓜基因组项目）及CAPS（根据黄瓜基因组项目Superscaff序列信息）筛选分析,共获得了AFLP标记6个、SSR标记8个、SCAR标记2个和CAPS标记1个,从而构建了M基因的精细遗传图谱。M基因侧翼最近的两个标记SSR23487和S ME8SA7与M基因之间的遗传距离均为0.1cM,最终将M基因定位在0.2 cM的遗传区间内。另外,通过染色体步移法开发出了一个SNP标记SN1,该标记在2,080个F2和751个BC1分离群体当中没有发生重组事件,也即SN1与M基因共分离。4.对近等基因系（WI1983G和WI1983H）的雌花和两性花cDNA分别进行EST测序,其中雌花199,032条,两性花176,139条。经Phred/Phrap/Consed软件包聚类拼接后共获得一致性序列（contig）23,627条,雌花和两性花的单一序列（singlet）分别是32,521和33,494条。经Blast比对（Nr非冗余数据库）、IDEG.6分析软件的卡平方检验,发现1,256个Unigene的表达量在雌花和两性花间存在显著性差异（p<0.05）。这些差异表达基因涉及到物质转运、能量代谢、信号转导及胁迫相关等诸多方面。此外,还有一些基因功能未知。对雌花和两性花间表达差异显著的1,256个Unigene进行功能注释和GO分类。它们被注释的基因为435个,按照GO分类结果如下：细胞组分,108条,占36%,主要涉及到细胞核、细胞膜、线粒体等6类组分；分子功能,155条,占51%,表现出7类分子活性；生物学过程,39条,占13%,涉及到信号转导、生物合成、代谢等过程。以近等基因系为材料,结合AFLP、SSR等多种分子标记构建了黄瓜性别决定基因M的精细遗传图谱,为后续图位克隆法克隆M基因奠定了基础。本文通过对WI1983G的雌花和WI1983H两性花约38条EST测序,分析了黄瓜性别相关转录表达谱,初步了解了这些基因参与的生物学过程。此外,本研究中获得的部分功能未知的EST,为我们进一步深入研究黄瓜性别表达的调控机理提供更多的相关候选基因资源。更多还原

【Abstract】 Sex differentiation in plants is a fundamental developmental process of economic importance since the sexual phenotypes of crops determine the processes of breeding and cultivation. The diverse sex types of cucumber allow this organism to serve as a model system for studying sex expression in flowering plants. The cucumber has three types of flowers:male, female, and bisexual. Morphologically, all cucumber floral buds are initially hermaphroditic with both male and female reproductive organs. Pistil development is then arrested in floral buds that develop into male flowers, whereas stamen development is arrested in floral buds that develop into female flowers. Bisexual flowers form from the buds in which neither pistil nor stamen development is arrested. Three primary genes that influence sex expression in cucumber have been described including F, M and A. In addition, sex expression is also influenced by environmental conditions and plant hormones. Long days, high temperatures, and gibberellic acid promote the formation of male flowers, whereas short days, low temperatures, auxins, and ethylene enhance the development of female flowers. The model of sex determination in cucumber is presumed that ethylene serves as both a promoter of the female sex and an inhibitor of the male sex. The model predicts that the F gene encodes a molecule that influences the range and gradient of ethylene production along the shoot, thereby acting to promote femaleness, whereas the M gene encodes a molecule that detects this ethylene signal and inhibits stamen development when ethylene levels reach a threshold. Recent studies have provided molecular evidence in favor of the ethylene model of sex determination in cucumber: the F gene encodes an ACC synthase, is cloned. The result is accord with above ethylene model of sex determination. In addition, the product of the M locus mediates directly the inhibition of stamen development by ethylene. For further testify the authenticity of the ethylene model, the M gene must be cloned and fine-mapping is the basic of map-based cloning. In this study, nearly isogenic cucumber lines WI1983G （gynoecious; MMFF） and WI1983H （hermaphrodite; mmFF）, F2 and BC1 individual plants were used to clone Mgene and analysis transcription expression profiling of female and bisexal flower. This study provides theoretic evidences for further understanding the ethylene fuction for sex determination in cucumber, revealing the evolutional history of sex expression in Cucurbitaceae and controling sex expression of other crops for speedup the utilization of heterosis by the way of gene engineering. The main conclusions are as follows:1. The mother plant WI1983G bears only pistillate flowers, whereas the mother plant WI1983H bears bisexual flowers. All F1 progeny that were derived from the cross between WI1983G and WI1983H lines bore pistillate flowers. This finding confirmed the complete dominance of the maternal sex type over the paternal sex type. In the 638 F2 individuals, 473 had only pistillate flowers and were scored as gynoecious （M_FF）. In addition,165 F2 individuals had only bisexual flowers and were, thus, scored as hermaphroditic （mmFF）. These results fit the Mendelian 3:1 ratio （x2= 0.38; p> 0.5）; In the 751 BC1 individuals, 373 had only pistillate flowers.378 individuals had only bisexual flowers. These results also fit the Mendelian 1:1 ratio（x2=0.03; p>0.95）, indicating the single M locus controlled the segregation of the sex expression in the F2 and BC1 progeny. The result is consonant with the conclusion of others’study.2. we obtain four AFLP markers were linked to the M locus using high-throughput AFLP technology. The local map spanned a genetic interval of 5.0 cM, which was defined by the AFLP markers PGGMCCC₄50/453 on one side and PGTMCTA₁85 on the other side. The M locus co-segregated with the CsEIL1 marker （no recombinants in 96 F2 individuals）, which developped according to the pivotal gene EIL1 in ethylene singal, and was flanked by the AFLP markers EACAMCAT₂02/203 and EATGMCAA₃80 that defined a 2.5 cM interval. The linkage map provides a solid basis for high-resolution mapping and ultimately for molecular isolation of the M gene.3. With flanking markers （PGGMCCC₄50/453 and PGTMCTA₁85） of the M gene, recombinant plants in M interval were identified from 1984 F2 and 751 BC1 segregating population and we obtain 6 AFLP,8 SSR,2 SCAR,1 CAPS markers by the way of screening 896 AFLP primer combinants,2000 SSR primers, relating to superscaff sequence information. A high-resolution genetic map of the M gene was constructed. The M gene was delimited into a genetic interval of 0.2 cM between SSR23487 （0.1 cM） and S_ME8SA7 （0.1 cM）. A SNP marker SN1, co-segregating with the M gene based on the linkage analysis （no recombinants in 2,080 F2 and 751 BC1 individuals）, was obtained by the way of chromosome walking.4.199,032 ESTs in female flower （WI1983G） and 176,139 ESTs in bisexal flower （WI1983H） were obtained from sequencing of cDNA. Sequence assembly by Phred/Phrad/Consed software revealed that 23,627 contigs were acquired except for 32521 singlet sequences in female flower （WI1983G） and 33494 singlet sequences in bisexal flower （WI1983H）.1,256 Unigenes, which have distinctly different expression （p<0.05） between female and bisexal flower, were ultimately found out using blast （Nr, non-redundant database） and chi test in the IDEG.6 software. These genes involved in several aspects such as substance transport, energy metabolism, signaling, stress response et al. In addition, there are mostly ESTs with unknown functions （No hits）.1,256 Unigenes, which have distinctly different expression （p<0.05） between female and bisexal flower, were done with blast, EST annotation and GO classification. By homology search and gene ontology analyses,435 identified ESTs, which were annotated, were mainly categorized as belonging to:Cellular Component （108,36% involving with nucleus, membrane, mitochondrion et al.）, Molecular Function （155,51% including seven kinds of molecular activity）, and Biology Process （39,13% involving with signaling pathway, biosynthesis, metabolization et al.）.The fine-mapping of M gene, which privides a basis for map-based cloning M gene, was constructed based on the nearly isogenic lines and AFLP, SSR molecular marker et al.By sequence of about 380,000 ESTs in female flower （WI1983G） and bisexal flower （WI1983H） and study of related transcription expression profiling for sex determination in cucumber, the biological processes in which these related genes for sex determination involved in came to be primary understood. In addition, partial unknown-fuction annotation ESTs were obtained in our study, in which provides more candidate genic resources for our further studies the regulative mechanism of sex expression in cucumber.更多还原