【作者】 李强；

【导师】 任仲海；

【作者基本信息】 山东农业大学 ， 蔬菜学， 2013， 博士

【摘要】 果实刺瘤是黄瓜（Cucumis sativus L.）重要的外观品质之一。组织学观察发现黄瓜表皮毛和果刺均为多细胞非腺体的柱状结构。黄瓜无毛突变体glabrous1(gl1)的茎、叶、卷须、花萼、子房均没有表皮毛，果实表面也没有果刺和果瘤，是研究黄瓜表皮毛及果实刺瘤形成机理的理想材料。遗传分析结果表明无毛基因（gl1）对控制果瘤性状的果瘤基因（Tu）存在隐性上位作用。因此，研究黄瓜表皮毛形成的分子机制有助于探明黄瓜果实刺瘤的形成机制。另外，表皮毛和果刺是由表皮细胞分化而来，而表皮细胞是研究植物细胞分化最好的模型，因此，对黄瓜表皮毛的形成机制的研究有助于丰富植物细胞分化理论。本研究用欧洲温室型黄瓜“壮瓜”与无毛突变体gl1杂交构建了F2群体，利用无毛表型的F2单株为定位群体，进行图位克隆以分离出无毛基因。同时，还利用生物信息学的方法分析了黄瓜中的R2R3MYB, bHLH，WD40-repeat和R3single repeat MYB基因家族的所有成员，并利用同源进化分析找出黄瓜中可能和表皮毛形成相关的同源基因。为了研究这些同源基因的功能，还进行了拟南芥和黄瓜的遗传转化。研究结果如下：1.共筛选了322对SSR标记，找到了在无毛突变体和壮瓜之间具有多态的SSR标记21个，并将无毛基因gl1定位在3号染色体上的SSR21054和SSR117之间。这两个标记之间的物理距离为3000Kb。根据无毛突变体的基因组重测序信息，在初步定位区间内找到了在双亲之间有多态的2个STS标记和3个CAPS标记。通过对2400棵无毛表型的F2单株进行标记分析，将gl1基因定位在STS-1和CAPS-1之间，物理距离约311K（28801842-29113539）的区间内，共有52个候选基因。通过对候选基因的注释及与拟南芥同源基因的比较分析发现，这52个基因中没有与细胞分裂、细胞周期或表皮细胞命运决定相关的同源基因。所以，黄瓜表皮细胞的分化机制很有可能与拟南芥有不同之处。2.通过数字基因表达谱（DGE）对无毛突变体及其亲本的叶片进行了差异表达基因分析。与亲本相比，在无毛突变体叶片中表达量下调的基因365个，上调的基因139个。其中差异倍数在20倍以上的基因共57个。GO功能富集分析表明，这些差异表达基因主要参与代谢作用、细胞过程、刺激应答、及细胞机构合成等相关生物学过程。3.在黄瓜全基因组中，利用BlastP的方法分析了R2R3MYB，bHLH，WD40-repeat和R3single repeat MYB基因家族，分别找到了46个R2R3MYB基因，138个bHLH基因，191个WD40-repeat基因及1个R3single repeat MYB基因。利用同源进化分析找到了拟南芥中TTG1，MYC1和TRY的同源基因，分别命名为CsTTG1，CsMYC1和CsTRY。另外，黄瓜的R2R3MYB基因家族中没有与表皮细胞命运决定相关的基因，但找到了一个与拟南芥GL1同源性最高的R2R3MYB基因，命名为CsGL1-like (CsGL1L)。序列分析表明，CsGL1L，CsMYC1， CsTTG1及CsTRY的CDS长度分别为783bp，1956，1002和249bp，分别编码260，651，333和82个氨基酸。4.为了分析CsGL1L，CsMYC1， CsTTG1及CsTRY的亚细胞定位情况，分别构建了CsGL1L，CsMYC1， CsTTG1及CsTRY与GFP的融合蛋白。共聚焦结果显示CsGL1L，CsMYC1， CsTTG1及CsTRY均定位在洋葱表皮的细胞核中。5.组织特异性分析结果表明，CsGL1L，CsMYC1， CsTTG1及CsTRY在黄瓜的各组织中均有不同程度的表达。其中CsGL1L主要在果实中表达，CsMYC1在根、茎、叶、花和果实中表达量均较高，只在卷须中表达量较低。CsTTG1在各个组织器官中表达量均较高。CsTRY主要在叶片，根和卷须中表达量高，在茎、雄花和果实中表达量较低。6.为了分析CsGL1L，CsMYC1， CsTTG1及CsTRY的功能，分别构建了这四个基因的过表达及RNAi载体。拟南芥的遗传转化实验表明，在拟南芥gl1突变体中过表达CsGL1L没有恢复gl1突变体有毛的表型。在野生型拟南芥Col中分别过表达CsGL1L和CsMYC1可以使转基因植株莲座叶上的表皮毛数量显著增加，但过表达CsTTG1后，转基因植株莲座叶上的表皮毛数量与野生型相比没有明显的变化。7.为了进一步分析CsGL1L，CsMYC1， CsTTG1及CsTRY在黄瓜中的功能，利用农杆菌侵染法进行了黄瓜的遗传转化。目前已经分别得到1棵PHB-TTG1和2棵PHB-CsMYC1的转基因植株。对过表达CsTTG1的转基因植株表型分析结果表明，转基因植株的花托和叶片上的表皮毛数量和长度均显著高于对照。转基因植株的果实上的刺瘤数量明显高于对照且出现了明显的棱。更多还原

【Abstract】 Warty fruit trait is one of the highly valuable external quality traits of cucumber(Cucumis sativus). Histology analysis showed that trichomes and fruit spines aremulti-cellular and non-glandular column-shaped structures. The glabrous1mutant (gl1) is agood material for studying the formation mechanism of the trichomes and fruit spines, whichshowed no trichome on stems, leaves, tendrils, receptacles and ovaries and there were nospine and tumor on cucumber fruit surface. The results of genetic analysis for gl1indicatedthat gl1gene is recessive epistatic to tuberculate gene (Tu). Studying the formationmechanism of trichome can lay the foundation of elucidating the molecular mechanism of thespines and tumors formation on cucumber fruit. In addition, the trichomes and fruit spines aredifferentiated from epidermis cells and the patterning of epidermal cell types in Arabidopsishas become one of the best models for studying the molecular basis of cell specification inplants. It is helpful for enriching the theories of cell differentiation to study the trichomeformation mechanism in cucumber.To mapping glabrous1gene (gl1), map-based cloning was used and we constructed F2segregate populations with ‘Zhuanggua’(Europe greenhouse type) and glabrous1mutant. F2glabrous progenies were used as mapping population. Bioinformatics approach was also usedto identify all of the members of R2R3MYB, bHLH, WD40-repeat and R3single repeat MYBfamily, respectively. Phylogenetic analysis was performed to find putative trichome formationrelated homologous genes in cucumber. To study the functions of trichome formationrelated homologous genes, we performed genetic transformation of Arabidopsis andcucumber, respectively. The results as follows:1.322SSR markers were screened and21polymorphic SSR markers were identified betweenglabrous1mutant and ‘Zhuanggua’. The gl1gene was delimited in the region between SSR21054and SSR117on chromosome3and their physical distance is about3000Kb.Based on primary mapping of gl1gene and re-sequence of gl1mutant,2polymorphic STSmarkers and3polymorphic CAPS markers were developed in the primary mapping region ofgl1gene. Markers analysis was conducted on2400individuals of F2population using thesenew STS and CAPS primers. Then the gl1gene was fine mapped within the region betweenSTS-1and CAPS-1. The physical distance of the STS-1and CAPS-1was311Kb and there are31candidate genes in this region. The results of genes annotation analysis compared toArabidopsis showed that there was not homologous gene related to cell division or epidermiscell fate determination genes. So the mechanism of cucumber epidermis cell division may bedifferent from Arabidopsis.2. Data gene expression profiles of leaves from gl1mutant and ‘Daqingba’ were performedand the results indicated that365genes down-regulated and139genes up-regulated in gl1mutant relative to ‘Daqingba’, respectively. Of these,57genes showed more than20-folddifference. Gene classification based on gene ontology (GO) for differentially expressedgenes in gl1mutant and ‘Daqingba’ showed that the genes related to metabolic, cellular,response to stimulus and cellular biosynthetic processes were abundant in the differentiallyexpressed genes.3. Whole-genome wide analysis of R2R3MYB, bHLH, WD40-repeat (WDR) and R3singlerepeat MYB family was performed using BlastP program, respectively. We identified46R2R3MYB,138bHLH,191WDR and1R3single MYB genes in cucumber genomedatabase, respectively. The homologous genes of Arabidopsis TTG1, MYC1and TRY wasidentified and named as CsTTG1, CsMYC1and CsTRY, respectively. It was worth to notethat there are not epidermis cell fate determination genes in cucumber R2R3MYB family.However, the gene most closely related with Arabidopsis GL1was identified and named asCsGL1L. Sequence analysis indicated that the CDS length of CsGL1L, CsMYC1, CsTTG1andCsTRY is783,1956,1002and249bp and encode260,651,333and82amino acids,respectively.4. To examine the subcellular distribution of the CsGL1L, CsMYC1, CsTTG1and CsTRY protein, CsGL1L, CsMYC1, CsTTG1and CsTRY was fused with GFP，respectively. Confocalimaging showed the four fusion protein localized exclusively in the nuclei of onion (Alliumcepa) epidermal cells.5. Tissue-specific expression of CsGL1L, CsMYC1, CsTTG1and CsTRY were performed andthe results showed that they expressed in different tissues to various degrees. CsGL1L showedhigher expression levels in fruits than any other tissues. CsMYC1showed high expressionlevels in roots, stems, leaves, male flowers and fruits but low levels in tendrils. CsTTG1highly expressed in all of the tested tissues. CsTRY showed higher expression levels in leaves,roots and tendrils and low levels in other tissues.6. To analyze the functions of CsGL1L, CsMYC1, CsTTG1and CsTRY, the over-expressionand RNAi recombinants vectors of these genes were constructed, respectively. The results ofgenetic transformation of Arabidopsis indicated that over-expression CsGL1L in Arabidopsisgl1mutant did not rescue the trichome formation of the gl1mutant. We over-expressedCsGL1L and CsMYC1in Col exhibited significantly increased trichome density to variousdegrees on rosette leaves compared to Col. However, over-expressed CsTTG1in wild typeCol, and the trichome number on rosette leaves of transgenic lines was indistinguishable fromthose in wild-type plants.7. To further study the functions of CsGL1L, CsMYC1, CsTTG1and CsTRY in cucumber, thegenetic transformation of cucumber were performed using Agrobacterium tumefaciens. Todate, we obtained1CsTTG1over-expression line and2CsMYC1over-expression lines. Thephenotype of over-expression CsTTG1line was also analyzed and the transgenic line showedsignificant increase in trichome number and length relative to control, particularly onreceptacles and adaxial surfaces of the leaves. In addition, there are more spines and tumorson the surface of transgenic fruits and more remarkable ribs were also observed.更多还原