黄瓜稀刺基因的定位、克隆及功能分析

发布时间：2018-04-27 11:39

本文选题：黄瓜 + 刺瘤密度　；参考：《山东农业大学》2016年博士论文

【摘要】：果实刺瘤密度是黄瓜(Cucumis sativus L.)重要的外观商品性状。然而到目前为止,还未见调控黄瓜刺瘤密度的相关基因被克隆的报道,黄瓜刺瘤密度形成的分子机制仍然未知。因此,对黄瓜稀刺基因定位、克隆,有助于研究黄瓜刺瘤密度形成的分子机制,同时为刺瘤密度分子育种提供理论依据和技术支持。本研究从华北型密刺黄瓜CNS2(wild type,WT)中分离出一个稀刺突变体,few spines 1(fs1)。fs1果实刺瘤数目显著少于WT,而茎、叶、卷须、花萼等其它部位的表皮毛形态、密度均无显著变化,是研究黄瓜果实刺瘤密度形成机理的理想材料。用华北型密刺黄瓜CNS2、9930分别与fs1杂交构建了2个F2群体,通过BSA(Bulk Segregant Analysis)法结合基因组重测序对fs1进行了初步定位,然后利用图位克隆技术分离出稀刺基因。同时,对密刺表型和稀刺表型果实混池进行转录组测序,找到了部分受稀刺基因影响的基因。为了研究稀刺基因的功能,还进行了黄瓜的遗传转化进行互补验证。具体研究结果如下:1.构建了稀刺突变体和其亲本的F2群体,取稀刺和密刺表型个体叶片混池,提取DNA后重测序,将获得的SNV进行关联分析,fs1被定位到黄瓜6号染色体上。2.由于fs1与WT的基因组相似度较高,在开发标记时未找到合适可用的标记来进行下一步的精细定位工作。因此,将fs1和9930进行杂交,构建了新的精细定位群体。利用黄瓜基因组重测序得到的SNP数据,找到了4个CAPS标记和1个SNP标记。用CAPS1和MM2对5400棵F2单株进行筛选,找到130棵交换个体。用CAPS3、CAPS4和MM2对32个稀刺表型交换单株进行分析,将稀刺基因fs1定位到了MM1和MM2标记之间,这两个标记之间的物理距离为110.4 kb,有25个注释基因。3.通过对候选区间内25个基因的编码区和启动子区域测序,将WT和fs1的序列进行比对,结果发现,Csa6M514870(CsHDZIV11/CsGL3/Tril)的启动子区域内发生了片段的替换,WT中10 bp的片段在fs1中被替换成了812 bp。Csa6M514870属于HD-ZIP IV亚家族,编码了一个PDF2相关蛋白。在开花当天的果实中,fs1中的Csa6M514870的表达显著高于WT。这表明,Csa6M514870是最佳候选基因。4.为了进一步分析CsHDZIV11在黄瓜中的功能,进行了黄瓜的遗传转化。目前已得到20棵再生植株,有待于进一步验证。5.对稀刺突变体的转录组数据进行分析并和已发表的黄瓜表皮毛相关转录组或表达谱数据比较,找到了四个可能参与黄瓜刺瘤密度形成调控网络的基因:Csa5M606310、Csa6M501990、Csa3M101810和Csa3M824990。6.根据CsHDZIV11的启动子区域内的片段替换,开发了一个分子标记MM3。经检测,这个标记和来自不同地区不同品种的果实刺瘤密度共分离。另外,这一片段也存在于野生黄瓜(C.sativus var.hardwickii)中,表明CsHDZIV11的启动子区域内的片段替换是黄瓜驯化过程中决定果刺密度的关键因素。本研究不仅定位、克隆了控制果刺密度的最佳候选基因,为研究黄瓜刺瘤密度形成的分子机制奠定了基础;而且找到了决定刺瘤稀密的关键因素,为黄瓜刺瘤密度分子标记辅助育种提供了一个有效的分子标记。
[Abstract]:The fruit prickle density is an important appearance of Cucumis sativus L.. However, up to now, there has not been a report on the cloning of the related genes regulating the density of cucumbers. The molecular mechanism of the formation of cucumber prickle density is still unknown. Therefore, the location and cloning of the cucumbers' dilute prickly gene can help to study the formation of cucumber tumor density. In this study, a rare spiny mutant was isolated from CNS2 (wild type, WT) in North China type, and the number of few spines 1 (FS1).Fs1 fruits was significantly less than WT, while the density of other parts of the stem, leaf, tendril, calyx and other parts were not significant. The change is an ideal material to study the formation mechanism of the density of the cucumber fruit. 2 F2 populations were constructed with the North China dense prickly cucumber CNS29930 and FS1, respectively. The preliminary location of FS1 was carried out by BSA (Bulk Segregant Analysis) method combined with genome sequencing, and then the thin prickly gene was separated by the technique of graphic cloning. The transcriptional group was sequenced by the phenotypic and dilute phenotypic fruit mixed pool, and some genes affected by the dilute prickly gene were found. In order to study the function of the dilute spiny gene, the genetic transformation of the cucumber was also carried out. The specific results were as follows: 1. the sparse thorn mutants and their parent F2 populations were constructed, and the individual leaves of dilute spines and prickles were obtained. In the mixed pool, the DNA was sequenced and the obtained SNV was associated. The FS1 was located on the cucumber chromosome 6 and.2., because of the high similarity between the FS1 and the WT genome, did not find the appropriate marker for the next step in the development of the marker. Therefore, the FS1 and 9930 were hybridized to a new fine positioning group. Using SNP data obtained from cucumber genome re sequencing, 4 CAPS markers and 1 SNP markers were found. 5400 F2 single strains were screened with CAPS1 and MM2, and 130 exchange individuals were found. CAPS3, CAPS4 and MM2 were used to analyze 32 barbaric phenotypes, and the dilute prickly gene FS1 was determined between MM1 and MM2 markers, and the two markers were between these markers. The physical distance was 110.4 KB, and 25 annotated genes,.3., were sequenced by the sequence of the encoding and promoter regions of the 25 genes in the candidate region. The sequence of WT and FS1 was compared. The results showed that the substitution of fragments occurred in the promoter region of Csa6M514870 (CsHDZIV11/CsGL3/Tril), and the fragment of 10 BP in WT was replaced with 812 bp.Cs in FS1. A6M514870 belongs to the HD-ZIP IV subfamily and encodes a PDF2 related protein. In the fruit of the day of flowering, the expression of Csa6M514870 in FS1 is significantly higher than that of WT., indicating that Csa6M514870 is the best candidate gene,.4. in order to further analyze the function of CsHDZIV11 in cucumbers, and to carry out genetic transformation of yellow gourd. At present, 20 plants have been regenerated, To further verify.5.'s analysis of the transcriptional data of the dilute mutants and compare with the published data of the transcriptional or expression profiles of the cucumber surface fur, four genes may be found that may participate in the regulation network for the formation of cucumber tumor density: Csa5M606310, Csa6M501990, Csa3M101810 and Csa3M824990.6. based on the start of CsHDZIV11 Fragment replacement in the subregion, a molecular marker MM3. was developed to detect the total separation of the fruit prickle density from different varieties of different regions. In addition, this fragment also existed in the wild cucumber (C.sativus var.hardwickii), indicating that the fragment replacement in the promoter region of the CsHDZIV11 was determined during the domestication of the cucumber. This study not only localizes, but also clones the best candidate genes to control the density of fruit thorn, which lays the foundation for the study of the molecular mechanism of the formation of cucumber prickle density, and has found the key factor determining the dilute density of the stab tumor, which provides an effective molecular marker for the molecular marker assisted breeding of cucumber tumor density.

【学位授予单位】：山东农业大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：S642.2;Q943.2

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