不结球白菜株型性状相关基因挖掘及分析

发布时间：2018-06-10 12:28

本文选题：Aux/IAA + ARF　；参考：《南京农业大学》2016年博士论文

【摘要】：白菜类作物隶属十字花科(Cruciferae)芸薹属(Brassica),原产自我国,栽培历史悠久且品种丰富,主要包括大白菜(Brassicarapassp.pekinensis)、不结球白菜(B.rapa ssp. chinensis; Non Heading Chinese Cabbage; NHCC)和芜菁(B. rapa ssp. rapa)。其中,不结球白菜是我国长江中下游广泛种植的大众蔬菜。不结球白菜包含了多个变种,具有丰富的形态多样性。在多个变种中,存在不同的株型性状,如直立、半塌地和塌地。而在直立株型中,又包含直立不束腰和直立束腰株型。在本试验中,利用简化基因组测序技术对直立束腰和半塌地品种杂交构建的F2分离群体进行测序,同时利用转录组和MeDIP测序技术对束腰品种进行束腰前后的株型差异基因挖掘。根据测序结果,对候选基因中生长素信号途径中的Aux/IAA及ARF家族在大白菜中进行鉴定和进化分析;对脱落酸信号途径中的SnRK2基因家族在不结球白菜中进行克隆和分析。主要研究结果如下:1.使用直立束腰品种和半塌地品种进行杂交,构建F2分离群体,来进一步测量束腰性状及其与其它性状之间的关系。结果证明,束腰性状并非质量性状,而是在植株成长过程中逐渐形成的数量性状。在两个组合的F2分离群体中,通过测量计算植株的腰粗和菜头粗的比值得到的束腰比呈正态分布。并且,植株的束腰比与植株的株高、叶柄长呈负相关。在束腰形成过程中,与低温处理的植株相比,处于18℃-24℃环境中的植株几乎不束腰,这说明低温积温与束腰性状的形成有关。2.以大白菜基因组为参考基因组,对两个株型性状表现极端的亲本构建的F2分离群体进行简化基因组测序,共获得78,723,756条有效reads, GC含量约42%,终获得具有1个或多个等位基因的SLAF标签62,978个,整体平均深度达414.26x,且SLAF标签在染色体上分布均匀。实验中定位到了 3个较可靠区域,包含8810个SNP标签,148条候选基因。根据不结球白菜变种转录组数据,筛选到25条表达差异较大的基因,其中11条是F-box基因。选定的25条基因在束腰形成前后的样品中表达差异较大。3.利用转录组测序和MeDIP测序方法,对不结球白菜束腰前后的基因进行全面系统的分析。在本研究中,转录组共得到103,846,926个高质量的双末端序列,比对到参考基因组上的大概约70%,得到上调差异基因1560条,下调基因1228条。MeDIP测序共测得223,380,998个长为49bp的双末端序列,与参考基因组的比对率分别是78.01%和78.13%。测序结果表明,在束腰过程中,基因组甲基化水平提高。两种测序联合分析后,共鉴定出基因主体负相关的基因123条,启动子负相关基因387条。其中代谢相关途径、次生代谢途径、植物激素转导途径以及植物病原互作途径所占比例较高。最终在植物激素信号转导途径中共选出14条候选基因并进行实时定量PCR验证。4.在不结球白菜束腰性状的转录组及甲基化联合分析中,鉴定出部分生长素响应基因Aux/IAA及转录调控因子基因ARF。本研究在大白菜中共鉴定出52条Aux/IAA和33条ARF基因,其中包括1条ARF类似基因,命名为AL。这些基因分化时间与大白菜三倍化时间基本一致。在全基因组加倍后,Aux/IAA基因被全部保留下来,远优先于ARF基因。在陆地植物进化过程中,ARF基因扩增平缓稳定,Aux/IAA基因家族扩增迅速且受到的选择压力较小。Aux/IAA基因家族表现出了更强的组织表达特异性,重复基因的功能发生了分化。在研究中鉴定出的AL基因广泛存在于植物中,并与ARF关系更近。5.转录组及甲基化联合分析中的候选基因SnRK2.6属于SnRK2基因家族。本研究结果表明该家族在进化过程中被优先保留,并在不结球白菜逆境文库中克隆到13条SnRK2基因。这些基因多数含有8个外显子,结构保守稳定。大部分基因,尤其是第三亚组参与了 ABA调控。除此之外,大部分基因还可响应冷处理。BcSnRK2.6a则是唯一可同时响应ABA与低温的BcSnRK2。BcSnRK2的表达模式说明其协同参与ABA和低温诱导的表达,且极有可能参与多种信号途径,进而参与不结球白菜的株型形成。
[Abstract]:Cabbage (Cruciferae) Brassica (Brassica), native to China, has a long history and rich variety, mainly including Chinese Cabbage (Brassicarapassp.pekinensis), non heading Chinese Cabbage (B.rapa ssp. chinensis, Non Heading Chinese Cabbage; NHCC) and turnip. Popular vegetables widely cultivated in the middle and lower reaches of the Yangtze River. Non heading Chinese cabbage contains many varieties and has rich morphological diversity. In many varieties, there are different plant type traits, such as erect, semi collapse and collapse. In the erect plant type, there are erect non girdle and erect beam waist plant type. In this experiment, simplified genome sequencing is used. The technology was used to sequence the F2 isolated population constructed by the vertical waist and the semi collapse varieties. At the same time, the plant type difference gene was excavated by the transcriptional group and the MeDIP sequencing technology. According to the sequencing results, the Aux/ IAA and the ARF family in the signal pathway of the candidate genes were identified and entered in Chinese cabbage. The SnRK2 gene family of the abscisic acid signal pathway was cloned and analyzed in the non heading Chinese cabbage. The main results were as follows: 1. using a vertical waist variety and a semi collapse variety to cross and construct a F2 separation group to further measure the relationship between the waist character and its characters. Non mass characters, but the quantitative traits that gradually formed during the growth of the plant. The ratio of the waist to the waist of the two F2 groups was measured by measuring the ratio of the waist to the head of the vegetable. And the waist ratio of the plant was negatively correlated with the plant height and the length of the stem. Compared with the treated plants, the plants in the environment of 18 -24 C were almost no waist, which indicated that the low temperature accumulated temperature and the formation of the waist character were related to the.2. with the genome of Chinese cabbage as the reference genome, and the F2 separation group constructed by the parents of two plant type traits was sequenced by the simplified base group, and 78723756 effective reads were obtained, GC The content was about 42%, and 62978 SLAF tags with 1 or more alleles were obtained, the overall average depth was 414.26x, and the SLAF label was evenly distributed on the chromosome. In the experiment, 3 more reliable regions were located, including 8810 SNP tags and 148 candidate genes. 25 expression differences were screened according to the data of the non heading Chinese cabbage variety transcriptional group. The larger genes, 11 of which are F-box genes. The selected 25 genes were expressed differently in the samples before and after the formation of the waist, and.3. was sequenced and MeDIP sequencing was used to systematically analyze the genes before and after the waist of the non heading Chinese cabbage. In this study, the transcriptional group obtained a total of 103846926 high quality double terminal sequences. By comparison to about 70% of the reference genome, 1560 of the differential genes were up-regulated, and 1228.MeDIP sequences of down regulated genes were measured to get 223380998 double terminal sequences with a length of 49bp. The ratio of the comparison with the reference genome was 78.01% and 78.13%. sequencing, respectively, indicating that the level of methylation in the genome was increased during the waist process. Two sequencing. After the joint analysis, 123 genes negatively related to the main body of the gene were identified and 387 of the promoter negative related genes were identified. Among them, the metabolic pathway, secondary metabolic pathway, plant hormone transduction pathway and plant pathogen interaction pathway accounted for a higher proportion. Finally, 14 candidate genes were selected in the plant hormone signal transduction pathway and were determined in real time. PCR verified.4. in the transcriptional group and methylation analysis of non heading Chinese cabbage, identified 52 Aux/IAA and 33 ARF genes in Chinese cabbage, including 1 Aux/IAA and 33 ARF genes, which were named AL., which were named AL.. The time of the three ploidy of Chinese cabbage is basically the same. After the whole genome doubles, the Aux/IAA gene is all retained, far superior to the ARF gene. In the process of terrestrial plant evolution, the ARF gene amplification is gentle and stable, the Aux/IAA gene family expands rapidly and the selection pressure is smaller than the.Aux/IAA based family showing a stronger tissue expression specificity. The function of repeat genes is differentiated. The AL genes identified in the study are widely found in plants, and the relationship with the ARF is closer to the.5. transcriptome and the candidate gene for the methylation analysis, SnRK2.6 belongs to the SnRK2 gene family. This study shows that the family is preferred in the evolution process and in the non heading Chinese cabbage adversity library. 13 SnRK2 genes are cloned into the gene. Most of these genes contain 8 exons, and the structure is conservative and stable. Most genes, especially the Sanya group, are involved in ABA regulation. In addition, most genes can also respond to cold treatment.BcSnRK2.6a as the only BcSnRK2.BcSnRK2 expression pattern that responds to ABA and low temperature simultaneously. And low temperature induced expression, and are likely to participate in a variety of signaling pathways, and then participate in the plant type formation of non heading Chinese cabbage.
【学位授予单位】：南京农业大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：S634.3;Q943.2

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