黄瓜表皮毛发育及其相关基因CsTTG1和CsGL2的功能分析

发布时间：2018-01-14 09:25

本文关键词：黄瓜表皮毛发育及其相关基因CsTTG1和CsGL2的功能分析　出处：《中国农业大学》2016年博士论文　论文类型：学位论文

更多相关文章：黄瓜 突变体 果刺 CsTTG1 CsGL2

【摘要】：果实作为黄瓜重要的经济器官,其外观商品品质能够决定其商品价值,而果刺(表皮毛)的覆盖能够影响黄瓜的外观品质。因此,探索黄瓜果刺的发育机理、阐明果刺发育调控途径对改善黄瓜商品品质和培育黄瓜新品种具有重大意义。本文利用密刺型黄瓜(3407)及其无毛突变体(tbh)观察其表皮毛的形态和细胞结构差异,利用数字基因表达谱分析筛选黄瓜果刺发育相关基因；研究了转录因子CsTTG1和CsGL2对黄瓜表皮毛形成的调控机制,以期丰富黄瓜果实表皮毛发育的理论基础。主要研究结果如下：1、肉眼观察下,3407的植株地上部分表面均覆有表皮毛,而其突变体tbh则表现为光滑无毛的表型；在扫描电镜(SEM)下,发现3407果实和叶片的表面存在两种类型的表皮毛,而tbh突变体的果实和叶片上均覆有发育不完全的表皮毛。通过石蜡切片观察,将果刺的发育分为起始、膨大和成熟三个阶段；流式分析表明,果刺细胞中DNA的含量与叶片细胞相比无明显差异；3407及其tbh无毛突变体的表达谱分析结果认为,在黄瓜果刺(表皮毛)的分化形成过程中可能有分生组织相关基因的参与调控。2、黄瓜CsTTG1属于WD40家族同源基因,通过时空表达分析发现,该基因在黄瓜的叶片、雄花芽和子房中表达量较高。子房时期,其表达主要集中在表皮,同时在果刺中亦有表达；亚细胞定位发现CsTTG1蛋白定位在细胞核和细胞膜上；与空载植株相比,35S:CsTTG1过量表达转基因黄瓜果实上果刺和有腺体表皮毛的数量明显增加,果刺的体积也明显变大,商品成熟期果实的刺瘤性状更明显；而CsTTG1-RNAi转基因黄瓜果实的果刺数目明显减少。在突变体csgll中过量表达CsTTG1,其发育不完全表皮毛的数量增加,但形态结构没有任何变化；酵母双杂交试验和双分子荧光互补试验结果证明CsTTG1和CsGL1能够直接互作。3、黄瓜CsGL2属于HD-ZIP家族同源基因,时空表达分析表明,CsGL2在黄瓜的幼嫩器官中有较强表达,而子房时期,CsGL2的表达主要集中在果刺和表皮中；亚细胞定位表明CsGL2蛋白主要定位于细胞核和细胞膜,但在细胞质中亦检测到较弱的荧光信号；将CsGL2转化到拟南芥gl2-8突变体中发现,CsGL2的异位表达不能恢复拟南芥gll2-8突变体表皮毛发育不完全的表型；另外,与对照植株相比,35S:CsGL2过量表达转基因黄瓜的子房和果实上的果刺密度和形态结构均没有发生明显改变。
[Abstract]:Fruit is an important economic organ of cucumber, its appearance commodity quality can determine its commodity value, and the covering of fruit prickles (epidermis) can affect the appearance quality of cucumber. Therefore, the development mechanism of cucumber fruit prickles is explored. It is of great significance to clarify the developmental regulation pathway of fruiting prickles in improving the quality of cucumber commodities and breeding new cucumber varieties. In this paper, we use the dense prickly Cucumber (Cucumis sativus) and its hairless mutant Tbh). The morphology and cellular structure of the epidermal hair were observed. The development related genes of Cucumber fruit thorn were screened by digital gene expression analysis. The regulation mechanism of transcription factors CsTTG1 and CsGL2 on the formation of cucumber epidermis was studied in order to enrich the theoretical basis of cucumber fruit epidermal development. The main results were as follows: 1, under naked eye observation. The surface of 3407 plants was covered with epidermal hairs, while the mutant tbh showed smooth hairless phenotype. Under scanning electron microscope (SEM), two types of epidermal hairs were found on the surface of fruit and leaf of 3407. On the other hand, the fruit and leaves of tbh mutants were covered with immature epidermal hairs. Through paraffin section observation, the development of fruiting spines was divided into three stages: initiation, expansion and maturation. Flow analysis showed that there was no significant difference between the content of DNA and leaf cells. The expression profiles of 3407 and its tbh hairless mutants showed that the meristem related genes might be involved in the process of differentiation and formation of fruit prickles (epidermis). Cucumber CsTTG1 belongs to the WD40 family homologous gene. It was found that the expression of Cucumber CsTTG1 gene was high in leaves, male flower buds and ovary by spatio-temporal expression analysis. Its expression was mainly concentrated in the epidermis and also expressed in the prickly fruit. Subcellular localization showed that CsTTG1 protein was located in nucleus and cell membrane. The number of fruit spines and glandular epidermal hairs on transgenic cucumber fruits was significantly increased and the volume of fruit spines was also significantly larger than that of non-loaded plants. The prickly characters of the mature fruit were more obvious. However, the number of fruit spines in CsTTG1-RNAi transgenic cucumber fruits was significantly decreased, and CsTTG1 was overexpressed in the mutant csgll, and the number of immature epidermal hairs increased. However, there was no change in morphological structure. The results of yeast two-hybrid test and bimolecular fluorescence complementary test showed that CsTTG1 and CsGL1 could interact with each other directly. Cucumber CsGL2 belonged to the homologous gene of HD-ZIP family. Spatio-temporal expression analysis showed that CsGL2 was strongly expressed in young organs of cucumber, while CsGL2 was mainly expressed in fruit prickles and epidermis at ovary stage. Subcellular localization showed that CsGL2 protein was mainly located in the nucleus and cell membrane, but weak fluorescence signals were also detected in the cytoplasm. When CsGL2 was transformed into Arabidopsis thaliana gl2-8 mutants, the ectopic expression of CsGL2 was not able to recover the phenotype of incomplete epidermal development of Arabidopsis gll2-8 mutants. In addition, there was no significant change in the density and morphological structure of fruit spines in the ovary and fruit of transgenic cucumber overexpressed by 35 S: CsGL2 compared with the control plant.
【学位授予单位】：中国农业大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：S642.2

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