本文關鍵詞：黃瓜表皮毛發(fā)育及其相關基因CsTTG1和CsGL2的功能分析　出處：《中國農業(yè)大學》2016年博士論文　論文類型：學位論文

  更多相關文章： 黃瓜 突變體 果刺 CsTTG1 CsGL2

【摘要】：果實作為黃瓜重要的經濟器官,其外觀商品品質能夠決定其商品價值,而果刺(表皮毛)的覆蓋能夠影響黃瓜的外觀品質。因此,探索黃瓜果刺的發(fā)育機理、闡明果刺發(fā)育調控途徑對改善黃瓜商品品質和培育黃瓜新品種具有重大意義。本文利用密刺型黃瓜(3407)及其無毛突變體(tbh)觀察其表皮毛的形態(tài)和細胞結構差異,利用數(shù)字基因表達譜分析篩選黃瓜果刺發(fā)育相關基因；研究了轉錄因子CsTTG1和CsGL2對黃瓜表皮毛形成的調控機制,以期豐富黃瓜果實表皮毛發(fā)育的理論基礎。主要研究結果如下：1、肉眼觀察下,3407的植株地上部分表面均覆有表皮毛,而其突變體tbh則表現(xiàn)為光滑無毛的表型；在掃描電鏡(SEM)下,發(fā)現(xiàn)3407果實和葉片的表面存在兩種類型的表皮毛,而tbh突變體的果實和葉片上均覆有發(fā)育不完全的表皮毛。通過石蠟切片觀察,將果刺的發(fā)育分為起始、膨大和成熟三個階段；流式分析表明,果刺細胞中DNA的含量與葉片細胞相比無明顯差異；3407及其tbh無毛突變體的表達譜分析結果認為,在黃瓜果刺(表皮毛)的分化形成過程中可能有分生組織相關基因的參與調控。2、黃瓜CsTTG1屬于WD40家族同源基因,通過時空表達分析發(fā)現(xiàn),該基因在黃瓜的葉片、雄花芽和子房中表達量較高。子房時期,其表達主要集中在表皮,同時在果刺中亦有表達；亞細胞定位發(fā)現(xiàn)CsTTG1蛋白定位在細胞核和細胞膜上；與空載植株相比,35S:CsTTG1過量表達轉基因黃瓜果實上果刺和有腺體表皮毛的數(shù)量明顯增加,果刺的體積也明顯變大,商品成熟期果實的刺瘤性狀更明顯；而CsTTG1-RNAi轉基因黃瓜果實的果刺數(shù)目明顯減少。在突變體csgll中過量表達CsTTG1,其發(fā)育不完全表皮毛的數(shù)量增加,但形態(tài)結構沒有任何變化；酵母雙雜交試驗和雙分子熒光互補試驗結果證明CsTTG1和CsGL1能夠直接互作。3、黃瓜CsGL2屬于HD-ZIP家族同源基因,時空表達分析表明,CsGL2在黃瓜的幼嫩器官中有較強表達,而子房時期,CsGL2的表達主要集中在果刺和表皮中；亞細胞定位表明CsGL2蛋白主要定位于細胞核和細胞膜,但在細胞質中亦檢測到較弱的熒光信號；將CsGL2轉化到擬南芥gl2-8突變體中發(fā)現(xiàn),CsGL2的異位表達不能恢復擬南芥gll2-8突變體表皮毛發(fā)育不完全的表型；另外,與對照植株相比,35S:CsGL2過量表達轉基因黃瓜的子房和果實上的果刺密度和形態(tài)結構均沒有發(fā)生明顯改變。
[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.
【學位授予單位】：中國農業(yè)大學
【學位級別】：博士
【學位授予年份】：2016
【分類號】：S642.2

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