發(fā)布時間：2017-12-26 21:22

  本文關(guān)鍵詞：大豆與胞囊線蟲互作中GmPRs的表達及胼胝質(zhì)沉積研究　出處：《沈陽農(nóng)業(yè)大學》2017年碩士論文　論文類型：學位論文

  更多相關(guān)文章： 大豆胞囊線蟲 病程相關(guān)蛋白 實時熒光定量PCR 亞細胞定位 胼胝質(zhì) 胼胝質(zhì)合成酶

【摘要】：為了明確大豆與大豆胞囊線蟲(Heteroderaglycine)互作中與抗線蟲相關(guān)的病程相關(guān)蛋白基因(GmPRs),本文選取大豆(Glycinemax)感病品種遼豆15和抗病品種灰皮支黑豆為供試豆種,通過實時熒光定量PCR的方法,檢測了大豆接種胞囊線蟲后,六種GmPRs基因的表達水平,分析這些基因分別在抗病品種和感病品種中的表達情況。明確了GmPR2((3-1,3-葡聚糖酶)和GmPR10與大豆抗胞囊線蟲相關(guān),并且構(gòu)建GFP與目的基因的融合表達載體,以探究GmPR2和GmPR10在亞細胞水平的分布情況。進一步對抗感品種以及抗感品種的雜交后代進行胼胝質(zhì)(β-1,3-葡聚糖)苯胺藍染色,研究了抗感品種中胼胝質(zhì)的沉積,并對四個大豆胼胝質(zhì)合成酶基因轉(zhuǎn)錄水平的表達情況進行測定。主要研究結(jié)果如下:1.接種大豆胞囊線蟲后,GmPR2(即β-1,3-葡聚糖酶基因)在抗病品種中,被強烈的誘導表達,在線蟲的侵染初期(2d)和發(fā)育期(10 d)表達量差異顯著,在感病品種中該基因表達量差異不顯著。GmPR3和GmPR10在線蟲侵染后的發(fā)育期(10 d)表達量達到最高值,表明這些基因可能與大豆抗胞囊線蟲發(fā)育的調(diào)控有關(guān)。2.構(gòu)建了 pCAMBIA-GmPR2和pCAMBIA-GmPR10瞬時表達載體,并通過農(nóng)桿菌轉(zhuǎn)化法使目的基因在煙草葉片瞬時表達,并使用正置熒光顯微鏡的FITC激發(fā)器激發(fā)GFP綠色熒光,分析目的基因在煙草表皮細胞中的定位。明確了 GmPR2主要分布于細胞膜,GmPR10主要分布于細胞質(zhì)和細胞核,推測病程相關(guān)蛋白表達產(chǎn)物與基因的轉(zhuǎn)錄調(diào)節(jié)、物質(zhì)運輸和胞外信息傳遞相關(guān)。3.通過對抗感品種以及抗感雜交后代的胼胝質(zhì)(即β-1,3-葡聚糖)進行苯胺藍染色研究,結(jié)果顯示,在線蟲侵染初期(3d),抗病品種產(chǎn)生大量胼胝質(zhì)沉積,感病品種中胼胝質(zhì)沉積非常少,并且抗性雜交后代中,肼胝質(zhì)的沉積量遠遠多于感性后代中胼胝質(zhì)的沉積量,即胼胝質(zhì)的沉積量與品種抗性呈正相關(guān)。在大豆胞囊線蟲侵染后的發(fā)育期(10 d),抗感品種中均未有明顯的胼胝質(zhì)沉積,說明胼胝質(zhì)的沉積隨著線蟲在根內(nèi)的發(fā)育而減少。推測肼胝質(zhì)的沉積抑制了線蟲發(fā)育,從組織水平上初步證明了胼胝質(zhì)參與了大豆抗大豆胞囊線蟲的侵染過程。4.在抗病品種和感病品種中,于接種后不同時間點對四個大豆胼胝質(zhì)合成酶基因(GmC4LS)的轉(zhuǎn)錄水平進行測定,結(jié)果顯示,在大豆與SCN互作中,胼胝質(zhì)合成酶基因受到不同程度的誘導。GmG4LS7、GmCALS10基因可能與胼胝質(zhì)的合成沒有必然聯(lián)系;GmCALS5基因在線蟲侵染后第3 d至5 d上調(diào)表達且表達量差異顯著,GmCAL S3基因在線蟲侵染后第4 d、5 d上調(diào)表達且表達量差異顯著,推測GmCALS3基因和GmC4LS5基因可能與胼胝質(zhì)的合成相關(guān)。
[Abstract]:In order to clarify the soybean and Soybean Cyst Nematode (Heteroderaglycine) interaction with nematode resistance related pathogenesis related protein gene (GmPRs), this paper selects soybean (Glycinemax) susceptible cultivar Liaodou 15 and resistant varieties of Huipizhi Heidou for test method of PCR seed by real-time fluorescence detection, the inoculation of soybean SCN, the expression of six GmPRs genes, these genes were analyzed in resistant varieties and cultivars in the sense of the expression. It is clear that GmPR2 (3-1,3- glucan) and GmPR10 are related to soybean cyst nematode, and the fusion expression vector of GFP and target gene is constructed to explore the distribution of GmPR2 and GmPR10 at subcellular level. Further hybrids resistant varieties and varieties of callose (-1,3- beta glucan) aniline blue staining of resistant and susceptible varieties of callose deposition, and the expression of four soybean callose synthase gene transcription level were determined. The main results are as follows: 1. after inoculating soybean cyst nematode, GmPR2 (i.e. -1,3- glucanase gene) in resistant cultivars, was strongly induced the expression of the early stage of the infection, the worm (2D) and the development period (10 d) was significantly different in the susceptible cultivar the gene expression difference no significant. The growth period (10 d) of GmPR3 and GmPR10 reached the highest value, indicating that these genes may be related to the regulation of the development of soybean cyst nematode. 2. constructed pCAMBIA-GmPR2 and pCAMBIA-GmPR10 transient expression vector, and the expression of target gene in tobacco leaf instantaneous by Agrobacterium transformation method, and the use of normal fluorescence microscope FITC laser excitation GFP green fluorescence analysis to locate genes in tobacco epidermal cells in. It is clear that GmPR2 is mainly distributed in cell membrane. GmPR10 is mainly distributed in cytoplasm and nucleus. It is speculated that the expression products of disease related proteins are related to transcriptional regulation, material transport and extracellular information transmission. 3. the resistant varieties and resistant and susceptible hybrids (i.e. -1,3- beta glucan callose) of aniline blue staining study, results showed that the nematode infection process (3D), resistant varieties produce large amounts of callose deposition, susceptible varieties callose deposition is very small, and the resistance of hybrids, the deposition amount of the deposition amount of hydrazine as the matter is far more than that in the offspring of callose sensibility, callose deposition and cultivar resistance was positively correlated. In the growth period of soybean cyst nematode infection after (10 d), resistant and susceptible varieties were not obvious that callose deposition, callose deposition decreased with the development of the root nematode. That callose deposition inhibited nematode development, preliminary evidence of the infection process of callose in soybean resistance to Soybean Cyst Nematode from tissue level. 4. in the resistant and susceptible cultivars, at different time after inoculation of four soybean callose synthase gene (GmC4LS) transcription level were measured, results showed that in soybean and SCN interactions, callose synthase gene was induced by different degree. GmG4LS7, GmCALS10 gene may have no relation to the synthesis and callose; GmCALS5 gene in nematode after infection third d to 5 D expression and expression of GmCAL gene significantly, S3 worms after infection fourth D, 5 D expression and the expression level was significantly different, suggesting that GmCALS3 gene and GmC4LS5 gene may be associated with callose synthesis related.
【學位授予單位】：沈陽農(nóng)業(yè)大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：S435.651

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