大豆与胞囊线虫互作中GmPRs的表达及胼胝质沉积研究

发布时间：2017-12-26 21:22

本文关键词：大豆与胞囊线虫互作中GmPRs的表达及胼胝质沉积研究　出处：《沈阳农业大学》2017年硕士论文　论文类型：学位论文

【摘要】：为了明确大豆与大豆胞囊线虫(Heteroderaglycine)互作中与抗线虫相关的病程相关蛋白基因(GmPRs),本文选取大豆(Glycinemax)感病品种辽豆15和抗病品种灰皮支黑豆为供试豆种,通过实时荧光定量PCR的方法,检测了大豆接种胞囊线虫后,六种GmPRs基因的表达水平,分析这些基因分别在抗病品种和感病品种中的表达情况。明确了GmPR2((3-1,3-葡聚糖酶)和GmPR10与大豆抗胞囊线虫相关,并且构建GFP与目的基因的融合表达载体,以探究GmPR2和GmPR10在亚细胞水平的分布情况。进一步对抗感品种以及抗感品种的杂交后代进行胼胝质(β-1,3-葡聚糖)苯胺蓝染色,研究了抗感品种中胼胝质的沉积,并对四个大豆胼胝质合成酶基因转录水平的表达情况进行测定。主要研究结果如下:1.接种大豆胞囊线虫后,GmPR2(即β-1,3-葡聚糖酶基因)在抗病品种中,被强烈的诱导表达,在线虫的侵染初期(2d)和发育期(10 d)表达量差异显著,在感病品种中该基因表达量差异不显著。GmPR3和GmPR10在线虫侵染后的发育期(10 d)表达量达到最高值,表明这些基因可能与大豆抗胞囊线虫发育的调控有关。2.构建了 pCAMBIA-GmPR2和pCAMBIA-GmPR10瞬时表达载体,并通过农杆菌转化法使目的基因在烟草叶片瞬时表达,并使用正置荧光显微镜的FITC激发器激发GFP绿色荧光,分析目的基因在烟草表皮细胞中的定位。明确了 GmPR2主要分布于细胞膜,GmPR10主要分布于细胞质和细胞核,推测病程相关蛋白表达产物与基因的转录调节、物质运输和胞外信息传递相关。3.通过对抗感品种以及抗感杂交后代的胼胝质(即β-1,3-葡聚糖)进行苯胺蓝染色研究,结果显示,在线虫侵染初期(3d),抗病品种产生大量胼胝质沉积,感病品种中胼胝质沉积非常少,并且抗性杂交后代中,肼胝质的沉积量远远多于感性后代中胼胝质的沉积量,即胼胝质的沉积量与品种抗性呈正相关。在大豆胞囊线虫侵染后的发育期(10 d),抗感品种中均未有明显的胼胝质沉积,说明胼胝质的沉积随着线虫在根内的发育而减少。推测肼胝质的沉积抑制了线虫发育,从组织水平上初步证明了胼胝质参与了大豆抗大豆胞囊线虫的侵染过程。4.在抗病品种和感病品种中,于接种后不同时间点对四个大豆胼胝质合成酶基因(GmC4LS)的转录水平进行测定,结果显示,在大豆与SCN互作中,胼胝质合成酶基因受到不同程度的诱导。GmG4LS7、GmCALS10基因可能与胼胝质的合成没有必然联系;GmCALS5基因在线虫侵染后第3 d至5 d上调表达且表达量差异显著,GmCAL S3基因在线虫侵染后第4 d、5 d上调表达且表达量差异显著,推测GmCALS3基因和GmC4LS5基因可能与胼胝质的合成相关。
[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.
【学位授予单位】：沈阳农业大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S435.651

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