【摘要】：蘋果輪紋病是我國蘋果產(chǎn)區(qū)的主要病害,屬于真菌性病害,主要危害枝干和果實,為果品生產(chǎn)造成重大威脅。因此研究蘋果果實與輪紋病菌之間的關(guān)系具有重要意義。我們通過研究和解析蘋果抗病及免疫機理,分析蘋果在受到病菌侵染后的各種生理活動和脅迫應(yīng)答來挖掘蘋果抗病基因和了解蘋果抗病機制。蘋果抗病基因的挖掘是進(jìn)行品種改良的基礎(chǔ),對于培育抗病品種以及高效生產(chǎn)優(yōu)質(zhì)蘋果具有重要的意義,同時該研究也對深入了解蘋果的抗病基因具有重要的意義。為蘋果優(yōu)新抗病種質(zhì)資源的培育提供科技支撐和依據(jù),服務(wù)我國蘋果產(chǎn)業(yè)發(fā)展。本實驗以‘富士’和‘金冠’兩種質(zhì)地不同的蘋果果實為材料,通過對蘋果接種輪紋病菌后,測定果實乙烯釋放量、硬度、基因相對表達(dá)量來進(jìn)行蘋果抗病性的系統(tǒng)研究,并對乙烯合成相關(guān)基因、細(xì)胞壁軟化基因和PR家族基因進(jìn)行深入研究。結(jié)果表明相關(guān)基因被病菌大量誘導(dǎo)表達(dá),兩個品種在抗蘋果輪紋病方面存在顯著差異,且蘋果在不同發(fā)育時期的抗病反應(yīng)非常復(fù)雜。針對病程相關(guān)基因在蘋果抗病中的表達(dá),我們從蘋果中篩選出MdPR5和MdPR8,通過植物RNA的提取、PCR技術(shù)和基因克隆等方法,獲得蘋果中基因的cDNA序列,并對之進(jìn)行了序列對比分析、基因表達(dá)和轉(zhuǎn)基因分析,通過分子生物學(xué)手段驗證不同種類蘋果中抗病基因的表達(dá)。主要研究結(jié)果如下:1.植物體在逆境脅迫下的活性氧積累情況。以‘嘎拉’組培苗為試材,對葉片和植物接種輪紋病菌和flg22處理后,用DAB染色法檢測活性氧的產(chǎn)生情況。結(jié)果葉片在受到輪紋病菌和flg22處理后有明顯的黃色沉淀顆粒,黃色顆粒越多說明活性氧產(chǎn)生的越多,植株在受到flg22處理后莖部發(fā)紅,說明葉片和植株都發(fā)生了逆境響應(yīng)。2.輪紋病菌接種富士和金冠兩個品種的蘋果果實,在幼果期、膨大期、成熟期三個發(fā)育時期的乙烯釋放速率、硬度變化、病斑大小差異明顯,且用1-MCP處理接菌的果實,抑制乙烯釋放的效果在不同品種的不同時期也不同,以金冠成熟期的變化最顯著,接菌后金冠果實的病斑和硬度都明顯小于富士的。對乙烯合成相關(guān)基因MdERF2、MdACS4、MdCTR1、MdACS5a、MdACS5b進(jìn)行qRT-PCR分析,結(jié)果表明在成熟期,MdACS5a、MdACS5b這兩個基因在輪紋病菌誘導(dǎo)下表達(dá)量明顯升高。研究1-MCP對基因表達(dá)量的影響,發(fā)現(xiàn)1-MCP對MdACS5a、MdACS5b表達(dá)量的升高有顯著抑制作用,1-MCP對MdACS4表達(dá)的抑制作用作用在金冠成熟期第六天較明顯。3.接菌的富士和金冠兩種蘋果果實,在不同發(fā)育時期對細(xì)胞壁軟化基因AFS1、PG1和PRs中的MdPR5、MdPR8、MdPR4進(jìn)行qRT-PCR分析。發(fā)現(xiàn)接菌后幼果期和膨大期細(xì)胞壁軟化基因相對表達(dá)量有明顯升高。PRs相關(guān)基因的表達(dá)量在病菌侵染下也明顯增高,這表明PRs具有抗病性。4.克隆MdPR5和MdPR8兩個病程相關(guān)基因,進(jìn)行序列分析和表達(dá)分析,并進(jìn)行原核誘導(dǎo)和SDS-PAGE分析。為蘋果抗病基因的挖掘和蘋果品種改良做出基礎(chǔ)性工作。并通過氨基酸序列對比分析發(fā)現(xiàn)MdPR5的氨基酸序列與蘋果、擬南芥、梨、i卙r的某些序列相似性很高。
[Abstract]:Apple rot is a major disease of apple in China. It is a fungal disease which mainly damages branches and fruits and poses a great threat to fruit production. The subsequent physiological activities and stress responses were used to discover Apple resistance genes and understand the mechanism of Apple disease resistance. Significance. To provide scientific and technological support and basis for the cultivation of new apple germplasm resources, and serve the development of apple industry in China. The results showed that the genes related to ethylene biosynthesis, cell wall softening and PR family genes were induced and expressed in large quantities by pathogens. There were significant differences in resistance to apple ring rot between the two cultivars, and the resistance responses of apples at different developmental stages were very complex. We screened out MdPR5 and MdPR8 from apple, obtained the gene cDNA sequence of Apple by the methods of plant RNA extraction, PCR and gene cloning, and carried on the sequence comparison analysis, gene expression and transgenic analysis, and verified the resistance of different apple species through molecular biological means. The main results are as follows: 1. Accumulation of reactive oxygen species (ROS) in plants under stress. The leaves and plants were inoculated with Rhizoctonia solani and flg22, and the production of ROS was detected by DAB staining. The more precipitated particles and yellow particles, the more reactive oxygen species produced, and the redder the stem was treated with flg22, indicating that both leaves and plants had undergone stress response. 2. The ethylene release rate and hardness of apple fruits inoculated with Fuji and Golden Crown were changed during the three development stages of young fruit, expansion and maturity. The effect of 1-MCP on inhibiting ethylene release was also different in different cultivars at different stages. The changes in the ripening stage of golden crown were the most significant. The plaque and hardness of golden crown fruit after inoculation were significantly less than Fuji. For the genes related to ethylene synthesis, MdERF2, MdACS4, MdCTR1, MdACS5a and MdACS5b, the qRT-ACS5b was used. PCR analysis showed that the expression of MdACS5a and MdACS5b increased significantly in the mature stage. The effect of 1-MCP on the expression of MdACS5a and MdACS5b was studied. It was found that 1-MCP significantly inhibited the expression of MdACS5a and MdACS5b, and 1-MCP significantly inhibited the expression of MdACS4 on the sixth day of golden crown. The relative expression of cell wall softening genes AFS1, PG1 and PRs, MdPR5, MdPR8, and MdPR4 in Fuji and Golden Crown apple fruits inoculated with bacteria were analyzed by qRT-PCR at different developmental stages. The results showed that PRs had disease resistance. 4. Cloning MdPR5 and MdPR8 genes, sequence analysis and expression analysis, prokaryotic induction and SDS-PAGE analysis were carried out. The basic work was done for the excavation of disease resistance genes and the improvement of apple varieties. The amino acid sequences of MdPR5 were compared with those of apple and Yunnan by amino acid sequence analysis. The sequence similarity of mustard, pear, I R is very high.
【學(xué)位授予單位】：山東農(nóng)業(yè)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2016
【分類號】：S436.611

【參考文獻(xiàn)】

相關(guān)期刊論文 前10條

1 田嘉;曾斌;羅淑萍;李秀根;李疆;;‘庫爾勒香梨’PsPL基因的克隆與表達(dá)分析[J];果樹學(xué)報;2015年06期

2 張鵬;林洋;李江闊;陳紹慧;;1-MCP處理時間對蘋果采后生理代謝的影響[J];中國食品學(xué)報;2014年05期

3 李雪姣;范偉;牛東東;關(guān)明俐;繆劉楊;史佳楠;竇世娟;魏健;劉麗娟;李莉云;劉國振;;水稻病程相關(guān)PR1家族蛋白質(zhì)在葉片生長及與白葉枯病菌互作反應(yīng)中的表達(dá)[J];植物學(xué)報;2014年02期

4 李佳;石琰t，

本文編號：2210882