【作者】 张旭东；

【导师】 冀瑞琴；

【作者基本信息】 沈阳农业大学 ， 蔬菜学， 2023， 硕士

【摘要】 根肿病是一种在世界范围内广泛传播的土传病害,专性寄生于大白菜等十字花科作物中,严重影响了大白菜的经济效益。挖掘关键抗病基因,选育新的抗性育种材料具有重要的研究价值。本课题组在前期通过差异转录组分析,初步筛选到一个在根肿菌接种处理后表达量显著上调的基因BraA10g022070.3C,该基因与拟南芥基因SAUR20(SMALL AUXIN UP RNA 20)高度同源,同源性达到83.51%,因此我们将其命名为BrSAUR20。本研究通过实时荧光定量分析了BrSAUR20基因在大白菜根肿病抗/感材料不同时期和不同处理中的表达量差异;通过原位杂交技术进一步明晰BrSAUR20基因在各时期和处理中的表达差异;通过亚细胞定位技术确定了BrSAUR20基因的表达部位;通过对拟南芥纯合突变体saur20进行抗根肿病分析,以及运用VIGS技术对大白菜BrSAUR20基因进行沉默及抗根肿病鉴定,研究了上述基因在根肿菌对大白菜侵染中的功能;利用酵母双杂交技术筛选BrSAUR20的互作蛋白。试验结果如下:1.实时荧光定量PCR结果显示,BrSAUR20基因在感病材料中的表达量高于抗病材料,而在感病材料中表达量受到根肿菌诱导而显著上调,且达到了5倍以上,特别是在接菌40 d后表达量上升更为显著;结果说明BrSAUR20基因可能在大白菜根肿菌侵染过程中发挥作用。2.原位杂交结果显示,在未接根肿菌的感病材料组织中,杂交信号较弱,而在接种14 d后,组织中的杂交信号明显增强,在接种40 d时,杂交信号达到最强。进一步表明BrSAUR20基因在大白菜根肿菌侵染过程中发挥作用。3.构建含BrSAUR20基因的GFP融合载体,在烟草中瞬时表达,通过激光共聚焦显微镜观察发现,注射空载菌液的烟草叶片中,荧光信号在细胞核、细胞膜和细胞质中均有出现,而注射含BrSAUR20的菌液时只有细胞核上出现荧光信号。说明生长素响应蛋白BrSAUR20定位在细胞核。4.利用三引物法筛选到拟南芥纯合突变体saur20,对突变体saur20和野生型WT接种根肿菌,显微镜观察根肿菌侵染情况发现,saur20突变体根毛中的根肿菌孢子侵染进程较野生型植株延缓,且根系的生长状况也更好。构建pTRV:BrSAUR20载体,利用VIGS技术将大白菜BrSAUR20基因沉默,在接种根肿菌后发现,沉默植株的发病程度较对照组植株减轻。结果表明BrSAUR20基因正调控根肿菌对大白菜的侵染。5.构建pGBKT7-BrSAUR20诱饵载体,通过酵母核文库筛选初步得到了13个互作蛋白,对上述蛋白基因进行转录组数据分析,发现其中3个互作基因RNC3、GeBP和PDV2,它们在接种根肿菌后以及在抗感材料中的表达量都发生显著的变化,RNC3、GeBP在转录水平调控基因表达,而PDV2参与细胞分离。更多还原

【Abstract】 Clubroot disease is a soil-borne disease which is widely spread in the world.It is an obligate parasitic disease in cruciferous crops such as Chinese cabbage,which seriously affects the economic benefits of Chinese cabbage.It is of great research value to explore key resistance genes and select new resistance breeding materials.In our previous study,a gene BraA10g022070.3C was screened using differential transcriptomic analysis,because its expression was significantly up-regulated after inoculation with Plasmodiophora brassicae.This gene was highly homologous to Arabidopsis SAUR20(SMALL AUXIN UP RNA 20),and the homology reached 83.51%.So call it BrSAUR20.In this study,the gene expressionof BrSAUR20 in different periods and different treatments of resistant/sensitive materials was analyzed using real-time quantitative PCR(RT-qPCR).In situ hybridization was used to further clarify the expression differences of BrSAUR20 gene at different stages and treatments.The expression site of BrSAUR20 gene was determined by subcellular localization technique.The homozygous mutant saur20 of Arabidopsis thaliana was analyzed and the VIGS technique was used to identify the BrSAUR20 gene of Chinese cabbage.It was found that these genes positively regulated the infection of Plasmodiophora brassicae to plants.The interacting proteins of BrSAUR20 were screened by yeast two-hybrid technique.The experimental results are as follows:1.Real-time quantitative PCR results showed that the expression level of BrSAUR20 gene in susceptible materials was higher than that in resistant materials,and the expression level in susceptible materials was significantly up-regulated by Plasmodiophora brassicae,and reached more than 5 times,especially after 40 days of infection.The results indicated that BrSAUR20 gene might play a role in the infection process of Plasmodiophora brassicae of Chinese cabbage.2.The results of in situ hybridization showed that the hybridization signal was weak in the tissue of susceptible material without inoculation,but the hybridization signal in the tissue of susceptible material was significantly enhanced 14 days after inoculation,and reached the strongest signal at 40 days after inoculation.The results indicated that BrSAUR20 gene play a role in the infection process of Plasmodiophora brassicae of Chinese cabbage again.3.GFP fusion vector containing BrSAUR20 gene was constructed and expressed instantaneously in tobacco.Laser confocal microscopy showed that fluorescence signals appeared in the nucleus,cell membrane and cytoplasm of tobacco leaves injected with no-loaded bacterial solution,while fluorescence signals only appeared in the nucleus when injected with fusion vector containing BrSAUR20.This indicates that the auxin response protein BrSAUR20 is localized in the nucleus.4.The homozygous mutant saur20 of Arabidopsis thaliana was screened by three primers method.The mutant saur20 and wild-type WT were inoculated with Plasmodiophora brassicae pTRV: BrSAUR20 vector was constructed,and the VIGS technique was used to silence BrSAUR20 gene in Chinese cabbage.After inoculation with Plasmodiophora brassicae,it was found that the disease degree of BrSAUR20 silenced plants was less than that of control plants.These results suggested that BrSAUR20 gene positively regulated the infection of Plasmodiophora brassicae to Chinese cabbage.5.The bait vector pGBKT7-BrSAUR20 was constructed,and 13 interacting proteins were preliminatively obtained by screening the yeast nucleolibrary.Transcriptome data analysis of the above protein genes showed that the expression levels of three interacting genes,RNC3,GeBP and PDV2,were significantly changed after inoculation and in anti-sensiti9 ve materials.RNC3 and GeBP regulate gene expression at the transcriptional level,while PDV2 is involved in cell separation.更多还原