立枯丝核菌AG1-IA诱导玉米差异表达基因的研究
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摘要
玉米纹枯病是由立枯丝核菌Rhizoctonia solani Kühn引起的真菌性病害。尽管该病主要发生在中国和东南亚地区，表现出一定的区域性，但是该病在近年内表现出逐年加重和快速蔓延的趋势，极有可能在未来几年传播到其他国家和地区，并成为一种世界性病害，在我国玉米主产区，该病已成为制约玉米产量高低的主要病害，深入研究玉米对纹枯病的抗病机制，是寻找培育广谱、高效、稳定、持久抗病性品种的有效途径。本研究利用电镜检测技术、抑制消减杂交(SSH)cDNA文库构建技术、反向Northern筛选技术、RT-PCR以及生物信息学分析方法相结合，通过建立相关抗病基因表达谱，从全基因表达的水平上探索玉米对纹枯病的系统抗病机制。
实验所用材料为本课题组多年筛选鉴定出的高耐玉米纹枯病材料R15，纹枯病菌为立枯丝核菌高致病性融合菌群AG1-IA。玉米种子经表面消毒，发芽，温室培育幼苗至五叶期后移入大田，拔节期时采用人工嵌入法将两粒布满菌丝的麦粒接种到植株下位第三叶鞘，接种后接种部位用塑料袋包裹以保温、保湿，接种6小时后取下塑料袋，分别取接种后12h、24h、36h、48h、60h、72h、84h、96h的玉米第三下位的叶片和叶鞘，电镜观察菌丝在接种叶鞘组织中的形态变化，提取叶片的总RNA构建SSH差减文库。获得的主要结果如下：
1．采用电镜检测病菌的侵染过程后发现，接种12h后，菌丝在叶鞘细胞表面伸长并生成少量分支；接菌后24h，菌丝交错生长，呈现多分支的网状结构；接菌后36h菌丝继续分支、交错、缠结，形成侵染垫雏形；接种后48h形成成熟侵染垫；接种后60h，侵染垫继续增大，形成团状或带状茵丝团；接种后72h、84h、96h，伴随新一轮的菌丝生长、分支、侵染垫形成等在叶鞘组织上扩展，从而完成对寄主的扩大侵染。另外，在接种24h后的各时间段均观测到菌丝通过侵染钉直接进入寄主或菌丝通过气孔侵入寄主，病原菌大面积侵入寄主是在接菌后36～48h。而在接种72h以后，在叶鞘组织的横断面内发现侵入的菌丝，说明菌丝侵入寄主后能在受侵细胞间扩展。
2．应用数码相机记录病斑扩展过程后发现，接种部位在接种后12h未出现病斑；在接种后24h有水浸状小病斑出现；接种后36h病斑丧失叶绿素而呈白色：接种48h后在形成的白斑周围出现新的水浸状病斑的大面积扩展；接种后60h扩大的病斑丧失叶绿素呈白色，并与之前白色病斑连在一起组成两个明显的病斑；接种后72h，水
Maize banded leaf and sheath blight (BLSB), caused by Rhizoctonia solani Kuhn, is a destructive disease that results in significant yield loss in most maize-growing areas in China. Although this is a regional maize disease mainly occurring in China and Southeastern Asia, it is possible that this disease may spread to other parts of the world in the future. To breed new varieties with efficient, broad-spectrum, stable resistance to the maize BLSB, the understanding of resistance mechanism is of great importance. In our research, several techniques were used such as electronic micrograph detection, cDNA library construction of suppression subtractive hybridization (SSH), reverse-Northern hybridization, RT-PCR and bioinfonnatics methods. The objective of the present research was to explore efficient methods to analyze high-throughout gene expression, and to obtain a primary gene expression profiling of the disease resistance in maize, and to understand the resistance mechanism against maize BLSB at global transcriptional level.
By use of SSH, a pathogen inducible expressing gene library was obtained from high tolerance maize inbreed lines of R15 and high pathogenic fungus anastomosis groups AG1-IA of Rhizoctonia solani Ktihn in Southwestern of China. At the jointing stage, two wheat seeds colonized by AG1-IA were placed into the third sheath of the maize plants. At 12 hrs, 24 hrs, 36 hrs, 48 hrs, 60 hrs, 72 hrs, 84 hrs and 96 hrs of the post-inoculation, the third sheath was picked down to detect the pathology varyment of the mycelium with the electronic micrograph methods. Total RNA from the inoculation and non-inoculation leaves were extract to construct the SSH library. The obtained main results were as follows:
1. At the different times of the post-inoculation, the mycelium was changed significantly. Following the inoculations time, the mycelium of the pathogen formed infection cushions, inoculums hoops and single appresoria. Infection pegs arose at the base of these cushions penetrated the cuticle. Also, the pathogenic penetrate through stomata without producing any infection structure at the time of 24 hrs, 36 hrs and 48 hrs after the inoculations. Further penetration was inter or intracellular after the 72 hrs of the
实验所用材料为本课题组多年筛选鉴定出的高耐玉米纹枯病材料R15，纹枯病菌为立枯丝核菌高致病性融合菌群AG1-IA。玉米种子经表面消毒，发芽，温室培育幼苗至五叶期后移入大田，拔节期时采用人工嵌入法将两粒布满菌丝的麦粒接种到植株下位第三叶鞘，接种后接种部位用塑料袋包裹以保温、保湿，接种6小时后取下塑料袋，分别取接种后12h、24h、36h、48h、60h、72h、84h、96h的玉米第三下位的叶片和叶鞘，电镜观察菌丝在接种叶鞘组织中的形态变化，提取叶片的总RNA构建SSH差减文库。获得的主要结果如下：
1．采用电镜检测病菌的侵染过程后发现，接种12h后，菌丝在叶鞘细胞表面伸长并生成少量分支；接菌后24h，菌丝交错生长，呈现多分支的网状结构；接菌后36h菌丝继续分支、交错、缠结，形成侵染垫雏形；接种后48h形成成熟侵染垫；接种后60h，侵染垫继续增大，形成团状或带状茵丝团；接种后72h、84h、96h，伴随新一轮的菌丝生长、分支、侵染垫形成等在叶鞘组织上扩展，从而完成对寄主的扩大侵染。另外，在接种24h后的各时间段均观测到菌丝通过侵染钉直接进入寄主或菌丝通过气孔侵入寄主，病原菌大面积侵入寄主是在接菌后36～48h。而在接种72h以后，在叶鞘组织的横断面内发现侵入的菌丝，说明菌丝侵入寄主后能在受侵细胞间扩展。
2．应用数码相机记录病斑扩展过程后发现，接种部位在接种后12h未出现病斑；在接种后24h有水浸状小病斑出现；接种后36h病斑丧失叶绿素而呈白色：接种48h后在形成的白斑周围出现新的水浸状病斑的大面积扩展；接种后60h扩大的病斑丧失叶绿素呈白色，并与之前白色病斑连在一起组成两个明显的病斑；接种后72h，水
Maize banded leaf and sheath blight (BLSB), caused by Rhizoctonia solani Kuhn, is a destructive disease that results in significant yield loss in most maize-growing areas in China. Although this is a regional maize disease mainly occurring in China and Southeastern Asia, it is possible that this disease may spread to other parts of the world in the future. To breed new varieties with efficient, broad-spectrum, stable resistance to the maize BLSB, the understanding of resistance mechanism is of great importance. In our research, several techniques were used such as electronic micrograph detection, cDNA library construction of suppression subtractive hybridization (SSH), reverse-Northern hybridization, RT-PCR and bioinfonnatics methods. The objective of the present research was to explore efficient methods to analyze high-throughout gene expression, and to obtain a primary gene expression profiling of the disease resistance in maize, and to understand the resistance mechanism against maize BLSB at global transcriptional level.
By use of SSH, a pathogen inducible expressing gene library was obtained from high tolerance maize inbreed lines of R15 and high pathogenic fungus anastomosis groups AG1-IA of Rhizoctonia solani Ktihn in Southwestern of China. At the jointing stage, two wheat seeds colonized by AG1-IA were placed into the third sheath of the maize plants. At 12 hrs, 24 hrs, 36 hrs, 48 hrs, 60 hrs, 72 hrs, 84 hrs and 96 hrs of the post-inoculation, the third sheath was picked down to detect the pathology varyment of the mycelium with the electronic micrograph methods. Total RNA from the inoculation and non-inoculation leaves were extract to construct the SSH library. The obtained main results were as follows:
1. At the different times of the post-inoculation, the mycelium was changed significantly. Following the inoculations time, the mycelium of the pathogen formed infection cushions, inoculums hoops and single appresoria. Infection pegs arose at the base of these cushions penetrated the cuticle. Also, the pathogenic penetrate through stomata without producing any infection structure at the time of 24 hrs, 36 hrs and 48 hrs after the inoculations. Further penetration was inter or intracellular after the 72 hrs of the
引文
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