摘要
以水稻纹枯病菌(Rhizoctonia solani AG-1IA)菌株GD118不同生长时期的总RNA为材料,以18SrRNA作为内参基因,根据实验室抑制消减杂交(suppression subtractive hybridization,SSH)已获得的立枯丝核菌基因片段的表达序列标签(expressed sequence tags,ESTs)设计引物,采用实时定量RT-PCR检测A、B、C、E、F基因(分别为双组分反应调节子、激活巨噬细胞糖蛋白、胞外金属弹力蛋白酶、亲环蛋白、内质网囊泡蛋白ERV29)的表达,以期找到水稻纹枯病菌菌核形成中的差异表达基因。结果表明:从菌核开始形成到菌核成熟的过程中,这5个基因的表达量逐渐降低,且菌核在成熟时表达量最低,但成熟后这些基因的表达量迅速恢复到之前的水平,之后表达量基本保持不变。提示这5个基因在水稻纹枯病菌菌核形成的不同时期的表达有显著差异,说明这5个基因可能受菌核形成过程中胁迫条件的诱导表达。
Summary Rhizoctonia solani is an important plant distribution, and the sclerotia are the main source of pathogenic fungus with a wide host range and worldwide infection for the diseases caused by this pathogen. The mechanism of formation and development of sclerotia of Rhizoctonia solan had been investigated by some researches, but they were mainly focused on the relationship between the sclerotium formation and the nutrition or environmental factors. There is still lack of the knowledge on the molecular basis of sclerotium formation of R. solani, and little is known about the sclerotium formation-related genes. Subtractive cDNA library of differentially expressed genes during the sclerotium formation of R. solani AG 1 IA （GI〉118 strain） was constructed previously by using suppression subtractive hybridization （SSH） technique in our laboratory. The hyphal and sclerotium mRNA was used as the driver and tester, respectively. In the screening, some specific sclerotium formation related gene fragments were identified and sequenced. According to the principle of hybrid SSH, the obtained gene fragments should be hyphal or sclerotium specific, but there were five gene fragments, we named them as A, B, C, E, F, respectively, detected both in hyphae and sclerotia. Bioinformaticanalysis showed that these five gene fragments were correspondent with the genes putatively coding two component response regulator, macrophage activating glycoprotein, extracellular elastinolytic metalloproteinase, Cyclophilin, and ER derived vesicles protein ERV29. In this study, real-time quantitative PCR （QRT-PCR） was applied to further detect the gene expressions of A, B, C, E, F in different growing stages of R. solani by using the total RNAs as the templates and the 18S rRNA as the reference gene. The five gene expressions were determined by the QRT-PCR and calculated by the relative quantitative method （2-△△CT ）. The results showed that： all of the five gene expressions decreased gradually from the initiation of sclerotium formation, but increased quickly at near maturation of sclerotia, then remained a constant level after maturation of sclerotia. Moreover, it was shown that these five gene expressions altered significantly among different growing stages of R. solani. According to the previous reports, the decrease of the expression of these five genes could repress the normal cell metabolism and result in the cell death, suggesting that the death of a large numbers of sclerotium outer cells during the sclerotium formation of R. solani may be associated with the expression levels of these five genes induced by external stresses. Obviously, complex metabolism and signal transduction pathways are involved in the cell death and the sclerotium formation, the detailed functions of these five genes in the signal transduction pathways leading to the sclerotium formation are still unclear and need to be studied deeply in the future.
出处
《浙江大学学报（农业与生命科学版）》
CAS
CSCD
北大核心
2013年第1期18-25,共8页
Journal of Zhejiang University:Agriculture and Life Sciences
基金
国家公益性行业(农业)科研专项资助项目(nyhyzx3-16)
关键词
水稻纹枯病菌
菌核形成
实时荧光定量RT
PCR
基因差异表达
Rhizoctonia solani
formation of sclerotia
real-time quantitative RT PCR （QRT-PCR）
differentialexpression of gene
