玉米温敏型核雄性不育基因差异表达分析

Analysis of Differential Expression of Thermo-Sensitive Genic-Male-Sterile Gene in Maize

以玉米温敏型核雄性不育近等位基因系琼42Qms(不育)和琼42(可育)为材料,通过跟踪解剖、显微观察确定了雄穗处于小花分化期为分离纯化mRNA的最佳时期,以琼42Qms的mRNA作检测样本(Tester)、琼42的mRNA作参照样本(Driver)进行抑制消减杂交,构建差异表达消减cDNA文库。从文库中选取10个cDNA片段作探针,与琼42Qms和琼42的小花分化期雄穗总RNA进行Northern杂交,获得一个仅在琼42Qms中特异表达的差异cDNA片段。以该片段作探针与琼42Qms小花分化期、抽穗成熟期的叶片和雄穗的总RNA进行Northern杂交,结果表明,该片段只在琼42Qms小花分化期的雄穗总RNA中有杂交信号,说明该片段是与核雄性不育相关的。

The flosculous differentiation phase of tested maize was determined. The passels of Qiong 42Qms and Qiong 42 in each differential stage were dissected and observed under the microscope. The passels of Qiong 42Qms and Qiong 42 developed to the flosculous differentiation phase were determined based on the observation of the number of leaves in each respective phase. The total RNA was extracted from the passels of Qiong 42Qms and Qiong 42 in the flosculous differentiation phase, and SSH (suppression subtractive hybridization) was made with the purified mRNAs of Qiong 42Qms and Qiong 42. Qiong 42 was used as control. A subtractive cDNA library of maize was established with the products of SSH. Northern blotting indicated that only one of DIG-labeled fragment probes gave positive signal in the flosculous differentiation phase of Qiong 42Qms and Qiong 42. Using this fragment as a probe, hybridization was done with the total RNA of leaves and passels extracted in different developmental phases including flosculous differentiation phase (a part of elongation booting stage) and headling stage, respectively. The results demonstrated that this special fragment was expressed only in the passels obtained in flosculous differentiation phase. The possible explanation is that this fragment is related to the fertility of maize.