Dfferential Expression Of Thermo-sensitive Genic-male-sterile Gene In Maize

Maize is one of the three principal crops in China. Because its planting area is unable to be enlarged, to increase the production of maize, the only way is improve its individual yield. As for the results of current research, making hybrid seeds using male-sterile line is the only way to increase its individual yield. But because of the lack in the heredity mechanism of male-sterility in maize, it is difficult to utilize male-sterile line.The purpose of this research is to separate differential cDNA fragments, thus lay a foundation for cloning full-length gene and analysing its function and heredity mechanism.The results as follows:Determination of flosculous differentiation phase of tested maize. The passels of Qiong 42Qms and Qiong 42 in each differential stage were dissected and visualized under microscope. Combined with the results of observation of the number of leaves in each respective phase, 1 determined that when the number of leaves reaches ll-13,breakes reaching 7.5-8.8, the passels of Qiong 42Qms and Qiong 42 develop to flosculous differentiation phase.Establishment of a subtractive cDNA library of maize. The total RNA was extracted from the passels of Qiong 42Qms and Qiong 42 in flosculous differentiation phase. SSH(suppression subtractive hybridization) was done 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.Plasmids were extracted using 22 randomly selected white colonies and were used for enzyme digestion. The results indicated that among the selected colonies, 20gave positive signals, which means fragments have been inserted into those positive colonies. 10 were picked out from the positive colonies and used in preparation of plasmids. After experienced enzyme digestion, the fragments were extracted and used for preparation of DIG-labeied probes.The results of Northern blotting indicated that only a 400bp fragment gave positive signal. Using this fragment as probe, hybridization was done with the total RNA of leaves and passels extracted in different development phases including flosculous differentiation phase (a part of elongation booting stage) and headling stage, respectively. The results demonstrate 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.The fragment was sequenced and the sequence obtained was analyzed by homology search in Genbank database. The results indicated that the fragment obtained has no homologous counterpart in Genbank database. The possible explanation is that the fragment is a part of a new gene.