Isolation And Expression Analysis Of Differentially Expressed Genes In The Buds Of Male Sterile Plants Induced By Chemical Hybridizing Agent

Rape is one of the most important oil crop in China. Rapeseed cultivation area in China accounted for40%in oil crop. Due to evidently heterosis of rape and the chemical hybridizing with freedom parental choice, easy hybrid preparation, hybrid Fl can multiple generation utilization etc, chemical hybridizing techniques has become one of the most important ways of heterosis utilization of rapeseed. However the researches on the mechanism of chemical hybridizing agent induced male sterility were usually found in the cell level and physiological and biochemical level.Firstly.this study obtained the differentially expressed gene fragments in the male sterility buds of R121which were induced by chemical hybridizing agent by using the technologys of suppression subtractive hybridization (SSH) and reverse northern dot blot. Secondly,we also analysis the quantitative expression of the fregments in the buds with different fertility and different length. Lastly.discussing the mechanism of male sterility induced by chemical hybridizing agent on the results of differential fragment homology comparison and quantitative expression. The study will provide strong theoretical guidance and lay a solid material foundation for the research of cheap, high efficiency and low pollution of chemical hybridizing agent. This research mainly get following results:(1) Using9ml7mg/ml chemical hybridizing agent "Hua-Sha-Ling WP1" sprayed on the whole R121plant,we can gained about more than98%completely sterile plants. The size, color and pistil development of sterile flowers are similar to the fertile flowers.There were no other significant differences but the stamen development.(2) Constructing a forward subtractive library which contains248monoclonal and reverse subtractive library which contains359monoclonal by suppression subtractive hybridization technique.Library monoclonal rates were higher than90%, and the strip is clear, fragment length was less than1OOObp.(3) Obtaining four differentially expressed gene fragments numbers1130,1252,1301,1319by reverse northern dot hybridization and sequencingg from the subtractive library.(4) Fragments Numbered1130was87%homology to the accession numbered9326790gene.The gene product has the function of GYF domain of protein, it is a proline-rich protein binding domain. On the expression of quantitative analysis of the gene in different fertility and different length buds.it was showed that when the bud length is0.00mm-2.50mm, the gene was higher expressed in male sterile flower buds, and when the bud length is3.00mm-4.00mm, the gene was lower expressed in male sterile flower buds. The differential expression of gene may lead to rape flower bud of proline-rich protein content changes, thereby causing the plants to resistance or abnormal development of male.(5) Fragments Numbered1252was87%homology to the accession numbered D10840.1gene.The gene product has the function of5.8S,25S,18S rRNA. On the expression of quantitative analysis of the gene in different fertility and different length buds.it was showed that when the bud length is0.00mm-2.00mm, the gene was higher expressed in male sterile flower buds, and when bud length is2.00mm-3.00mm, the gene was lower expressed in male sterile flower buds, when bud length is3.00mm-3.50mm, the gene was higher expressed in male sterile flower buds, and when bud length is3.50mm-4.00mm, the gene was lower expressed in male sterile flower buds. The differential expression of the gene may influence the normal function of the ribosome in the buds, resulting in male sterility at last.(6)Fragments Numbered1301was91%homology to the accession numbered BT021929.1gene.The gene product is a protein of unknown function. On the expression of quantitative analysis of the gene in different fertility and different length buds.it was showed that when the bud length is0.00mm-2.00mm, he gene was higher expressed in male sterile flower buds, and when bud length is2.00mm-4.00mm, the gene was lower expressed in male sterile flower buds.(7) Fragments Numbered1319was100%homology to the accession numbered DQ658218.1gene.The gene is polygalacturonase gene (MF6) which was pollen expressed specificly. On the expression of quantitative analysis of the gene in different fertility and different length buds,it was showed that the gene was lower expressed in male sterile flower buds through out the entire development process, and the differences were most pronounced at the length of1.00mm-1.50mm and3.50mm-4.00mm. The differential expression of MF6gene may be associated with stamen development and filament elongation growth.