Thermo-sensitive Male Sterile Gene Cloning And Over-expression Vectors Construction Of Chinese Pink

Chinese pink(Dianthus chinensis) is a kind of perennial herbaceous flower with good ornamental character, cold-resistant and drought-tolerant features. It is often used in flower bed, flower border and ground cover in large area. Plant material D. chinensis ’Yingying’is a hybrid with excellent ornamental character got by our research team. It is male sterile when the average temperature is below 18℃, but it is fertile when the average temperature is above 18℃. In this experiment, anther semithin section and pollen scanning electron microscope of D. chinensis’Yingying’under different temperature treatment were observed to look for the cytogenetic reason of thermo-sensitive male sterile phenomenon. At the mean while, transcriptome and expression profile sequencing and semi-quantitative analysis of flower bud of D. chinensis ’Yingying’ under different temperature were selected to seek different expression genes about thermo-sensitive male sterility. At last, the over-expression vectors of different expression genes about thermo-sensitive male sterility were constructed to explore the reason of thermo-sensitive male sterility. The main content and result are as follows:1.The observation of anther development process. The anther semithin section and pollen scanning electron microscope of D. chinensis ’Yingying’ under different temperature were made to observe the anther development process. The result showed that anther development process of D. chinensis’Yingying’contained 5 stages:the division of archesporial cell, microspore mother cell, tetrad, mononuclear pollen and mature pollen stage. There was significant difference in mononuclear pollen and mature pollen stage, while the difference in the first 3 stages was not significant. In mononuclear pollen, compared with the normal temperature,tapetum at low temperature presented inflated and could’t degrade normally. At mature pollen stage, pollen at low temperature was few, chamber was empty and turned to be a dumpling of cells at last.2.The analysis of transcriptome data. The anther development period was firmed by the semithin section observation, and I made the transcriptome and expression profile sequencing of each period buds at different temperature.65535 Unigene were obtained and the number belonged to molecular function process, biological process and cell component were 13813,33227 and 17397 respectively. Blasting these Unigene with KEGG data bank,17250 Unigene had Pathway annotation,containing 131 classes like metabolism, genetic information processing, cell process, environmental signal processing and so on.3.The selection of thermo-sensitive male sterile genes.22 genes of anther development were selected to make analysis of transcriptome digital different expression. The result indicated that only BAM2, EXS, MPK6, RPK2, SAP, TDR genes had significant difference, which accorded with the result of semi-quantitative analysis. Combining with relative articles and gene function,6 genes APRT, UGP, EXS, RPK2, SAP, TDR were firmed to have an important relationship with thermo-sensitive male sterility.4.The over-expression vectors construction of thermo-sensitive male sterile genes. With existing technology in our library,the vector pCAMBIA2300s was selected to construct the over-expression vector of genes APR T, UGP, EXS, RPK2, SAP, TDR, which layed a good foundation for the next gene transformation.