Tapetum Development And Its Gene Regulation During Anther Development In An Androdioecious Tree,Tapiscia Sinensis

Androdioecy is an extremely rare and unusual breeding system present in angiosperms.Recently,artificial pollination experiments and paternity analysis all suggested that T.sinensis is a functionally androdioecious species.This special populations contained both male and hermaphrodite individuals,and hermaphroditic flowers could self-fertilize,but the pollen viability of which was significantly lower than that of males.Furthermore,combining the analysis results of histochemical staining and gene expression related to sugar transport and metabolism,we found that the sugar transport and metabolism of hermaphroditic flowers were abnormal compared with the males.At vacuolate microspore(VM)stage,polysaccharide of hermaphroditic anthers was accumulated in the connective tissues,but could not be transported into anther chambers for the pollen development.As the key part of transport and synthesis of nutrients for pollen development,compare with the males,whether the development process of the hermaphroditic tapetum in the hermaphrodite flowers is normal?whether the expression of the key genes related to the development of the hermaphrodite tapetum is normal?What the interaction and regulation relationship among these genes are?what the similarities and differences between T.sinensis and the model plant Arabidopisis thaliana are?In order to address these questions,we compared the development process of tapetum and microspore between males and hermaphrodites in T.sinensi,s,analyzed the expression of the core genes related to the tapetum development,and preliminarily explored the interaction and regulation relationship among these genes.The main research results of this Master's Degree thesis are as follows:(1)The comparison results of anther transverse sections between males and hermaphrodites at various developmental stages showed that when individual young microspores were released from the callose wall,the cytoplasm of hermaphroditic tapetum became dense,the cytoplasm of early uninucleate microspores was gradually agglomerate,and pollen wall was defective.(2)Ultrastructural observation indicated that compare with the males,normal biosynthesis and secretory function of hermaphroditic tapetum were hindered,the programmed cell death(PCD)process of hermaphroditic tapetum was delayed,the tectum structures of hermaphroditic sexine were stuck together,nexine was defective,and the intine was not formed all the way.(3)The results of qRT-PCR indicated that there was no expression in vegetative tissues or fruits for TsDYT1,except the flowers,and at Tetrad(Tds)and Young microspore(YM)stages,TsDYT1 showed significantly low expression in hermaphroditic flowers compared with male flowers;RNA in situ hybridization showed that a strong hybridization signal was detected in the tapetum and microspores;Subcellular localization analysis indicated that TsDYTl was mainly localized in the nuclear region.(4)Compared with the males,core transcription factors of tapetum development,including TsTDF1,TsAMS and TsMSl,all showed significant difference expression in hermaphroditic flowers.(5)The gene sequences of TsDYT1,TsTDF1,TsAMS and TsMSl and their promoter sequences were cloned successfully from T.Sinensis.The results of gene structure analysis showed that the number of exons for TsDYT1,TsTDF1,TsAMS and TsMS1 were 4,3,8 and 3 respectively,and their promoter sequences all contained E-box cis-acting elements;The comparison of the amino acid sequence among T.sinensis,A.thaliana and Oryza sativa demonstrated that these four genes of T.sinensis all had the highest amino acid sequence similarity with their corresponding orthologous genes in A.thaliana;Phylogenetic tree analysis indicated that these four genes all had the relatively distant relationship with A.thaliana,and had the farthest relationship with O.sativa.(6)Our yeast two-hybrid(Y2H)assays showed that TsDYT1 interacted with TsAMS,but there was no detectable protein interaction between TsDYT1 and TsTDFl;Yeast one-hybrid(Y1H)assays confirmed that TsDYT1 protein successfully binded to the promoter sequence of TsTDFl and the binding site was located in 616 to 720 bp upstream of the the start codon ATG;However,TsDYT1 could not bind to the promoters of TsAMS(787 bp upstream of the start codon ATG)and TsMSl(1176 bp upstream of the start codon ATG).Taken together,our results suggested that the abnormal development of the hermaphroditic tapetum caused the defective pollen wall,resulted in the lower pollen vitality and ultimately promoted the androdioecious population maintenance of T.sinensis.The abnormal development of the hermaphroditic tapetum was closely related to the abnormal expression of the core genes for tapetum development,including TsDYT1,TsTDF1,TsAMS and TsMS1.In addition,there are both similarity and difference for gene regulation pathway of tapetum development between T.sinensis and A.thaliana.