Functional Analysis Of Male Fertility Related Genes In Arabidopsis Thaliana

Controlling male fertility is an important goal for plant reproduction and selective breeding.Further understanding of the male sterile molecular mechanisms will provide an effective way to regulate male fertility and hybrid generation.ms606 is an male sterile mutant which is identified in the previous work.The ms606 anther displayed altered endothecium secondary thickening,which impacted on anther dehiscence.Transcription factor MYB26/MS35 plays a regulatory role in endothecium lignification as wall thickening is not observed in the endothecial cells of the myb26 mutant and displays indehiscent anthers.In this study,we generated an ms35ms606 double mutant where homozygous ms35 lines were used for crosses with ms606 plants.The genotypic,phenotypic and gene expression analysis were performed in the F2 generation.One homozygous ms35ms606 double mutant were obtained,this will provide a valuable material for further analysis of the relationship between MS606 and MYB26/MS35 in the regulation of secondary thickening in the endothecium.Auxin plays an important role in stamen development.To investigate whether MS606 regulates the development of stamens by auxin pathway,in this dissertation work,first the flower organs of wild type and mutants were treated with different concentrations of IAA;second,we constructed and identified a DR5:GUS plant with ms606 background,where ms606 lines were used for crosses with DR5:GUS plants.In addition,GUS staining was performed on the anther to detecte the IAA content of flower buds in different periods and the expression of auxin synthesis genes(YUC2 and YUC6)and transport genes(PIN2,PIN7 and AUX1)were analyzed.These results showed that different concentrations of IAA could not restore the anther dehiscence defect.GUS staining indicated that the expression of auxin was at the same time in anthers of wild-type and ms606,which began to express in the stage 10 of flower development and didn't display GUS signal in late stage 12.But in the stage 11 of flower development,the GUS activity in mutant anthers was dramatically stronger compared to the wild type.And the content of IAA in stage 10-11 buds was significantly higher than that of wild type.RT-PCR and qRT-PCR analysis showed that the expression of auxin synthesis genes YUC2 and YUC6 in flower buds of ms606 decreased remarkably.The expression of PIN2 and PIN7 was also changed in the mutant buds,but there was no difference in the expression of influx transporter AUX1.These results indicate that MS606 gene can affect the genes of auxin pathway in the flower buds to a certain extent.In addition,the expression of genes involved in jasmonate induced pathway(MYB21,MYB24,MYB108)and tapetum development(AMS,ABCG26)in ms606 buds altered.It can be seen that the MS606 dysfunction affect several pathways,but the detailed mechanism needs to be further analyzed.