| Callose is a polysaccharide in the form of ?-1,3-glucan and it exists in the cell walls of a wide variety of higher plants.It plays important regulatory roles during a variety of processes in plant growth and development.Abnormal callose deposition or degradation will lead to pollen abortion,resulting in male sterility in plants.In order to discuss deeply molecular regulatory mechanism related callose deposition,GMS NIL 10L03 was used as materials and cytology,biochemistry and molecular biology were applied to systematically studying callose deposition during microspores abortion to attempt to provide some theoretical reference for revealing abortion mechanism of microspores in GMS Chinese cabbage.The main research results were as follows:1.Combined aniline blue staining with microscopy callose deposition dynamics was investigated during the development of microspore abortion.Results showed that to tetrad stage,the fluorescence response of sterile microspores was consistent with the normal fertile microspores.But the appearance of tetrad was obviously irregular.Then the fluorescence reaction of fertile anthers gradually weakened,indicated that the callose around tetrad was degraded and the mononuclear microspore was released and finally there was no fluorescence reaction in the anther cavity.Then fluorescence in pollen wall was detected,indicated that with the microspore development callose deposited in the exine wall of pollen.While the fluorescence of the male sterile plants was continued,we founded the uninucleate microspores was not released,indicated callose degradation around tetrad was abnormal,then microspores was not able to be released,failed to further development,leading to abortion.2.DNS and aniline blue methods were employed in examining activity of BG and GSL in different developmental stages of microspore.Results showed that during the microspore abortion,the change trend of GSL activity in different developmental stages of sterile buds was consistent with the fertile buds,and gradually decreased with the development of microspore.But GSL activity in the meiosis period was significantly higher than that of fertile plants while it was more significantly higher than that of fertile plants in the uninucleate stage,and the change of BG activity was small in the progress of microspore abortion,which was significantly lower than that of fertile plants.In the microspore abortion process,callose synthesis was enhanced while degradation was declined,resulting in abnormal callose deposition in sterile buds.3.Real-time PCR was applied to analysing genes expression of GSL and BG related callose deposition.Results showed that BcGSL1,BcGSL2,BcGSL8 and BcGSL10 were highly expressed in anthers.However,the expression of these genes in meiosis to early microspore of sterile plants was lower than that of fertile plants,which proved that these genes had no relationship with the delayed degradation of callose.Although the expression of BcGSL12 gene in the sterile microspore was higher than that of the fertile microspore in tetrad,the activity of GSL in this period was still lower than that of fertile plant.Therefore high expression of BcGSL12 gene in tetrad did not increase the total synthesis of callose.BcENBG7 and BcENBG12 genes were also highly expressed in anthers,but the expression of these genes in tetrad of sterile plants was higher than that of fertile plants,so regulation level of these two genes was not decreased.The expression of BcA6 gene in sterile plants was significantly lower than that of fertile plants in tetrad,led to the lower enzyme activity during this period,then effected the degradation of callose.Comprehensively analysing the results of cytology,biochemistry and molecular biology,the delayed degradation of callose on microspores is an important reason of microspores abortion in GMS Chinese cabbage and low expression of BcA6 led to the lower activity of BG in sterile than fertile buds,which maybe have relation to the delayed degradation of callose on microspores. |
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