| Male sterility is a common phenomenon in higher plants.It is of great significance to study plants about their fertility.As a widely used male sterility type of brassica crops,ogu-CMS is used for the research on the mechanism of nucleus cytoplasmic interaction and production for hybrid seeds.As a widely cultivated vegetable in Jianghuai area,Wucai is a variant of Chinese cabbage,which is known for its extremely resistant to low temperature and abundance of high vitamin C.However,there are few studies on male sterility of Wucai.To improve efficient breeding technology,further research on the mechanism of male sterility in Wucai is necessary.In this experiment,we compared the pollen development process in ogu-CMS and its maintainer line,and analyzed the reasons which cause differenees in fertility by cloning TDF1 gene and verifying its related functions.The main results are as follows:1.The flower buds of ogu-CMS and its maintainer line were successfully graded,divided into 10 periods,and cytological observations were compared.It was found that the abortion of microspore in ogu-CMS was caused by two aspects.On the one hand,most microspores cannot be released from the tetrad,resulting in complete degradation.on the other hand,the microspores were squeezed by the tapetum,which were abnormal vacuolated vacuolation,resulting in final abortion.2.The temporal and spatial expression patterms of tapetum-related genes DYT1,TDF1 and AMS in Wucai were analyzed by RT－PCR.The expression levels of three transcription factors were not significant and low in roots,stems and leaves of fertile and sterile plants.However,the expression levels of DYT1,TDF1 and AMS in fertile lines in inflorescence tissues were significantly higher than those in sterile lines,which were 2-4 folds higher than that in sterile lines.During the anther development period,the expression level of DYT1 in stage 3-5 ogu-CMS was significantly lower than that in the maintainer line.In contrast,DYT1 was expressed higher in ogu-CMS during the 8-9 period.Except for stage 5,the expression level of the AMS gene in the fertile line at all developmental stages was significantly higher than that of the sterile line.In addition,the expression level of AMS was so low that it was almost undetectable during stage 8-9 in ogu-CMS.The expression of the TDF1 gene that regulates the AMS gene was lower than that of the fertile line in all stages of the sterile line.3.TDF1 gene was cloned successfully from ogu-CMS and its maintainer line.BLAST analysis showed the highest similarity with TDF1 gene of Brassica campesis,named BcTDF1.The gene length vas 972 bp,and a total of 323 amino acids were encoded.4.The results of online prediction indicated that BcTDF1 gene was located in the nucleus.Meanwhile,PBI121-35S::BcTDF1-GFP expression vector was successfully constructed with the designed primers,and the subcellular localization of onion epidermal cells mediated by Agrobacterium EHA105 was used to confirm its intracellular expression5.RT-PCR analysis of A6 and EXYPB5 genes,which are directly regulated by TDF1 during anther development,showed that the expression of A6 gene in fertile inflorescence was much higher than that in sterile inflorescence.In addition,the expression of A6 gene in fertile lines was significantly higher than that of sterile lines in stages 3-4,however,the results were contraiy in stages 5-6,while the expression in fertile lines and sterile lines was very low in stages 7-9,which was almost difficult to detect.The expression of EXPB5 was low in all stages of anther,which could hardly be detected except stage 6 in maintainer line.In order to analyze the function of BcTDF1,the plant overexpression vector pBI121-35S::BcTDF1 was constructed successfully,and Agrobacterium tumefaciens was used to mediate successful infection and screened for 4 TO generation plants of 35S::BcTDF1-1 transgene.A total of 35 plants with TDF1/TDF1 homozygous lines were obtained in the T3 generation by antibiotics,GUS staining and molecular marker screening.The results of Alexander staining showed that there were normal mature pollen grains in the anthers of transgenic plants,and their pod development was also tending to be normal gradually.RT－qPCR experiments showed that the expression levels of AMS,A 6 and EXPB5 genes in transgenic plants were also increased compared with the mutant,but did not recover to wild-type expression levels. |
