The Molecular Mechanisms Of BnaA08.SPATULA And BnaA02.NIP6;1a In Regulation Of Silique Growth And Development In Brassica Napus

Brassica napus is one of the major oil crops in China,it can provide edible oil for human beings,rich protein for livestock,and serve industrial production as biofuel.The silique is an important production organ in rapeseed,which is very important for photosynthesis and seed development during the reproductive period.Therefore,it is of great significance to study the molecular mechanism of silique development and its application in rapeseed production.In this study,the experiments were performed to investigate the molecular mechanisms of transcription factor BnaA08.SPATULA and boron transporter BnaA02.NIP6;1a in regulating silique growth and development in Brassica napus.The main results are as follows:1.In order to explore the similarities and differences between SPATULA homologous genes in Brassica napus,four SPATULA homologous genes were found in the genome database of Brassica napus.The four homologous genes were analyzed by bioinformatics,and the results showed that the protein sequences of four SPATULA homologous genes in Brassica napus have highly conserved domains and are highly homologous with At SPATULA,indicating that the SPATULA homologous genes in Brassica napus may have similar functions in regulating silique growth and development as At SPATULA.2.To explore the expression pattern of BnaA08.SPATULA in Brassica napus,sequence analysis of the promoter of BnaA08.SPATULA found that it contains a large number of cis acting elements related to light,stress,auxin,gibberellin and abscisic acid response.BnaA08.SPATULA was localized in the nucleus and highly expressed in the flowers and the developing siliques(especially in the middle stage of the silique development).Overexpressing BnaA08.SPATULA in spt-12 mutant of Arabidopsis thaliana could restore the phenotype of plant height,silique number,silique length and 1000 seed weight to wild type.3.In order to explore the molecular mechanism of BnaA08.SPATULA regulating the early development of the silique in Brassica napus,RNAi transgenic lines of BnaA08.SPATULA in Brassica napus were obtained by RNAi interference technology.Phenotypic analysis showed that the silique development of the RNAi lines was significantly hindered,and the number of seeds per silique was significantly reduced.It was found that the number of pollen tubes in RNAi interference lines was significantly lower than that in wild type.Furthermore,BnaA08.SPATULA could interact with HEC family proteins related to pistil development.In Arabidopsis thaliana,At SPATULA may affect the development of the pistil by directly acting on the promoter of At GA3OX1,At IAA7,At NGA3,At HEC1 and At MYB107.4.In order to explore the molecular mechanism of BnaA08.SPATULA in regulating silique dehiscence in Brassica napus,the experiments showed that BnaA08.SPATULA was mainly expressed in the dehiscence zone of the silique,and BnaA08.SPATULA could affect the formation of lignified cells and small cells in the separation layer.Furthermore,yeast two-hybrid test,Bi FC and Co-IP showed that BnaA08.SPATULA could interact with BnaA03.IND,which is a key protein related to silique dehiscence in Brassica napus.Taken together,BnaA08.SPATULA interacts with BnaA03.IND and regulates the process of silique dehiscence by influencing the cell differentiation of silique dehiscence zone in Brassica napus.5.In order to explore the mechanism of BnaA02.NIP6;1a regulating silique development in Brassica napus,this study found that BnaA02.NIP6;1a was strongly expressed in roots,stems,leaves,especially in the buds and flowers of Brassica napus,and its subcellular localization was in cell plasma membrane and cytoplasm.The rapeseed BnaA02.NIP6;1a RNAi lines were obtained by genetic transformation.Under boron-deficient condition,the seedling growth of RNAi lines was seriously hindered,and the boron concentration in the seedlings was significantly lower than that of wild-type plants.In reproductive stage,RNAi lines showed a range of typical B deficiency symptoms,including exposed stigma,flat and dry papilla cells,smaller and fragile stamens,abnormal development of stylar canal cell and smaller seeds.In addition,the boron concentration in the flower of RNAi lines was significantly lower than that of wild type.Furthermore,the expression level of BnaKDOPS related to RG-II synthesis was significantly increased in the pistils and stamens of RNAi lines.Taken together,BnaA02.NIP6;1a encodes boron transporter protein,which is required for the development of flower organs and fertility under boron deficiency in Brassica napus.