The Function Mechanism Of Rice Male Sterile Gene BM1 And Seed-setting Rate Regulatory Gene LSSR1

It is of great importance to study the fertility regulation of rice spikelets.In this study,we got a no-pollen type sterile mutant baymax1?bm1?with a Zhonghui8015 background by treating with ⁶⁰Co-?ray.The sterility causes of bm1 mutant were explored through the observation of anther semi-thin section,SEM,TEM,etc.In addition,the function of this male sterile gene was studied through map-based cloning,complementary experiment,knockout vertification,TUNEL assay,etc.The main results were listed as follows:1.Mutant bm1 showed normal vegetative growth,but with no visible pollen grains in its anthers.2.The semi-thin section and TEM analysis of bm1 anthers showed that the secretion transition of bm1 tapeum in stage 9 failed.In bm1 tapeum,the organelles almost completely degenerated in stage10,which did not synthesize Ubisch body.As a result, the pollen wall could not form,and the microspore degraded.3.According to the SEM observation,the outer surface of bm1 anthers displayed abonormal cuticle structure,and there was no granular structure on the anther inner surface and pollen wall surface in bm1 mutant.These results suggest that the tapetum in mutant bm1 develops and degenerates abnormally,which can not synthesize Ubisch bodies,resulting in nutrition deficiency for microspore development and the degradation of microspores.4.BM1 were finely mapped to a 17.9 kb region between 4MD-6 and 4MD-11 on chromosome 4.Based on the gene annotation and sequencing analysis,mutant bm1 carries a single nucleotide substitution?A?T?in the second exon of the forth ORF ?LOC_Os04g39470?,resulting in a residual change from glutamic acid to valine.The results of complementary experiment and knockout vertification confirmed that it is the mutated BM1?namely OsMYB103?that accounts for the male serility in mutant bm1.5.The expression profile of BM1 indicates that the 5?-terminal promoter of BM1 promotes its specific expression in anthers from stage 5 to later stage 10,and the strongest signals were observed in stage 6 anthers.The results of sequence alignment and phylogenetic analysis showed that BM1 is a R2R3 MYB transcription factor, whose homologs in several plant species control the anther development.The transcriptional activation assay suggests that BM1 does have transcriptional activation,and the 64 amino acids in the C-terminal is the main activation domain.6.According to the results of TUNEL detection and DAPI staining of anther paraffin sections,BM1 negatively regulates the degeration of tapetum,which is cinsistent with its homologous AtMYB103.However,the callose observation results suggest that BM1 does not involve in the synthesis and degeneration of callose during microsporogenesis,while AtMYB103 does.These indicate that the functions of BM1 are both conserved and variable during the evolution process.7.Combining the qRT-PCR results with the phonotypic defects in the loss-of-fuction mutants of some related genes,we speculate that BM1 is upstream of MSP1 and UDT1 in the regulation pathway for anther development.In addition,BM1 may be parallel with GAMYB and TDR to antagonistically regulate the expression of downstream genes for tapetum degeneration and synergistically promote the genes for pollen wall formation.On the other hand,low seed setting rate1?lssr1?mutant lines with a low seed-setting rate were obtained in this study through reverse genetic method.The reason of the low seed-setting rate in lssr1 lines was also analyzed.The main results were listed as follows:1.LSSR1 is predominantly expressed in anthers during the microsporogenesis stage,including the premeiosis,meiosis,and single-cell pollen stages.LSSR1 encodes a putative GH5 cellulase,which has 12 active sites,including the two putative function-conserved glutamines?an acid/base and a nucleophile on?-strands 4 and 7,respectively?as GH5 hydrolases.In addition,it contains a signal peptide at the N-terminal.2.Loss-of-function mutants of LSSR1 were created through the CRISPR-Cas9 system,which showed a significant decrease in rice seed setting rate.Co-segregation analysis, genetic analysis and selecting multiple sites for single knockout of LSSR1 were conducted.The results confirmed that it is the mutated LSSR1 that results in the low seed-setting rate in lssr1mutant lines.3.The morphology of the vegetative and reproductive organs appears normal in lssr1 mutant lines.In addition,lssr1 pollen grains could be normally stained by I₂-KI solution.Cytological results demonstrate that the blockage of fertilization mostly accounted for the low seed setting rate in lssr1 mutant lines,which was most likely caused by abnormal pollen grain germination,failed pollen tube penetration,and retarded pollen tube elongation.Together,our results suggest that LSSR1 plays an important role in rice fertilization,which in turn is vital for maintaining rice seed setting rate.The two genes and their function exploration in this study will be helpful to improve our understanding in the molecular mechanism of rice fertility,and lay a theoretical foundation for the maintenance and improvement of rice yield,as well as heterosis utilization.