Map-based Cloning Of Male Sterile Gene OsNP2 In Rice

Rice is an important food crop,and it is the staple food for 50%of the world’s population.Rice yield is an important indicator of rice production,and the heterosis is one of the theoretical basis for improving rice yield.The use of male sterility is the best way to achieve heterosis.Although many of male-sterility genes have been reported,few genes are suitable for heterosis utilization,and the mechanism of male sterility in rice has not been fully elucidated.In this study,a no-pollen male-sterile mutant osnp2（Oryza sativa L.no pollen 2）found in Minghui 63 was firstly studied in detail through semi-thin sectioning and scanning electron microscopy.Then,through molecular markers,combined with the improved MutMap method,map-based cloning of OsNP2 was performed to identify candidate genes.Subsequently,CRISPR/Cas9was used to verify the candidate gene,and the expression pattern of OsNP2 was analyzed by RT-qPCR.The specific research results are as follows:1.Characterization features of osnp2 mutantsCompared with wild-type Minghui 63,osnp2 showed no obvious change in the panicle type and spikelets shape,and the spikelet structure was intact.The anther could protrude normally.There was no significant difference in the color of the anthers.The anthers were smaller,and there was no mature pollen in the anther.The abortion for mutant pollen appeared at stage 10 stage,and the main differents were the abnormal structure of the outer epidermis of the anther,delayed anther tapetum degradation,the irregular development of the Ubisch body,and the defective formation of the pollen exine.The microspores were degraded from the 10th stage,and finally resulted in male sterility without pollen.2.Mapping of OsNP2Using molecular markers,OsNP2 was located on chromosome 3 between SSR marker 0305 and In Del marker C03D3.The genetic distance between the two markers was 0.41 c M and 0.82 c M respectively,and the physical distance between the two markers was 0.7 Mb.3.Determining the candidate of OsNP2 geneUsing the improved MutMap method,a candidate gene MH03g0072500,Gramene accession number LOC＿Os03g07140 and gene name DPW（Defective Pollen Wall）,was found in the C03D3 and 0305 mapping interval.The mutant had G-A base substitutions in the 5’UTR of the gene.4.OsNP2 gene structure and OsNP2 protein analysisAccording to Gramene’s prediction,the length of OsNP2 gene was 4112 bp,with a total of 9 exons,encoding a protein product with a length of 609 aa.After Uni Prot KB query,it was found that OsNP2 had Fatty acyl-CoA reductase（FAR）activity,participated in lipid metabolism pathway,and played an important role in the formation of pollen exine.The protein contained two domains,the NAD-binding 4domain located at 135-437 aa and the sterile domain located at 543-613 aa.5.Expression pattern analysis of OsNP2The spatiotemporal expression pattern of OsNP2 was analyzed by RT-qPCR,and it was found that the gene was expressed in stems,leaves,flowers and seeds,but not in roots.The expression of this gene at anther stage 9 reached the highest level,and was also expressed to a certain extent in other stages,but relatively less.The gene mutation made its expression up-regulated in the mutant anther.6.Gene editing of OsNP2OsNP2 was knocked out by CRISPR/Cas9 technology,and the molecular detection and phenotype observation of transgenic progeny were carried out.It was found that the homozygous/double allele mutant plants showed sterile anthers with no-pollen.The fertility segregation ratio in F₁ generation,cas9-osnp2/cas9-osnp2×OsNP2/osnp2,is 1:1,which suggested LOC＿Os03g07140 was the gene of interest controlling the male-sterility phenotype in osnp2.