Phenotypic Analysis And Gene Cloning Of Two Male Sterile Mutants Osfla1 And Osgalt In Rice(Oryza Sativa L.)

Male sterile materials is one of the most commonly used means in rice production.Male reproduction is an extremely complex cytological process,and a large number of genes are involved in the regulation.The abnormalities in the process often lead to male sterility.At present,the breeding method of combining genetic engineering with male sterile mutants has been reported,which can participate in seed production by transgenic means,and the seeds do not contain transgenic components.It can be predicted that the recessive male sterile gene is of great value in basic and applied research,so the research on the function and regulation mode of related mutants is particularly important.First,this study performed phenotypic identification and functional analysis of osfla1 mutant.osfla1 is a complete male-sterile mutant whose mapping region has been determined,but the target gene has not yet been determined.In this study,the phenotype of the osfla1 mutant was analyzed by I₂-KI staining,calcium fluorescent white staining,sudan red 7B staining and callose staining;Then the target gene of Os FLA1 was verified by complementary verification and CRISPR/Cas9 gene knockout,and encodes a fasciclin-like AGP;Finally,the function of Os FLA1 was analyzed by Os-PCR,mRNA in situ hybridization,AGPs detection,subcellular localization,yeast two-hybrid,and dual-luciferase.Secondly,the phenotype analysis and gene cloning of osgalt mutant were also carried out.osgalt mutant is a male sterile mutant screened from ethyl methane sulfonate（EMS）mutant library.The phenotype of osgalt mutant was determined by stereoscopic observation,scanning electron microscope observation,semi-thin section observation and callose staining;Through gene mapping and sequencing analysis,Os Gal T candidate gene（Os Gal T-cg）was identified and encodes aβ-1,3-galactosyltransferase;Moreover,the AGPs were also tested for the osgalt mutant;Finally,the subcellular localization and Os-PCR analysis of Os Gal T-cg were carried out.1.Phenotypic identification and functional analysis of osfla1 mutant1.1 Phenotype analysis of osfla1 mutantI₂-KI staining of osfla1 pollen showed that osfla1 pollen was shriveled,wrinkled and inactive,showing complete abortion.Calcium fluorescent white staining（CFW）was used to detect the cellulose of osfla1 pollen intine.It was found that osfla1 pollen had no fluorescence signal,indicating that there was defect in osfla1 pollen intine.osfla1 pollen was not stained with sudan redn 7B,indicating that the lipid deposition on the surface of osfla1 pollen was also affected.The callose of osfla1 mutant was detected by aniline blue fluorescence staining.It was found that the fluorescence signal of osfla1 mutant was not significantly different from that of wild-type anther,indicating that the synthesis and metabolism of callose in osfla1 anther were normal.1.2 Complementation and gene knockout verification of Os FLA1According to the existing fine mapping results,the target gene was initially locked as LOC_Os04g48490.The p FLA1:FLA1-p CAM1301 complementary vector was constructed,and the positive complementary transgenic plant osfla1-COM obtained by transforming the osfla1 mutant callus.Compared with the osfla1 mutant,the pollen morphology of osfla1-COM was plump and round,and the fertility was restored.Using CRISPR/Cas9 gene editing technology to knockout the LOC_Os04g48490 gene in wild-type plants,two mutant LOC_Os04g48490^-Cas9 plants were obtained,both of which showed a similar sterile phenotype to osfla1 mutant.Based on the results of complementation and gene knockout,LOC_Os04g48490 is the target gene of Os FLA1 and encodes a fasciclin-like AGP（FLA）.1.3 Expression analysis of Os FLA1Os-PCR and mRNA in situ hybridization were performed on wild-type anthers at various stages of anther development.The results were showed that at stage 7-8b,Os FLA1 was highly expressed in microspores and tapetum.1.4 Arabinogalactan proteins detection of osfla1 mutantUsing the monoclonal antibody JIM13 to detect the content of arabinogalactose proteins（AGPs）in osfla1 anther,it was found that compared with the wild type,the fluorescence signals of osfla1 anther and microspore at stage 8b were significantly weakened,indicating that the osfla1 anther and microspore were significantly reduced.This results showed that the AGPs synthesis of osfla1 anthers and microspores was abnormal.1.5 Subcellular localization of Os FLA1The fusion expression vectors of Os FLA1-GFPM with different plasmid backgrounds were constructed respectively.The rice protoplasts and Nicotiana tabacum were transformed for transient expression.The results showed that Os FLA1 was localized on the cell membrane.1.6 Analysis of the regulatory relationship between Os FLA1 and PTC2The CRISPR / Cas9 vector of PTC2 was constructed to transform wild-type callus,and ptc2^-Cas9 transgenic plants were obtained.The ptc2^-Cas9 mutant showed phenotypes such as pollen exine defect and complete pollen abortion,were similar to the osfla1 mutant.The results of Os-PCR,yeast two-hybrid and dual-luciferase experiments indicated that PTC2 was not directly regulate Os FLA1.1.7 Expression analysis of genes related to pollen wall formationOs-PCR results showed that in osfla1 spikelets,the expression of PTC1,ABCG26,TDR,TIP2,EAT1 and TIP3 were increased to varying degrees;the expression of Os C6 was significantly decreased;AP25,AP37,PTC2,WDA1 and CYP704B2 also appeared disordered expression patterns to varying degrees.These results suggest that Os FLA1 may regulate pollen wall formation by affecting the expression of other genes.2 2.Phenotypic analysis and gene cloning of osgalt mutant2.1 Phenotypic analysis of osgalt mutantCompared with the wild type,the osgalt mutant showed shorter,irregularly curved and pale yellow anther.And osgalt mutant exhibit complete male sterility.Scanning electron microscope showed that osgalt anther was shrunken and shriveled,the surface of anther wall was smooth,and there was no obvious waxy and cuticle structure.Ubisch bodies on the inner surface of anther wall was narrowly spaced,and some of them were abnormally stacked together.The pollen grains were cohesive and aggregated together,and each pollen grain showed a shriveled and shrunken shape,and the germination holes protruded outwards,and the shape was abnormal.It was found that from stage 7 the tapetum of osgalt anther was significantly thicker than that of wild type,and there was no slight concentration and cavitation until stage 11.In addition,the osgalt microspores remained aggregated and did not form independent free microspores.Aniline blue fluorescence staining showed that the degradation of osgalt callose was abnormal,and the callose signal could still be detected around the microspore of osgalt at stage 9.Therefore,it is speculated that the abnormal expansion of tapetum and the delay of cell programmed death lead to the abnormal degradation of callose and serious developmental defects of microspores,finally resulting in pollen abortion.2.2 Genetic analysis of osgalt mutantThe osgalt mutant and ’Jinhui 10’ were crossed to generate F1 generation,and the F1 generation was normal.The F1 generation was self-crossed to generate the F2 generation,and the segregation of normal and sterile traits appeared in the F2 generation population.Chi-square test showed that the ratio of the normal phenotype to the mutant phenotype in the F2 population was in line with the Mendelian segregation ratio of 3:1（χ²=0.539 < χ²0.05,1=3.841）,indicating that the sterility of osgalt mutant is controlled by a pair of recessive nuclear genes.2.3 Gene mapping and candidate gene determinationGene fine mapping locates the osgalt mutation site between R13 and R19 with a physical distance of 39 kb.There are 9 annotated genes in the mapping region.After sequencing and comparison,it was found that only one base substitution occurred in the sixth exon of ORF6 among these annotated genes,resulting in a translation error.Therefore,ORF6 was identified as a candidate gene for Os Gal T.2.4 Expression analysis of Os Gal T-cgOs-PCR results showed that the Os Gal T-cg was expressed in root,stem,leaf,sheath and spikelet,and was highly expressed in spikelet.From stage 6-12 of anther development,the Os Gal T-cg was expressed in all stages,and highly expressed at stage8 a,8b,9 and 12.In situ hybridization showed that the Os Gal T-cg was expressed in the anther tapetum at stages 6-8a and in microspore at stages 8b-9.Taken together,the Os Gal T-cg was specifically expressed in the anther tapetum and microspore.2.5 Arabinogalactan proteins detection of osgalt antherUsing the monoclonal antibody JIM13 to detect the content of AGPs in osgalt anther,it was found that compared with the wild type,the fluorescence signals of osgalt anther and microspore at stage 8b and 9 were significantly weakened,indicating that the osgalt anther and microspore were significantly reduced.This result showed that the synthesis of AGPs in anthers and microspores of osgalt mutant was abnormal,and indirectly proved that Os Gal T-cg was the target gene.2.7 Subcellular localization of Os Gal T-cgOs Gal T-cg encodes β-1,3-galactosyltransferase,and the subcellular localization results of Nicotiana tabacum and rice protoplasts indicated that Os Gal T-cg was localized in the Golgi apparatus.2.8 Expression analysis of genes related to tapetum development and degradationOs-PCR showed that the expression of the tapetum developmental gene UDT1 was significantly increased in osgalt anther at stage 6.At stages 8,9 and 12,the expression levels of TDR,EAT1 and Os TDD involved in the tapetum apoptosis cascade were significantly reduced.These results indicate that the Os Gal T is likely to be involved in the regulation of tapetum development and degradation.