Functional Study Of OsLG And OsGT43B Genes In Rice(Oryza Sativa L.)

Spikelet is an important organ in the process of rice reproductive growth,affecting the yield andthe quality of rice.The identification and functional analysis of spikelet mutant is beneficial to further elucidate the molecular mechanism of spikelet development in rice.In this study,a long sterile lemma mutant oslg was obtained and and analyzed through EMS treatment of jopanica rice Napponbare.The sterile lemma of oslg is similar to the lemma,instead of the normal sterile lemma,showing a tendency to lemmatization.Two pieces of sterile lemma were evolved from two sterile flowers of the lemma.The OsLG gene was mapped between SSR markers AT117 and AT4 within a physical interval of 95.6 kb on chromosome 7 by using a BC1F2 population.In this region,there was a G1 gene reported previously that inhibited sterile lemma into lemma.Sequence alignment showed the G1 alleles mutated in four varieties with long sterile lemma(oslg,FeiJiDao,XiaoFengGu,ShuangErDuoHongGu),including oslg mutant,that caused the changes of encoded protein.Therefore,the OsLG was considered an allele of G1 that encodes a member of DUF640 family,unique in plants.The OsLG-GFP fusion protein was localized in the nucleus of both rice protoplasts and tobacco cells.Transcriptional assay revealed that OsLG transactivated the expression of a reporter gene in the yeast GAL4 system.Dual-luciferase reporter assay showed the expression level of LUC activated by OsLG was more than four times that of the control in rice.RNA-seq analysis indicated that OsLG regulates the expression of the MADS-Box genes and DUF640 domain genes and it was further confirmed by qRT-PCR analysis.KEGG analysis indicated that the MADS-Box genes and DUF640 domain genes involved in the MAPK signaling pathway.It suggests OsLG might play an important role in the development of sterile lemma through MAPK signaling pathway.The glycosylation of glycosyltransferase in plants can produce a cascade effect that changes the biological activities and functions of plant compounds,thus affecting plant growth and development.We identified the function of the OsGT43B(Os03g0287800)gene in the glycosyltransferase family(GT,EC 2.4.x.y)and investigated the regulation mechanism of OsGT43B on grain shape and yield.Compared with the wild type(Dongjin),the grain length,grain width,grain thickness,1000-grain weight and brown rice rate of osgt43b mutants decreased significantly.We hypothesized that the rice glycosyltransferase gene OsGT43B affects rice yield by affecting grain length and grain width.qRT-PCR analysis showed that OsGT43B was highly expressed in rice grain,followed by panicle and root.Subcellular localization analysis showed that the OsGT43B::GFP fusion protein was mainly located in the Golgi apparatus.The upstream gene of OsGT43B,OsKNOX is a transcription activator located in the nucleus.Transcription factor OsKNOX can activate up-regulated expression of OsGT43B gene.Using agrobacterium-mediated genetic transformation,we constructed 4 types of transgenic rice plants with overexpression of OsGT43B in the background of indicarice varieties 93-11 and 93·11-gl3 NIL and fixed-point editing materials obtained by CRISPR/CAS9,respectively.