Fine-mapping Of QTGW7b For Grain Weight And Functional Analysis Of OsFTL6 In Rice(Oryza Sativa L.)

Rice(Oryza sativa L.)is an important food crop,and a high and stable yield has been the goal of breeders.Grain weight is an important component of yield,and exploring of new QTLs controlling grain weight and functional analysis of genes are essential to improve grain yield.At present,mapping and cloning of grain weightrelated QTLs are still major ones,and little research has been conducted on minor QTLs.In addition,PEBP family genes play an important role in rice growth and development,but the study of its family members is not yet complete.With the development of functional genomics and molecular genetics,especially the application of CRISPR/Cas9 technology,it has become possible to perform targeted modifications of genes and dissect gene functions.In this study,a set of chromosomal fragment substitution lines with Nipponbare as the recipient parent and 9311 as the donor parent was constructed in the previous study,and the grain size of the substitution line N83 was found to be slightly larger than that of the recipient parent Nipponbare.N83 was backcrossed with Nipponbare.The secondary separation population was constructed for fine mapping of QTL qTGW7b.PEBP proteins are a multigene family of proteins that are involved in several growth and developmental processes,including plant flowering,plant architecture,and seed germination.In this study,a rice FT-like gene OsFTL6(LOC_Os04g41130)was the target,and the knockout mutants were constructed by CRISPR/Cas9 approach.We screened to obtain transcriptional regulatory proteins of OsFTL6 and analyzed the role of these genes in rice seed germination regulation by molecular genetics and plant physiology method.The main results are as follows.Part 1 Fine-mapping of qTGW7b for grain weight in rice1.Compared with Nipponbare,the grain length,grain width and 1000-grain weight of N83 at maturity was significantly higher than those of Nipponbare,and there was no difference in grain thickness.Scanning electron microscopy of the seed glumes revealed that the N83 glume cells became longer and wider without differences in cell number,which was presumed to be the direct cause of the larger grain size.2.The BC9F2 segregating population was constructed by backcrossing N83 with Nipponbare.The mean values of grain length,grain width and 1000-grain weight of the Nipponbare homozygotes in the segregating population were significantly lower than those of the 9311 homozygotes.3.Eleven pairs of molecular markers within the mapping interval were developed,and qTGW7b was primarily mapped between markers G7-31 and G7-32 with a physical distance of about 651 kb by creating introgressive segments lines and map-base cloning.The BC9F2:3 population was used to screen for substitution lines,and combined with phenotypic and genotypic analysis,qTGW7b was finally fine mapped between markers G7-72 and G7-32 at a physical distance of about 86.2 kb.4.According to RGAP rice gene annotation analysis,there were 14 genes in the interval.Sequencing analysis revealed that mutations in the coding regions of ORF3,ORF6,ORF9,ORF10,ORF11,and ORF14 in 9311 resulted in altered amino acid sequences compared to Nipponbare.The expression analysis of the candidate gene revealed that the transcription of ORF3,ORF9,ORF10,ORF11,and ORF14 was highly significantly different between Nipponbare and N83.Part 2 Functional Research of OsFTL6 in Rice1.There are 19 PEBP genes in rice,including 13 FT-like genes,4 RCN genes,and 2 MFT-like genes.Phylogenetic tree,protein structural domain,and gene structure analysis showed that the 19 PEBP proteins were divided into 3 subfamilies,among which 13 FT-like proteins were divided into 3 branches.OsFTL6,like the other 13 PEBP proteins in rice,contained a conserved PEBP structural domain with 4 exons and 3 introns,and the promoter contained more hormone and stress response components.2.The qRT-PCR analysis revealed that OsFTL6 had the highest expression in the leaf sheaths,and higher expression was also detected in the stem and leaves,with lower expression in the roots and spikes.The tissue GUS staining results and qRT-PCR results were consistent,indicating that OsFTL6 was constitutively expressed.OsFTL6 protein was distributed in both cytoplasm and nucleus.OsFTL6 was induced by hormones and abiotic stresses.ABA and NaCl could induce up-regulation of OsFTL6 gene expression3.Under normal conditions,the germination rate of osftl6 mutant was lower than that of wild type,and under ABA treatment conditions,osftl6 mutant was not sensitive to ABA and showed no difference in germination rate from wild type.Meanwhile,the NaCl treatment results were consistent with those of ABA.Starch gel assays and expression analysis of α-amylase-related genes showed that OsFTL6 positively regulated the expression of Ramy1C,Ramy3D,and Ramy3E to affect seed germination.4.The expression trends of OsbZIPs and OsFTL6 during germination were opposite,and the expression of OsFTL6 decreased and that of OsbZIPs increased with increasing ABA concentration.Dual luciferase reporter system showed that OsbZIP10 and OsbZIP23 could combine to negatively regulate the expression of OsFTL6.Yeast one-hybrid,EMSA and ChIP-qPCR assay demonstrate that this interaction is through binding to the ABRE motif of the OsFTL6 promoter.OsbZIP10/23 could not interact with OsFTL6,while OsbZIP10 could interact with OsbZIP23.5.Compare with the wild type,the germination rate of OsbZIP23-OX and OsbZIP10-OX was lower,and that of the osbzip23 mutant was slightly higher than that of the wild type under normal conditions,while under ABA conditions,the osbzip23 mutant showed insensitivity to ABA with a higher germination rate than that with the wild type,while the OsbZIP23-OX and OsbZIP10-OX were ultrasensitive to ABA and the gap of the germination rate with that of the wild type was reduced.The above results indicated that ABA could activate the expression of several OsbZIP genes including OsbZIP10 and OsbZIP23,binding and negatively regulating the expression of the downstream gene OsFTL6,which acts as a positive regulator of seed germination and participates in positively regulating the expression of α-amylaserelated genes to regulate the seed germination process.