Functional Study On The Rice DOF Family Genes And Grain Size Gene WG7

The flowering time of rice determines the regional distribution,adaptability and yield potential of rice varieties.Panicle architecture and grain size directly determine the component traits of rice yield: spikelet number per panicle and 1000-grain weight.Functional analysis of genes regulating flowering time,panicle architecture and grain size of rice and exploitation of more excellent alleles are helpful for directional genetic improvement and molecular design breeding of rice.Only three of the rice 30 Dof genes have been confirmed to be involved in the regulation of heading date.In order to further explore whether Dof family genes have more heading date genes and excellent gene resources,the first part of this study focused on the function of rice Dof family genes.Grain size directly determines 1000-grain weight,and hence grain yield.The second part of this study focuses on the functional analysis of a rice grain size regulator gene WG7.The main results are as follows:1.Using the heading date of 529 rice germplasm resources and genotype data on Rice Var Map,the haplotype level association analysis of 30 rice Dof family genes was carried out.A total of 22 Dof genes were associated with heading date.The 22 Dof genes are classified into three types.The first type had eight Dof genes(Os Dof 6,7,10,11,13,16,20 and 21).Significant differences in heading dates were detected between/among major haplotypes of these eight genes in both indica and japonica accessions under long and short-day conditions.Os Dof1 and Os Dof12,which belong to the second type,had significant effects on heading dates in both indica and japonica rice under under long-day conditions,whereas no significant effects were observed under short-day conditions.Four Dof genes(Os Dof23,25,29 and 30)in the third type had significant effects in japonica accessions,but not in indica rice under long and short-day conditions.There was no significant difference in haplotypes of the remaining eight Dof genes.2.CRISPR/Cas9 gene editing technique was used to verify the function of 30 Dof genes.It was found that the CRISPR/Cas9 mutants of 11 genes and 9 genes had significant effects on heading date under long-day conditions and short-day conditions,respectively.Other members of the Dof family had no effect on heading date.In addition,mutants of Dof family genes also affect plant height.3.Nucleotide diversity analysis showed that the ratios of πc /πw of six genes were less than 0.5,indicating that they underwent selection during domestication and genetic improvement.Among the six genes,Os Dof2,16 and 24 regulate heading date.Tajima’s D of Os Dof7 and Os Dof21 was negative in wild rice(P < 0.05),indicating that Os Dof7 and Os Dof21 had undergone purification selection in wild rice.In addition,the Fu and Li’s D values of eight Dof genes(Os Dof 5,8,12,14,17,18,22 and 29)in cultivated rice and three Dof genes(Os Dof3,5 and 7)in wild rice significantly deviated from neutrality(P < 0.05),suggesting that these genes have undergone natural selection in wild rice and artificial selection in cultivated rice.4.We found that the CRISPR/Cas9 mutant of Os Dof15 was significantly smaller than that of wild type Zhonghua 11,especially plant height and panicle architecture.The T-DNA insertion mutant of Os Dof15 promoter has smaller plant size and shorter panicle length,so we named the gene SP3(Short Panicle 3).Transgenic complementation and CRISPR/Cas9 knockout experiments further confirmed that SP3 is Os Dof15.5.Real-time quantitative PCR and RNA in situ hybridization showed that SP3 was specifically expressed in young panicles,especially in branch primordia(including primary and secondary branches).Subcellular localization showed that SP3 was a nuclear protein,and dual luciferase transcriptional activity assay showed that SP3 was a transcriptional activator.Real-time quantitative PCR and RNA in situ hybridization showed that SP3 could regulate the expression of APO2/RFL.6.The down-regulation and up-regulation expression of genes respectively involved in cytokinin biosynthesis and degradation in sp3 mutant resulted in the decrease of endogenous cytokinin content in sp3 mutant,which was also the main reason for the shorter panicle in sp3 mutant.7.We found a T-DNA insertion mutant,named wg7(wide grain 7),with smaller plants and narrower grain size.Sequencing results showed that T-DNA was inserted into the seventh exon of LOC_Os07g47360.Genotype analysis showed that T-DNA insertion was co-segregated with grain width.Overexpression and CRISPR/Cas9 knockout experiments confirmed that LOC_Os07g47360 was the gene underlying WG7.8.The results of cross-section of spikelet hull and scanning electron microscopy showed that the difference of cell number was the main reason for the difference of grain width,and the expression level of cell cycle-related genes was also significantly different between wg7 mutant and wild type.9.Yeast one-hybrid and electrophoretic mobility shift assay showed that the CATTTC was the core cis-element for WG7 binding,and Os MADS1 is a direct downstream target gene of WG7.Dual luciferase transcriptional activity assay demonstrated that WG7 activates Os MADS1 expression by directly binding to the CATTTC element of Os MADS1 promoter.The CRISPR/Cas9 knockout of the core ciselement CATTTC confirms the authenticity of this regulatory relationship.10.WG7 encodes a cysteine-tryptophan(CW)domain-containing zinc finger protein.Chromatin immunoprecipitation and real-time quantitative PCR results show that the histone H3K4me3 level in the promoter region of Os MADS1 is significantly lower than that in the wild type.These results suggest that WG7 regulates the expression of Os MADS1 by mediating the H3K4me3 modification of Os MADS1 promoter.