Cloning And Functional Analysis Of Grain Weight Genes ZmGW1 And ZmZFP2 In Maize

Maize(Zea mays L)is one of the important crops,mainly providing people with starch,protein,and industrial energy.High corn yield plays an important role in ensuring national food security and socio-economic development.Grain weight is one of the factor determaining grain yield in maize.Cloning of related genes that control grain weight helps provide a theoretical basis for high and stable corn yields.In our previous studies,three generations(RIL,F2:3 and BC2F2)derived from a dent corn inbred Dan232 with large grain and a popcorn inbred N04 with small grain was constructed to QTL with grain weight.A100-grain weight QTL q GW1 located on chromosome 1 with the highest contribution rate of24.6% was detected.In other previous researches QTLs related to grain weight were also located in this interval,reflecting that GW1 was stable across different backgrounds and environmentals.Zm ZFP2,encoding RING-C4HC3 zinc finger protein,was cloned according to EST sequences electronically mapped from four suppression extractive hybridization libraries in our previous research.This study mainly analyzes the genetic mechanism of grain weight regulation from two directions: map-based cloning and function analysis of maize 100-grain weight major gene Zm GW1;function analysis of C4HC3-RING zinc finger protein Zm ZFP2 in regulating grain size.Map-based cloning and functional analysis of grain weight major QTL GW1:1.Fine-mapped q GW1 and BC5F2 populations with 9143 individual plants and screened75 with individual plants exchange,combined with the genotype and phenotype of the exchange individual plants,q GW1 was finely mapped to the 42.1 kb region between the two In Del markers M202 and M1-93,in which the only candidate gene named Zm GW1 was found.The two selected haplotypes exchanged individual plants were backcrossed to BC6F1,and continued to self-bred BC6F2 to isolate GW1 near-isogenic lines NIL-N04 and NIL-Dan232 with the background of N04.The grain traits of NIL-Dan232 were compared with NIL-N04.The grain weight increased by 16.8%,and the grain length,grain width and grain thickness increased by 7.28%,9.29% and 4.54% respectively.2.Sequencing and comparison of near isogenic lines found that the SNP variation in the coding region of the allele Zm GW1 did not cause changes in amino acid sequence.The promoter contained 5 In Dels and 22 SNPs,and the 1.3 kb upstream of ATG contained a1033 bp indel mutation.In order to determine whether the promoter variation affects the yield,the results of association analysis of natural populations with 505 inbred lines showed that the promoter variation was related to grain weight.The sequence linkage disequilibrium(LD)analysis of 111 inbred line promoters in the associated population showed that In Del-1033 was closely linked to grain weight.The LUC/REN system promoter activity test showed that the promoter activity of the NIL-N04 haplotype was higher than that of NIL-Dan232,and the deletion of In Del-1033 reduced the promoter activity.Expression pattern analysis showed that the expression level of Zm GW1 in NIL-N04 was higher than that of NIL-Dan232 in grains at different developmental stages RNA in situ hybridization showed that Zm GW1 had the strongest signal in endosperm.3.Compared with the wild type,the three promoter T-DNA inserted mu mutants with W22 background showed that reduced expression of Zm GW1 and increased grain weight by13.59%,17.37% and 12.65%,respectively.The results of overexpression and CRISPR-Cas9 knockout verification showed that the grain weight of the three overexpression homozygous lines decreased by 14.89%,13.51%,and 11.76%,respectively;CRISPR editing screened 3different site editing knockout lines,their grain weight increased by 13.18%,13.58% and11.14% respectively.Both the result of T-DNA inserted mutants and transgenic verification lines indicated that Zm GW1 negatively regulated grain weight and seed size.4.The comparison of the dry and fresh weight of the near-isogenic lines showed that the difference between NIL-N04 and NIL-Dan232 occurred in the early stage of grain development.Comparing both cell number and cell of endosperm in the early stage of grain development,the cell number and cell size of NIL-Dan232 were significantly higher than that of NIL-N04.Zm GW1 influences grain weight and seed size through regulating cell division and expansion.Quantification of protein and starch content of mature seeds showed that the protein content in NIL-Dan232 was lower and the starch content was higher than in NIL-N04.5.Zm GW1 encoded the SINA protein,which contains the N-terminal RING-type zinc finger domain and the C-terminal SBD domain.The phylogenetic tree analysis of maize Zm GW1 had the highest homology with rice Os DIS1.Zm GW1 is subcellularly located in the nucleus,and in vitro ubiquitination indicates that Zm GW1 has E3 ubiquitin ligase activity.Yeast two-hybrid and luciferase complementation verified that Zm GW1 forms a dimer through SBD domain interaction.6.In order to reveal the molecular mechanism of Zm GW1 regulating grain weight and seed size,we used near-isogenic transcriptome and ubiquitination quantitative proteomic analysis.The results showed that the m RNA level of Zm GW1 was mainly involved in carbohydrate metabolism and small molecule metabolism,and protein level The ubiquitination pathway mainly affects the glycolysis pathway and sugar metabolism and synthesis processes.The yeast two-hybrid library screening with Zm GW1 interacting proteins showed that Zm GW1 interacted with other members of the SINA protein family,as well as the glycolytic pathway enolase and glycosyl hydrolase.Function analysis of zinc finger protein Zm ZFP2 in regulating grain size:1.Mutant zfp2 was screened in the mutant library.Zm ZFP2 encodes C4HC3-RING zinc finger protein.Compared with the wild type,the mutant had smaller grains and a34.84% reduction in grain weight.The endosperm of mutant zfp2 was significantly smaller than wild-type paraffin sections at 10 and 20 days after pollination.The starch granules in mutant zfp2 were irregularly arranged compared with wild-type,and the number of protein bodies around starch granules was reduced.Zm ZFP2 affects endosperm development and filling.The temporal and spatial expression pattern of Zm ZFP2 by RT-q PCR indicated that Relative expression level of Zm ZFP2 varied drastically among different tissues.RNA in situ hybridization showed that expression signals were detected in the endosperm,basal endosperm transfer layer BETL and embryos.2.To validate the function of Zm ZFP2,we over-expressed Zm ZFP2 and knocked out Zm ZFP2 using CRISPR-Cas9.The grain weight of three overexpression lines increased5.93%,9.82% and 6.64% compared with the negative control,and their grain width also increased significantly.Compared with the negative control,the grain weight of two Cas9-free knockout lines reduced 27.76% and 23.39% respectively,and the seeds was smaller.The results indicated that Zm ZFP2 positively regulates grain weight and seed size.3.Phylogenetic tree analysis showed that Zm ZFP2 has low similarity with GW2 in rice and maize.The C4HC3-RING domain was conserved in different crops,however the protein N-terminal was significantly different.Zm ZFP2 protein locatized in the nucleus,and Zm ZFP2 had RING-dependent E3 ubiquitin ligase activity in vitro ubiquitination.4.Transcriptome analysis of mutant zfp2 and wild-type and ubiquitination quantitative proteome analysis of CRISPR knockout mutant zfp2-cr2 and negative control were analyzed.The results showed that differentially expressed genes at m RNA level were mainly enriched in starch and sucrose metabolism,phytopathogenic bacteria interaction,MAPK signaling pathway,glycolysis pathway,etc.The protein level Zm ZFP2 influenced the ribosome,RNA transport and spliceosome pathways.For Zm ZFP2 screening the yeast two-hybrid library showed that ribosomal proteins S20-1,S3 a,L44 and transcription factors MADS1,MADS8,AP2 and other interaction proteins related to grain development.