Cloning And Functional Study Of Dense And Short Panicle Gene DSP1 In Rice(oryza Sativa L.)

China is an important food production base in the world,and the production level of rice(Oryza sativa L.)is closely related to people’s quality of life.Grain number per panicle,1000-grain weight and effective panicle are three key factors of rice yield.In addition,excellent plant type is also crucial to the improvement of rice yield.For example,appropriate plant height and stem strength can enhance the lodging ability of rice,and appropriate tiller angle can increase the photosynthesis of plants.Therefore,high-yield and high-quality varieties have become the breeding target of breeders.CRISPR/Cas9 a novel genome editing method,has gradually entered the limelight,it has a broad application prospect in plant genetic improvement and breeding research.In previous work,we identified a small panicle and grain mutant spg1 through Ethyl Methan Sulfonate(EMS)mutagenesis screening.Further evolutionary analysis revealed that there is a homologous gene LOC＿Os04g19140 of SPG1 in rice.To better investigate the functions of this gene in rice,we used gene editing technology CRISPR/Cas9 to perform targeted editing of the gene LOC＿Os04g19140 and obtained its knockout mutant,which we named dsp1(dense and short panicle 1)according to the phenotype.Afterwards,we carried out phenotypic observation and agronomic character analysis,and used modern biological techniques for explored and identified its function in rice.The main findings are as follows:1.Bioinformatics analysisGene evolution analysis found that DSP1 and SPG1 belonged to homologous genes in rice.The sequence of DSP1 was analyzed by the protein function analysis website Inter Pro Scan 5,and it was found to have a Ribonuclease H-like(RNase H-like)domain that locates in the 367-586 range of its amino acid sequence,and it is a member of the RNase H-like protein family.According to the analysis results of the structural domain,we used the website http://www.bio-soft.net/sms/ to conduct multiple sequence alignment,and found that the RNase H-like domain has a certain conservatism.2.Phenotypic observation and agronomic character analysisField observation and statistics showed that compared with wild type,the panicle length of dsp1 was shorter,the primary branch had no difference,but the secondary branch and the number of grains per panicle increased significantly,and there was no significant difference in the number of effective panicles,and the yield per plant of dsp1 increased.In addition,dsp1 had no difference in grain length and grain width compared with wild type,but its grain thickness decreased,leading to a decrease in 1000-grain weight.Observation statistics found that the stem of dsp1 was thicker.At the same time,the tiller angle of dsp1 increased.We took the wild-type and mutant tiller bases at heading stage to observe by SEM and found that the cells on the near side of dsp1 were significantly elongated compared with the far side,and the cells on the near side of dsp1 were significantly longer than those of the wild type.Compared with the wild type,the leaf surface of dsp1 was smoother during growth and development.SEM was used to observe the leaf trichome number of wild-type and mutant at maturity stage,and found the trichome number of dsp1 leaf was significantly reduced.RT-qPCR analysis found that the expression levels of WOX3 B,HL6,and SDG714,which positively regulate rice trichome development,were significantly down-regulated in dsp1.3.Histocytology AnalysisPhenotypic observations revealed that dsp1 was more upright compared to wild type after maturation.Paraffin section statistics of the stem showed that the number of cell layers of dsp1 significantly increased,the cells became smaller and the number of cells increased.In addition,in view of the decrease of grain thickness of dsp1,we conducted paraffin sections on the glume of spikelet at booting stage,and the results showed that the number of glume cells of dsp1 was not significantly different from that of the wild type.4.RT-qPCR analysis of genes related to grain number and grain developmentIn view of the significant increase in grain number of dsp1,we extracted RNA from panicle to conduct RT-qPCR analysis of grain number-related genes,and found that compared with the wild type,the expression levels of Ghd7,Gn1 a and qEPD2 genes were extremely significantly increased,while the expression levels of lax panicle gene LAX2 were significantly decreased.RT-qPCR of grain development-related genes found that,except for the auxin primary response gene BG1,other grain development-regulated genes including GS5,GW2,GW5,GW7,TGW2,PGL1,and PGL2 were not different.5.Analysis of expression patternsThe wild-type RNA was extracted from each tissue site at the heading stage.RT-qPCR analysis showed that DSP1 was expressed in rice roots,stems,leaves,sheaths,and panicles,and the highest expression was in young panicle(panicle length < 0.5 cm).The results of GUS staining showed that GUS signals could be detected in roots,stems,leaves,sheaths,tiller buds,branch stalks,panicle developmental stages,and grain developmental stages,which also indicated that DSP1 was a gene expressed in multiple sites.Subcellular localization found that DSP1 was located in the nucleus,and it was also found that DSP1 was expressed in the nucleus at the subcellular level by detecting the fluorescent signal of the young root of the p TCK303-DSP1-GFP positive transgenic line.6.Determination of auxin content and RT-qPCR analysis of auxin pathway related genesThe auxin content of young panicles of dsp1 was significantly higher than that of wild type,and the auxin synthesis related genes YUCCA1,YUCCA2,YUCCA3 and YUCCA4 in young panicles of dsp1 were significantly higher than that of wild type of the same period.The expression levels of auxin transport-related genes PIN1 d,PIN2,PIN5 b,PIN8,PIN10 a and PIN10 b were also significantly higher than those of wild type,and the auxin signal response genes ARF1 and ARF11 were also significantly different.7.Detection of DSP1 self-activation and toxicity to yeastDSP1 does not have the ability to activate itself and is not toxic to yeast cells.