Screening And Functional Verification Of Extra-heavy Panicle 1 Related Genes

Rice grain number per panicle is one of the key factors determining the yield.Compared with cultivated rice,the wide rice has a significantly higher grain number per panicle.Therefore,identification of the genes related to grain number per panicle in wild rice has great significance to elucidate the molecular mechanism of the formation of rice grain number.In the previous study,using wide rice and cultivated rice 93-11 as the donor and recipient parent,respectively,by hybridization,embryo rescue culture and backcross selection,a stable line with 700 gains in its main panicle?designated as extra-heavy panicle 1?was obtained.In this study,using extra-heavy panicle 1 and cultivated rice 93-11as materials,we employed RNA-sequencing to screen the gene related to extra-heavy panicle 1?EHP1R?and then clone and verify the function of the EHP1R.The results are listed as follows:?1?Results of the RNA-sequencing:Compared with the cultivated rice 93-11,2532genes were up-regulated and 1245 genes were down-regulated in extra-heavy panicle 1.GO enrichment,pathway enrichment and KEGG enrichment for these differentially expressed genes showed that the photosynthetic pathway and the photosynthesis signal transduction pathway are the most significantly enriched.Gene-sets enrichment analysis for the up-regulated and down-regulated genes by PlantGSEA showed that 10 up-regulated genes reached significant enrichment levels in photosynthetic pathways.qPCR results indicated that their expression levels were higher in extra-heavy panicle 1 than those in the cultivated rice 93-11.?2?Analysis of photosynthetic characteristics:The net photosynthetic rate,Soil and Plant Analyzer Development?SPAD?value,chlorophyll fluorescence parameters,and leaf morphology-related traits of extra-heavy panicle 1 were significantly higher than that of the cultivated rice 93-11.The intercellular CO₂ concentration was lower in extra-heavy panicle 1 than invariety 93-11.There was no significant difference in stomatal conductance and transpiration rate between extra-heavy panicle 1 and 93-11.The dry weight of fresh spikes of extra-heavy panicle 1 was significantly higher than that of rice 93-11 at the booting stage and heading stage but has no significant difference at the filling stage.The gain number per panicle was significantly correlated with the morphological characteristics of the leaf.?3?Cloning and functional verification of EHP1R gene:Sequence alignment of EHP1R between extra-heavy panicle 1 and cultivated rice 93-11 revealed 7 single nucleotide polymorphisms in the cDNA sequence.Two out of the 7 single nucleotide polymorphisms resulted in amino acid substitution.Then the over-expression vector of EHP1R was constructed and genetically transformed into the rice 93-11.the CRISPR/Cas9gene editing vector of EHP1R and EHP1R-pro::GUS fusion vector were constructed and genetically transformed into the extra-heavy panicle 1.35S::EHP1R:eGFP vector was constructed and injected into tobacco epidermal cells,and observation by confocal microscopy showed that EHP1R protein was distributed in the chloroplasts.