Molecular Mechanism Of Chloroplast Development And Chlorophyll Biosynthesis Mediated By DGP1 Gene In Rice

Photosynthetic efficiency is an important factor affecting rice yield.Previously research in this laboratory,found a dark green panicle mutant dgpl in rice,its panicle color and leaf color were dark green,and the photosynthetic efficiency was significantly increased.The DGP1 gene was isolated by map-based cloning and the function of the encoded protein was unknown.On this basis,this study used RNA-Seq and bioinformatics analysis to screen downstream genes that were regulated by the DGP1 gene and RT-qPCR and Western blot were used to detect the expression of photosynthetic related genes and protein accumulation,respectively.Then the chloroplast development was analyzed by chloroplast autofluorescence and electron microscopy.The transcriptional activity of DGP1 promoter was also analyzed.The molecular mechanism of DGP1 gene-mediated chloroplast development and chlorophyll synthesis was described.First,by using RNA-Seq analysis technique and statistical analysis of bio-information,genes that were up-or down-regulated in the dgpl mutant were screened for and compared to genes that were up-or down-regulated in wild-type.Comparing the two,190 candidate differentially expressed genes were obtained.Among them,125 genes had the highest expression in dgpl,followed by wild-type and lowest in overexpression lines.65 genes were the lowest in dgpl,the wild type higher,and the highest in overexpression lines.In order to further confirm the function of candidate differentially expressed genes and the involved biological metabolic pathways,after GO analysis,it was found that candidate differential genes were mainly concentrated in photosynthesis-related sites such as photosystem(I and II),chloroplasts,and thylakoids;After KEGG analysis,the highest enrichment rate of photosynthetic genes was found.Then,the screened photosynthetic related genes were verified by RT-qPCR.The results showed that the expression of 28 photosynthetic related genes were the same as that of RNA-Seq analysis.The protein accumulation of photosynthetic proteins was detected by Western blotting.It was found that the photosynthetic proteins Lhcal,Lhca3,Lhca4,Lhcb2 and so on had a high protein accumulation in the dgpl mutant,compared to the wild type accumulation,and the overexpression accumulation was lower than that of wild type.In order to study the effect of DGP1 gene on chloroplast development,the protoplast morphology of rice was observed by laser confocal microscopy imaging.The dgpl mutant had more chloroplasts,larger morphologies,and when compared with wild-type chloroplasts.The numbers of chloroplasts in overexpression were only a few and the morphology was small.At the same time,chloroplast electron microscopy analysis showed that the dgpl mutant had thicker capsules in the body,the wild-type internal capsules were sparsely packed,and the over-expression line had relatively thin internal capsules.In the coleoptile,dgpl forms chloroplasts earlier than wild-type and over-expression lines.The transcriptional activity of the DGP1 promoter fragment was analyzed,with the promoter fragment being analyzed first.The fragmented DGP1 and Green fluorescent protein(GFP)fusion expression vectors were constructed separately,and green fluorescence was observed in tobacco leaf epidermal cells.It was found that the DGP1 promoter has transcription activity from-195 to-1352 bp.It lays the foundation for the subsequent screening of DGP1 gene transcription factors.In summary,mutation or overexpression of the DGP1 gene results in significant changes in the expression and protein accumulation of photosynthetic related downstream genes,and the development of chloroplasts is also affected.These results laid the foundation for the molecular mechanism study of DGP1 gene and highlight efficiency breeding of plants.