Functional Analysis Of Seed Specific Expression Genes RAG2 And OsEn97 And Genome-wide Analysis Of Endosperm Differential Genes In Rice

Rice is one of the most important foods for human.It is mainly from the endosperm of rice seeds.The development processes of endosperm were composed of different phases including mitosis,endoreduplication,accumulation of many nutrients,as well as the programmed cell death(PCD)of starch.So far,some genes related to rice endosperm development have been reported.However,the mechanism of molecular regulation of endosperm development and the accumulation of storage protein are still limited in rice.In this study,nine genes with high expression in rice seeds(endosperm)were selected,and the transgenic materials of target genes were obtained by RNAi,CRISPR and overexpression techniques.This study focused on the function of seed allergen protein RAG2 and AUX/IAA protein OsEn97 in the development of endosperm.In this paper,the differential expression analysis of rice endosperm development-related genes was carried out to study the evolution origin and function of rice endosperm differential expression genes by comparing with endosperm expression genes in other plants.The main results are as follows:1.Creation of genetically modified material.We obtained RNAi materials of 9 genes,CRISPR materials of 3 genes and overexpression materials of 3 genes.Meanwhile we gained promoter connect GFP reporter gene of 2 genes.2.Study on the function of rice seed allergen gene,RAG2.The RAG2 protein contains 10 cysteine residues,and 19 amino acid residues homology with plant lipid transfer protein.The results of qRT-PCR and in situ hybridization showed that RAG2 was specifically expressed in the developing seeds and the highest expression was observed in 14 days after flowering.In T2 RAG2-overexpression lines,we found the seed size and 1000-grains weight were increased significantly compared with the WT,mainly include grain length,grain width and grain thickness.In T2-5 RAG2-RNAi lines,the seed setting rate and 1000-grain weight were reduced notable.The grain length,grain width and grain thickness were decreased.We also observed that RAG2-RNAi lines grains displayed higher chalkiness.Scanning electron microscopy(SEM)showed that the packaging of starch granules in the endosperm of RAG2-RNAi lines were loose,round and smaller.qRT-PCR detected that storage protein related genes(GluA,GluB,GluD and RM1)were commonly unregulated in RAG2-overexpression lines,and were reduced in RAG2-RNAi lines;lipid synthesis related genes(Os08g0510400 and Os09g0505300)were increased and decreased in RAG2-overexpression and RAG2-RNAi lines,respectively.The results demonstrated that RAG2 was critical important not only for protein and lipid metabolism but also for starch granules morphology in the endosperm.3.Study on the function of rice endosperm-specific expression gene,OsEn97.OsEn97 encodes an AUX/IAA-like protein.The analysis of qRT-PCR,in situ hybridization and pOsEn97:gfp transgenic plant showed that OsEn97 was specifically expressed in seeds and the highest expression was observed 7 days after flowering.In T2 OsEn97-RNAi lines,we found that the seed size,seed setting rate and 1000-grain weight were significantly reduced compared with the WT,and a certain percentage of shrunken seeds were produced.The results of cytological observation showed that the number of endosperm cells was significantly decreased from 3 to 7 days after flowering,and a certain percentage of abnormal embryos or large embryos appeared.Flow cytometry showed that the number of endosperm cells in 7DAP was reduced.Evan's blue detected PCD(Programed Cell Death)process was occurred earlier.The reduced expression of OsEn97 resulted in the expression of cell cycle(E2Fs)and storage(GluA?GluB and GluD)related genes were down,and the PCD related genes were up.Six candidate proteins interacting with OsEn97 were obtained through the yeast two-hybrid library screening.Those results indicate that OsEn97 may be involved in the development of rice seed and the accumulation of endosperm storage substances.4.Analysis of differentially expressed genes in endosperm of rice.Based on the rice chip data CREP,439 differentially expressed genes were screened,including 213 specific highly expressed genes and 226 specific lower expressed genes.Basic bioinformatics data of these genes including TIGR accession number,KOME number,SMART/Pfam domain,chromosomal location,ORF length,and BAC/PAC names were obtained from MSU,KOME and Pfam.Chromosomal localization found that the distribution of 439 genes present disorder.Blast of rice endosperm differentially expressed genes was carried out in Arabidopsis,poplar,maize,sorghum and wheat.The results showed that the number of homologous genes in monocotyledon plants,including maize(319 homologous genes),sorghum(339 homologous gene)and wheat(304 homologous genes)were significantly more than those of the dicotyledon plants,such as Arabidopsis(175 homologous genes)and poplar(195 homologous genes).These 439 genes can be roughly classified into 3 major types:conserved endosperm genes,monocots endosperm-specific genes and rice endosperm-specific genes according to the distribution of homologous genes.Analysis of the expression profiles of homologous genes in Arabidopsis and maize showed that endosperm development-related genes changed during the evolution of monocotyledons and dicotyledonous plants.A part of genes functioned conservatively to control the development of endosperm in mono-and dicotyledonous plants,and another part of genes has generated new function.In rice,some new genes may be involved in the regulation of endosperm development during evolution.