| Rice is one of the most important crops in the world.Moreover,rice is the staple food of more than half of the popuLation in China.Seed dormancy and germination are two key physiological processes in the life cycle of seed plants,which are reguLated by various environmental factors and endogenous signals.Good seed germination can ensure that the growth of seedlings is consistent and robust,and also with a better tolerance to biotic or abiotic stresses.Thus it will lay an important foundation for the high yield of crops.Pre-harvest sprouting(PHS)is a special germination phenomenon,which means seeds germinate on the mother plant before harvest.PHS is a big problem in crop production all over the world.Once the temperature and humidity is high,the phenomenon of PHS will be more serious,thus leading to a great loss in rice yield and quality.At present,the research progress of the molecuLar mechanism on rice PHS is still limited,and elite genes that can be used to improve rice tolerance to PHS are still rare.Therefore,it is important to clone novel genes with PHS resistance and further dissect its molecuLar mechanism,which will be helpfuL for the cuLtivation of new rice varieties with PHS resistance.Previously,our group screened and selected a number of important genes with specific or high expression in rice seeds,and their corresponding mutants were generated via CRISPR/Cas9 technology.In 2018,the phenomenon of rice PHS was serious in Yangzhou.Interestingly,the PHS of oserf44 mutant was less serious than that of the wild type control in the field.Based on the fact that oserf44 mutant has an impressive PHS resistance and also OsERF44 is a novel gene,this study focused on revealing its expression and function as well as its molecuLar mechanism on reguLating rice PHS and seed germination.In addition,the effects of OsERF44 on rice yield and quality are also explored.The detailed resuLts are as follows:1.Bioinformatics analysis shows that OsERF44 protein contains a conserved AP2 domain,which belongs to the DREB subfamily of plant-specific AP2/ERF transcription factor family.The resuLts of promoter analysis showed that the OsERF44 promoter contained several cis-acting elements related to plant hormone reguLation,endosperm development and stress resistance.2.OsERF44 showed a constitutive expression pattern in rice.In more detail,its expression was low in the root,stem,leaf and sheath of rice,but high in the developing seeds.Transcriptional activation activity assay in yeast showed that OsERF44 protein had strong transcriptional activation activity.Then the OsERF44 protein was truncated into two parts,OsERF44△N and OsERF44ΔC.Further analysis indicated that the self-activation activity of OsERF44 depended on its C-terminal.3.Several homozygous oserf44 mutants were obtained by screening and identification of the transgenic rice with OsERF44 gene edited.In this study,three representative mutants,including erf44-2,erf44-3 and erf44-5,were selected and used the following analysis.After ripening of rice,the major panicles of the erf44 homozygous mutants and its wild type control were collected and used for PHS assay in the artificial climate room with high temperature and humidity.The resuLts were consistent with that of the previous PHS assay in the field.In addition,the seed germination of the mutants was also significantly slower than the control under normal germination conditions.In addition,we also created the OsERF44 overexpression lines and 10 independent T0 transgenic seedlings have been obtained.4.The resuLts of agronomic traits analysis showed that OsERF44 mutation had no significant effect on plant height,tiller number,length and width of flag leaf,but notably reduced panicle length and seed setting rate,and also smaller grains.The resuLts of rice quality analysis showed that the physicochemical properties of rice also remarkably changed,including increased AC,decreased GC and altered DSC.5.Hormone treatment experiments showed that ABA inhibitor couLd restore seed germination of erf44 mutant better than GA.At the same time,a large number of ABA-related genes were identified in the co-expression network of OsERF44,including OsABI5,the core element of ABA signaling pathway.Further analysis of the co-expression network of OsABI5 and OsERF44 showed that there were 132 common genes.Based on this,we specuLate that OsERF44 and OsABI5 may work in the same reguLatory pathway.In consideration that a large number of ABRE binding sites in response to ABA exist in the OsERF44 promoter,we proved that OsABI5 can inhibit the expression of OsERF44 by using the tobacco dual fluorescence system.6.In order to further uncovering the mechanism of OsERF44 in reguLation of seed germination,we first analyzed the expression of genes related to ABA synthesis,metabolism and response in erf44 mutants by qRT-PCR.The resuLts showed that the expression of all these genes increased in the mutants,suggesting that OsERF44 reguLated seed germination is at least partially mediated by ABA.In addition,we also identified OsERF44 interacting proteins using the yeast-two-hybrid library system.So far,we have obtained nearly 200 positive yeast clones,which will be sequenced and verified for protein interaction in vivo and in vitro later. |
