Preliminary Study On NAC Transcription Factors Involved In Regulating Rice Grain Filling And Seed Germination

Grain filling is one of the most important physiological processes of cereal crop rice,which directly affects the yield and quality of rice.The NAC transcription factor is a unique transcription factor in plants and one of the largest transcription factor families in plants.Its N-domain end is composed of about 140 highly conserved amino acids.NAC transcription factors bind to DNA,interact directly or indirectly with other proteins,and thus participate in the regulation of gene expression during plant life.Existing research on the function of NAC transcription factors mainly focuses on the role in stress response.The previous research of the research team proved that NAC transcription factors might be involved in the regulation of rice filling process.This study further found that NAC transcription factors are involved in the regulation of rice seed dormancy and germination.In this study,we used reverse genetics techniques to construct OsNAC2,OsNAC25 and OsNAC26 overexpression plants and CRISPR/cas9 knockout mutants,and preliminarily studied the molecular mechanism of NAC transcription factors regulating rice grain filling and seed germination.The research conclusions are as follows:(1)The results of q PCR gene expression analysis of wild-type rice Zhonghua 11 at different developmental stages showed that among the 9 NAC transcription factors discovered in the early stage of this study,OsNAC2,OsNAC25 and OsNAC26 were specifically expressed in full-head stage grains.In order to further study the functions of these NAC transcription factors,we used genetic transformation technology to construct and obtain homozygous overexpression strains OEOsNAC2,OEOsNAC25,OEOsNAC26 and knockout mutant strains cr-OsNAC2,cr-OsNAC25,cr-OsNAC26.(2)Phenotypic analysis of homozygous overexpression lines and knockout mutants showed that overexpression of OsNAC2,OsNAC25 and OsNAC26 genes can significantly inhibit grain weight and plant height.Knockout mutants OsNAC2,OsNAC25 and OsNAC26 genes Significantly increase the grain weight and plant height.These results indicate that OsNAC2,OsNAC25 and OsNAC26 negatively regulate rice plant height and grain weight.(3)The test results of seed germination traits showed that overexpression of OsNAC2,OsNAC25 and OsNAC26 genes caused a decrease in seed germination rate,and the germination rate of knockout mutants OsNAC2,OsNAC25 and OsNAC26 was not significantly different from that of Zhonghua 11.In addition,GA hormone treatment of OsNAC2 overexpression and knockout mutant lines made the seed germination rate of each line reach 100%,while ABA hormone treatment reduced the seed germination rate of each line.The results indicate that OsNAC2 is upstream of GA hormone regulation in the seed germination regulation network.(4)We performed chromatin immunoprecipitation sequencing analysis using seeds germinated for 48 hours and found that OsNAC2 was mainly enriched at the transcription initiation site,its first binding motif was AAAGAAGGTA,and the target site of the target gene was in the promoter To the 3 kb region of the transcription start site,and it may participate in the regulation of hormone metabolism pathway.In the detection of ABA hormones in seeds,the ABA content of the seeds of the overexpression line was 64.33%higher than that of the Zhonghua 11 seeds,and the ABA content of the knockout line was28.66% lower than that of the Zhonghua 11 seeds.Analysis of the gene expression patterns of OsNCED3,a gene related to ABA synthesis and OsABA8ox2,a gene related to ABA metabolism,shows that OsNAC2 positively regulates the expression of OsNCED3 gene and negatively regulates the expression of OsABA8ox2 gene.It is speculated that it may affect seed germination by adjusting the balance of ABA metabolism/ABA synthesis.To sum up,this research has passed the cloning and preliminary functional verification of OsNAC2,OsNAC25 and OsNAC26.It is proved that OsNAC2,OsNAC25 and OsNAC26 are also involved in the regulation of rice plant height and grain weight.The molecular network of OsNAC2 regulating the germination of rice Zhonghua 11 grains was preliminarily constructed.Provide new evidence for the molecular mechanism of NACs involved in regulating rice seed germination.