Functional Analysis Of Seed Dormancy Gene Sdr4 And Gene Cloning And Functional Analysis Of Dormant Mutant H470 In Rice(Oryza Sativa L.)

Seed dormancy and germination is a complex and important agronomic character and controlled by the major genes+multiple minor genes,involving many physiological processes and energy metabolism pathways.The high dormancy of rice will lead to low and irregular seedling emergence in the field,which is not conducive to the growth of direct seeding cultivation population.However,if the dormancy level is too low,the phenomenon of pre-harvest sprouting is easily caused by cloudy and rainy weather during the maturity period,which reduces the yield and quality of rice,seriously affected the seed value.Therefore,it is meaningful to study the molecular mechanism of seed dormancy and to cultivate rice varieties with moderate dormancy.In this study,two genes which controlled seed dormancy were studied.The first part is "Functional analysis of seed dormancy gene Sdr4 in rice".The second part is "Gene cloning and functional analysis of rice dormant mutant h470".Part I:Sdr4(Seed dormancy 4)gene specifically in rice embryos regulates seed dormancy.Previous studies have only cloned the gene,but the Sdr4 gene encodes proteins still unknown,and its regulatory mechanism for seed dormancy has not been studied.In this study,the function of Sdr4 was deeply explored,and the main results are as follows:1.In N22,a highly dormancy indica cultivar,cr-sdr4 was obtained by using CRISPR/Cas9 gene editing technology,which knocked out Sdr4.There were two knocked out genotypes,both of them caused early termination of protein translation.Compared with the wild type,the dormancy of cr-sdr4 was basically lost and the embryo became significantly larger.The alpha-amylase activity was significantly increased.2.Compared with the wild type,cr-sdr4 was not sensitive to low concentrations of exogenous ABA.The expression level of ABA metabolism related genes significantly reduced,which resulted in a significant increased of endogenous ABA.3.The amino acid sequence and homologous protein evolutionary tree analysis of Sdr4 showed it did not contain known domain and had low conservatism with proximal species.The results of subcellular localization showed that it was located in the nucleus,and the transcriptional activation experiments showed that it had no transcriptional activity,might not be a transcription factor.4.Further identification and analysis of the reported dormancy-related genes revealed that the expression of VP1(Viviparous 1,ABI3 transcription factor,participated in the ABA signaling pathway)was significantly reduced in cr-sdr4.The analysis of full length 2 Kb promoter region of Sdr4 shows that it contains seven ABRE elements,which reported are the binding elements by VP1.The transgenic material cr-VP1 was obtained by using CRISPR/Cas9 technology which knocked out VP1.The phenotype of cr-VP1 was consistent with cr-sdr4,which dormancy was greatly reduced.5.The yeast one-hybrid and luciferase assay(LUC)was used to demonstrate that VP1 can bind to full length 2 Kb promoter,particularly bound to 2 Kb promoter’s P1 and P6 segments,positively regulate the expression of Sdr4.6.Screening of the yeast library identified a protein that may interact with Sdr4,named OsERF2(Ethylene response factor 2,a downstream component of ethylene signaling pathway).The interaction among Sdr4 with OsERP2 was confirmed by yeast two-hybrid.Previous studies have shown that the expression of OsERF2 is positively correlated with sugar contents.We suspected that Sdr4 may interact with OsERF2 to regulate the accumulation of energy and carbohydrate in seeds,finally affected seed dormancy.The second part:The main results of mutant h470 are as follows:1.A weakened dormant mutant h470 was screened from the 400Gy 60Co radiation-induced N22 population,its dormancy was significantly reduced.At different stages of seed development and storage,the germination rate of h470 was higher than that of wild type.2.Compared with the wild type,the sensitivity of h470 to exogenous GA and ABA was no significant difference.The endogenous GA4 and ABA levels of h470 were significantly increased,and the expression levels of related genes were consistent with the results.3.The reciprocal cross F2 population was constructed by using N22 and h470,30 plants with high germination and 30 plants with low germination were selected for the whole genome sequencing of mut-map.The results showed that there was a T base deletion on Os02g0202400 gene of chromosome 2.Combined with the analysis of bioinformatics and NCBI database sites,Os02g0202400 encodes an ADP-glucose transporter protein which containing 425 amino acids,annotated as OsBT1.The mutation site is located on the first exon and causes early termination of translation.OsBT1 was the cause of the decreased dormancy of h470 by the germination experiments of transgenic complementary,RNA interference and allele mutant osbt1 materials.4.In h470,the expression of some dormancy genes were analyzed,found that the expression level of alpha-amylase genes were significantly increased.The determination of alpha-amylase activity of h470 showed that the alpha-amylase activity was also significantly increased.5.OsBTl itself is an ADP-glucose transporter that affects starch synthesis in seeds.So,metabolomics analyses showed that most sugar components were higher and starch was significantly decreased in h470 seeds.We speculated that OsBT1 influenced the glycometabolism,and then regulated seed dormancy.