Functional Verification And Transcriptional Regulation Of Seed Dormancy Gene SD7-1 In Rice(Oryza Sativa L.)

The seed dormancy of rice is an important agronomic characters.too strong or too weak dormancy is not conducive to rice production.The molecular mechanism of rice seed dormancy is still unknown.Previously,the S10 with stable phenotype of seed dormancy was found in previous work.On this base,we constructed F₂population by S10 and background huajingxian 74（W0）.Bulked segregant analysis was used to mapped candidate genes,and the mutant was used to validate function of target gene.RNA-seq and q PCR was performed for researching gene transcription regulatory networks of SD7-1,and verified the breeding value of SD7-1 by pyramiding breeding.The main results are as follows:1.BSA mapping were carried out by four mixed pools constructed by S10,W0 and plants with extreme phenotype in F₂population.The region of the target gene was reduced to 1.46 Mb in chromosome7,including 33 genes with different sequences.The target gene was Os07g0170800,which was determined by referring to the region of fine mapping of SD7-1 in previous research.2.The mutant of Os07g0170800 was used to verify gene function.There is an insertion about 7.5 kb in the 5’UTR region,which resulted in gene no expression.The germination rate of the mutant was significantly higher than that of the wild type in both field and artificial simulation,and the dormancy of the mutant seeds was weakened.In addition to the seed dormancy,other agronomic characters has changed such as plant height,ear length,seed setting rate due to no expression of the gene.3.In order to study the transcriptional regulation mechanism of SD7-1 affecting seed dormancy,RNA-seq was performed on the seeds of S10 and W0 imbibed for 24 h after heading for 30 days.KEGG enrichment analysis showed that the differential genes were mainly concentrated in carbon metabolism,amino acid metabolism and so on.Furthermore,quantitative PCR was used to detect the expression of differentially expressed genes in the transcriptome at 0,12,24,36,48,60 and 72 hours after seed germination.In the pathway of amino acid metabolism,the expression patterns of lactate dehydrogenase gene（Os02g0105400）and cysteine synthase genes（Os04g0165700 and Os06g0149700）are consistent with the transcriptome,and the expression in S10 during seed germination is significantly lower than the control W0.In the pathway of carbohydrate metabolism,transcriptome results showed that the expression ofα-amylase precursor（Os08g0473600）,α-amylase（Os08g0473900）,β-amylase（Os07g0543100）andβ-glucosidase（Os09g0490400）closely related to seed germination were significantly higher in W0 than in S10,but no similar results were detected by quantitative PCR.In the phytohormone signaling pathway,the ethylene receptor gene Os ETR4 and the PP2C gene Os PP108 in the ABA pathway were significantly higher in S10 than in W0.The expression level of auxin aminosynthetic enzyme gene Os GH3.13 in S10 was significantly lower than that in W0,which was similar to the transcriptome results.4.To evaluate the breeding value of SD7-1,the single segment substitution line material KH92 containing LTG3-1 and KH30 containing SD7-1 were polymerized to obtain double segment homozygous KH1.The dormancy of seeds was estimated during harvesting,and it was found that the germination of KH1 was significantly lower than that of KH92,indicating that the introduction of SD7-1 can reduce the pre-harvest sprouting of low temperature resistant materials.After 50°C for 7 days to break dormancy,seeds were germinated at normal temperature（30°C）,low temperature（15°C）,and osmotic stress（1%and 3%mannitol solution）conditions.The germination rates of KH1 were significantly higher than that of KH30 in all conditions,which proved that the introduction of LTG3-1could improve seed germination speed.In summary,this study conducted a functional verification and transcription mechanism study on the seed dormancy gene SD7-1,identified Os07g0170800 as a gene controlling rice dormancy,and initially revealed the transcriptional regulation mechanism of the SD7-1 affecting seed dormancy.The breeding value of SD7-1 was evaluated,and new materials with both pre-harvest sprouting resistance and low-temperature germination resistance were cultivated.