| Glyoxalase system is a ubiquitous group of enzymes in prokaryotes and eukaryotes,which plays an important role in defense against abiotic stresses.Our laboratory group using the method of comparative proteomic,screened a rice seed storability-related protein glyoxalase I(OsGLYI3),suggesting that it might play an important role in seed storage.Hence,molecular biology,genetics and bioinformatics studies were comprehensively applied to identify the functions of OsGLYI3,and study its relationship with seed longevity and vigor,and abiotic stress tolerance such as salt and drought.This study will provide new candidate gene resources for further select and breed rice varieties with storability,salt and drought stress tolerance.The results were as follows:1.Analysis of the transcriptional level of OsGLYI3 by q RT-PCR and GUS staining observation revealed that OsGLYI3 was highly expressed in the seeds,suggesting that OsGLYI3 was a seed-specifically gene and might be involved in seed development.The YFP sequence was fused to the C-terminal of OsGLYI3,OsGLYI3-YFP fusion protein was visualized under a confocal microscope and showed that OsGLYI3 protein was localizated in the cytoplasm.2.Full-length cDNA of OsGLYI3 was cloned into the p Cambia1390 expression vector to generate 16 transgenic rice lines with overexpressed OsGLYI3(TR-OE).Screening for three independent overexpression transgenic T2 homozygosis lines(OE#6,OE#11,and OE#13),showed that OsGLYI3 was upregulated by 7.5-,3.6-,and 14.4-fold,respectively,compared to WT plants.The vector construct of the OsGLYI3 CRISPR/Cas9 were introduced into Agrobacterium tumefaciens strain EHA105 and transferred into the WT,and21 lines(TR-C9)were obtained.Sequencing analysis showed that the editing efficiency of CRISPR/Cas9 gene was 66.7%,and CR#1,CR#11,and CR#15 chosen from TR-C9 homozygous knockout plants with different editing sites for further study.3.Compared to wild-type plants,overexpressed transgenic rice lines exhibited increased GLYI activity and decreased methylglyoxal(MG)levels,while TR-C9 lines showed decreased glyoxalase I activity and increased MG levels,suggesting that rice OsGLYI3 confers MG detoxification.Overexpression of OsGLYI3 increased seed germination rate and vigor index,while TR-C9 lines accelerated seed vigor loss in TR-C9 lines.After seed aging treatment,SOD and POD activities of TR-OE seeds were higher than WT seeds,while SOD and POD activities of TR-C9 seeds were lower than WT seeds,suggesting that OsGLYI3 regulated seed germination percentage may be related to the antioxidant enzyme system.4.The transcription level of OsGLYI3 was analyzed by q RT-PCR,and the results showed OsGLYI3 was up-regulated under the stress such as Na Cl and Mannitol.TR-OE seedlings displayed higher growth rates than WT based on shoot height and fresh weight data when they germinated and grown on MS media with salt and mannitol stress treatment.Inversely,seedlings of TR-C9 showed lower growth rates in shoot height and fresh weight than in WT in response to the salt and mannitol stress treatment.Meanwhile,MDA content was significantly lower in TR-OE lines but higher in TR-C9 lines compared to WT plants.Under leaves salt treatment,TR-OE compared to WT showed increased salt stress tolerance,and higher levels of chlorophyll contents;while TR-C9 compared to WT showed more sensitive to salt stress andsignificantly lower chlorophyll contents.OsGLYI3 overexpressed lines increased salt stress tolerance under soil salt treatment.CRISPR/Cas9 knockout lines were more sensitive to salt stress.Under soil salt or drought treatment,OsGLYI3 overexpressed lines increased the salt/drought stress tolerant,CRISPR/Cas9 knockout lines were more sensitive to salt/drought stress.The results showed that OsGLYI3 was related to salt and drought tolerance.In summary,our results provide evidence that OsGLYI3 is specifically expressed in rice seed and contributes significantly to seed longevity and abiotic stress tolerance. |
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