| The giant embryo rice is a mutant with a larger embryo tissue.Because of its rich in γ-aminobutyric acid(GABA),vitamin E,unsaturated fatty acids and minerals and other nutrients,now the world has been concerned about,and has a very high market value and development prospects.At the same time,the generation of giant embryo mutant phenotype also provides a good research material for exploring the development of rice embryos from molecular mechanism.It has been found that auxin plays an important role in the development of plant embryos and organs.The content of endogenous auxin(IAA)in the seed of different developmental stages of ‘Shangshida No.5’ rice(giant embryo mutant)and ‘Chao 2-10’ rice(control rice)was studied in this study group.The results showed that the IAA content of the seeds was higher than that of the control ‘Chao 2-10’ from 6 day after flowering to 18 day after flowering.The IAA content of two rice largest difference in 10 day after flowering(10 DAF),and then the difference gradually became smaller,until the IAA content of the ‘Shangshida No.5’ was reduced to the same level as the control in 18 day after flowering(18 DAF).At present,there are reports on cell morphology,identification of nutrient and gene cloning of giant embryo rice at home and abroad,but there are few reports on the reason of IAA content change during seed development of giant embryo.In this study,we analyzed the causes of changes in the content of endogenous auxin in the early and middle development stages of the seed of giant embryo rice(giant embryo mutant ‘Shangshida No.5’ rice)and its normal embryo control(‘Chao 2-10’ rice),and the role of auxin-early response gene in the formation of giant embryos.The main findings are as follows:By Heatmap gene expression profiles cluster analysis and q RT-PCR of auxin synthesis-related genes,the expression levels of Os YUCCA9,Os YUCCA11 and Os TAR1 were higher in the two tested rice seeds.The results further confirmed that these three genes were important genes for IAA synthesis in rice seed development.Comparing the two rice q RT-PCR results showed that the expression levels of Os YUCCA11 and Os TAR1 were significantly up-regulated in the ‘Shangshida No.5’ seeds at 6 DAF to 10 DAF,and the expression levels of these two genes in 10 DAF were significantly higher than Normal embryo ‘Chao 2-10’ control rice.Furthermore,the embryos and endosperm were isolated from two rice cultivars.Although the relative expression level of the Os YUCCA1 gene was lower in the whole seed,q RTPCR analysis showed that the Os YUCCA1 gene was higher in the embryo than the endosperm,and ‘Shangshida No.5’ in the expression level was significantly increased;Os YUCCA9,Os YUCCA11 and Os TAR1 were mainly expressed in endosperm,and the expression trend was consistent with that of whole seed.According to the results of the analysis of auxin synthesis-related gene expression,it was speculated that the significant increase in IAA content in the ‘Shangshida No.5’ seed of 6 DAF to 10 DAF may be due to the up-regulation of Os YUCCA1,Os YUCCA11 and Os TAR1 expression levels.In order to explore the reason that the IAA content of ‘Shangshida No.5’ seed decreased gradually from 10 DAF to 18 DAF until the same level as that of normal embryo rice,the GH3 gene family(auxin-early response gene)members were analyzed by q RT-PCR.The results showed that the expression levels of GH3-2,GH3-4 and GH3-8 in the seed of ‘Shangshi No.5’ were significantly up-regulated,indicating that some GH3 gene family members has responded to high IAA content of the ‘shangshida No.5’ seed.The GH3-8 gene encodes an amino synthase that binds to free IAA and reduces endogenous IAA levels.The expression trend of GH3-8 gene was highly consistent with the trend of IAA content in 10 DAF to 18 DAF seeds of ‘Shangshida No.5’.The GH3-8 expression was significantly up-regulated at 10 DAF,and was significantly higher than that of other GH3 genes.The results showed that the decline of IAA content in ‘Shangshi No.5’ seed from 10 DAF to 18 DAF could be due to the role of GH3 gene family members in balancing the endogenous somatotropin mechanism of giant embryo rice seed,which may be the GH3-8 is a relatively more important member of the gene.The Aux/IAA gene family(auxin-early response genes)and the auxin response factor(ARF)gene family are important participants in the auxin signaling pathway.In this study,31 Aux/IAA gene family members and 25 ARF gene family members were analyzed by Heatmap cluster analysis.Twelve Aux/IAA genes and eight ARF genes were screened out,and further expression analysis was performed using q RT-PCR.The results showed that IAA4,IAA9,IAA26,ARF11,ARF15 and ARF17 were upregulated in ‘Shangshida No5’,while IAA14,IAA29 and ARF14 were down-regulated in "Shangshi No.5".This suggests that the high IAA content in "Shangshida No.5" 10 DAF seed may have caused auxin signaling pathway involved.Interestingly,the IAA29 gene of the two tested rice plants had the highest relative expression in all the selected Aux/IAA genes,and the expression level of the IAA29 gene was significantly decline in the ‘Shangshida No5’ seed.It is speculated that IAA29 may play an important role in the formation of rice giant embryos.In this study,we further performed the transgenic experiment for exploring the function of Os IAA29 and Os GH3-8 by CRISPR-Cas9 gene editing technique.On the other hand,we carried out related bioinformatics analysis on these two genes.Os IAA29 and Os GH3-8 were all highly expressed genes in the seeds,and Os IAA29 was specifically expressed in the endosperm;The subcellular localization of Os IAA29 was predicted in nuclear and it a nuclear localization gene,and Os GH3-8 was predicted in the cytoplasm.The sequence comparison of 31 Aux/IAA proteins in rice showed that the Os IAA29 protein had no domain I,complete domains III and IV,and the domain II did not appear to be completely absent;The phylogenetic tree analysis of Aux/IAA protein family of Arabidopsis,maize,sorghum and rice showed that Os IAA29 was closely related to Os IAA28,followed by maize Zm IAA29,but there was no direct homologous gene in Arabidopsis thaliana.;The phylogenetic tree analysis of 13 GH3 proteins in rice showed that the Os GH3-8 protein belongs to class II,which is consistent with the reported results.Now,two CRISPR positive transgenic regeneration seedling of Os IAA29 genes have been obtained.The results obtained in this study have laid an important foundation for the further study of the relationship between auxin-related genes and rice giant embryos phenotype. |
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