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- The OsAGO7 gene is the first isolated gene controlling upward curling of leaves in rice. It belongs to the AGO family because of its PAZ and PIWI domains（Fig2）. AGO is a large protein family that constitutes essential components of RNA-induced silencing complexes (RISCs), and their proteins are characterized by two unique domains, PAZ and PIWI (Carmell et al. 2002)
- In order to analyze the biological function of the OsAGO7 gene, Shi et al. joined a 223 bp fragment from the PAZ domain and a 510 bp fragment from the PIWI domain to the p1301UbiNos vector, in which there would be anti-sense segments expressed by the strong,Ubi promoter. The two resulting vectors—pAntiPAZ and pAntiPIWI—were transformed into the wild-type Zhonghua No. 11 by agrobacterium-mediated transformation.They obtained a total of 107 T0 transformants,with 80
being derived from pAntiPAZ and 27 from pAntiPIWI. In these transformants, we did not detect any obvious phenotype modiWcations in the T0 generation and the T1 generation.
- Since no phenotype changes in those anti-sense transformants were detected, we constructed an over-expression vector pCod, in which the whole OsAGO7 ORF was inserted into the 3’ end of the Ubi promoter. After pCod was transformed into the wildtype Zhonghua No. 11, we found 58 T0 transformants, 52 (90%) of which exhibited the rolled-leaf trait. Furthermore, LRIs in the transformants were generally over 45 (Fig. 5a). Some exceeded 100, that is, the two margins of the leaves turned inside to overlap each other so that the whole leaf formed a cylinder (Fig. 5b).
- To verify that the transformants were derived from different transformation events, we sampled several transformants with the rolled-leaf trait and isolated the Xanking sequences of inserted T-DNA left border by TAIL-PCR. This analysis conWrmed that T-DNA was inserted into diVerent sites in diVerent transformants. In order to further investigate if the insertion of T-DNA co-segregated with the rolled-leaf trait in the OsAGO7 over-expression transformants, we sampled three lines with the rolled-leaf trait and diVerent T-DNA integration sites. Seeds were planted and raised to the T1 generation, and individuals were analyzed through GUS staining and PCR detection, where two pairs of primers—P3/Ubip and GusP+/GusP—were used (Fig. 3b). The results showed that the rolledleaf trait completely co-segregated with the integration of T-DNA.
- RT-PCR analysis of the OsAGO7 gene expression :Seedling leaves from the wild type and the mutant R05 were used in semi-quantitative RT-PCR analysis to check the expression of the OsAGO7 gene. The samestage regenerated leaves of T0 generation of overexpression and anti-sense transformants were sampled.The expression of OsAGO7 in mutant R05 was higher than in the wild type (Fig. 5c), although expression of OsAGO7 in both samples was low and Northern blot showed no signal. In the transformants derived from the vector pCod, the expression of OsAGO7 was much higher in plants with rolled leaves than in those without.In the transformants derived from anti-sense vectors pAntiPAZ and pAntiPIWI, the expression of OsAGO7 was also low. Thus, it was apparent that the expression of OsAGO7 was correlated with the rolledleaf trait
- Function of OsAGO7 gene and its expression. a The transformants of OsAGO7 over-expression showed the rolled-leaf trait. b Some overexpression transformants showed the cylinder leaves.c RT-PCR analysis of the OsAGO7 gene expression.Different primers were used in‘a’ and ‘b’ groups (see method);‘WT’ refer to the wild type; 1–5 refer to the pCod transformants: agcod19,agcod1, agcod2, agcod4, agcod5,respectively, among which agcod1 was the Xat-leaftrait and the others showed the rolled-leaf trait; 6 and 7 refer to pAntiPAZ and pAntiPIWI transformants: antipaz1, antipiwi15, respectively,both of which show the Xat-leaf trait; 8 refers to the wild-type genomic DNA as the control of PCR
- OsAGO7 gene is an ortholog of Arabidopsis ZIPPY(ZIP/AGO7)：We searched the Uniprot database (UniProt Release7.5) for the homologues of the rolled-leaf protein and found 18 proteins of rice with the PAZ and PIWI domains. We suggest that there were at least 18 members of AGO family in rice. In Arabidopsis, the AGO family has ten members (Fagard et al. 2000; Carmell et al. 2002). Based on the alignment of the amino acid sequences, we analyzed the evolutionary
relationships among rice and Arabidopsis AGO proteins. Phylogenetic analysis reveals that the whole proteins fall into three clades. The rolled-leaf protein was involved into the clade III. It was high similar to ZIP from Arabidopsis, and we could not Wnd another protein grouped into this subclass. This suggests that the protein may represent orthologs of Arabidopsis ZIP; therefore we called its gene OsAGO7. In Arabidopsis,the function of ZIP is to regulate the juvenile to adult transition in leaf development (Hunter et al. 2003).
Labs working on this gene
Labs working on rice argonaute gene（not just OsAGO7 ）
- National Key Laboratory of Plant Molecular Genetics,Institute of Plant Physiology and Ecology,Shanghai Institutes for Biological Sciences
- Interdisciplinary Centre for Plant Genomics and Department of Plant Molecular Biology, University of Delhi South Campus
- State Key Laboratory of Genetic Engineering, Institute of Plant Biology, School of Life Sciences, Fudan University
- Department of Biology, The Huck Institutes of the Life Sciences, Pennsylvania State University
- Cold Spring Harbor Laboratory
- Program in Genetics, State University of New York at Stony Brook
- 1. ZhenYing Shi;Jiang Wang;XinShan Wan;GeZhi Shen;XinQi Wang;JingLiu Zhang.Over-expression of rice OsAGO7 gene induces upward curling of the leaf blade that enhanced erect-leaf habit. Planta, 2007, 226(1): 99-108
- 2. Carmell MA, Xuan ZY, Zhang MQ, Hannon GJ (2002) The Argonaute family: tentacles that reach into RNAi, developmental control, stem cell maintenance, and tumorigenesis. Genes Dev 16:2733–2742
- 3. Hunter C, Sun H, Poethig RS (2003) The Arabidopsis heterochronic gene ZIPPY is an ARGONAUTE family member.Curr Biol 13:1734–1739
- 4. Nonomura K I, Morohoshi A, Nakano M, et al. A germ cell–specific gene of the ARGONAUTE family is essential for the progression of premeiotic mitosis and meiosis during sporogenesis in rice[J]. The Plant Cell Online, 2007, 19(8): 2583-2594.
- 5.Kapoor M, Arora R, Lama T, et al. Genome-wide identification, organization and phylogenetic analysis of Dicer-like, Argonaute and RNA-dependent RNA Polymerase gene families and their expression analysis during reproductive development and stress in rice[J]. BMC genomics, 2008, 9(1): 451.
- 6.Carmell M A, Xuan Z, Zhang M Q, et al. The Argonaute family: tentacles that reach into RNAi, developmental control, stem cell maintenance, and tumorigenesis[J]. Genes & development, 2002, 16(21): 2733-2742