Analysis Of The Expression Of MtNSN1 In Medicago Truncatula And Its Roles In Regulating The Growth Of The Transgenic Plants

Nucleostemin(NS),a nucleolar GTP-binding protein widely present in animals,is involved in embryogenesis and cell proliferation,which are heavily dependent on cell cycling.Arabidopsis Nucleostemin-like1(NSN1)has been reported to function in both shoot and root apical meristem by maintaining cell cycling.Loss-of-function mutant nsn1 exhibited severe growth retardent in root and aerial organs,causing a significant loss of biomass.To investigate the effect of Medicago truncatula Nucleostemin(Mt NSN1)on yield,this thesis mainly focused on the expression pattern of Mt NSN1 and its function in the transgenic plants by bioinformatics and molecular biology.The main results are as follows:1.Cloning and bioinformatic analysis of Mt NSN1 from M.truncatula: The full-length c DNA of Mt NSN1(2193 bp)was cloned with an open–reading-frame of 1800 bp encoding 599 amino acids of 133.7 k Da.The secondary structure of Mt NSN1 predominantly consists of alpha helix(44.57%)and random coil(41.57%).Phylogenetic analysis showed that Mt NSN1 is closely related to NSN1 in Glycine max with an identity of 66.67%,and 63.53% identical to Arabidopsis NSN1.Mt NSN1 was predicted to locate in nucleus,and it was grouped into the Rbg A/Ylq F/Yaw G GTPas family due to the presence of an MMR/HSR1 domain.2.Expression analysis of Mt NSN1: Transcriptional analysis of Mt NSN1 showed that the highest transcript level was detected in flowers,followed by roots,and the lowest in leaves,which is about 10% of the flowers.44 Mt NSN1 signal was also observed in the apical meristem and the elongation zone of root.The expression pattern of Mt NSN1 implies that Mt NSN1 may involve in the maintenance of shoot and root apical meristem of M.truncatula,which is crucial for the development of both underground and aerial organs.3.Transformation of Mt NSN1 and the phenotypic analysis of the transgenic Arabidopsis: Morphologic analysis using scanning electron microscopy(SEM)showed that constitutive expression of Mt NSN1 in nsn1 mutant partially rescued the phenotype of fewer leaf primordiain in nsn1.Mt NSN1 overexpressing plants had longer primary roots,and the transgenic plants possessed more leaves than the non-transgenic ones,resulting in slightly improved growth of the seedlings.These results suggested that the expression of Mt NSN1 in meristem may contribute to its roles in regulating plant growth.4.Mt NSN1 affected the expression of genes regulating cell cycling and meristem maintenance: Realtime q PCR analysis showed that cell cycling marker genes including CYCA2;3,CYCB1;1,CYCD3;1,H4 and RNR were significantly up-regulated in Mt NSN1 overexpressing plants.Treatment with the genotoxic agents demonstrated that the length of the primary roots of Mt NSN1 overexpressing plants was longer than that of the non-transgenic control,indicating weaker repression by the reagents on the transgenic plants.Moreover,compared with nsn1 mutant,the transcriptional level of WUS and CLV3,two meristem marker genes,were slightly up-regulated in partially complemented nsn1 plants.These findings suggested that ectopic expression of Mt NSN1 altered the transcriptional level of both the cell cycling genes and meristem maintenance genes,implying the critical roles of Mt NSN1 in plant growth and development by regulating cell cycling.