Molecular Characterization, Expression, And Genetic Transformation Of Isopentenyl Transferase Gene From Rice And Mitogen-Activated Protein Kinase Kinase Gene From Cotton

Senescence is a sequential process of the function decline during the natural growth in plants. The initiation of senescence and the corresponding progression in plants largely determine the life span of the organs and the whole plant, which further exert potential affection on the yield and quality of the crops. So far, the studies of isopentenyl transferase (IPT) gene and mitogen-activated protein kinase kinase (MKK) gene that derived from plant species have been reported fewer. In this study, an IPT gene from rice and a MKK gene from cotton, designated as OsIPT6 and GhMKK9, respectively, have been investigated. The main results were as follows:1. An IPT gene form rice referred to OsIPT6 has been identified based on the NCBI website search. The cDNA full length of OsIPT6 is 885 bp, encoding 294 aa with a molecular weight 32.5 kDa and a isoelectric point 6.06. Phylogenetic analysis suggested that OsIPT6 had higher similarities with OsIPT7(AB239804),OsIPT8(AB239805)and maize ZmIPT8 (EU263131).2. With the growth progression of the leaves, the expression of OsIPT6 was increased. This suggested that OsIPT6 was regulated by the growth phase and senescence degree. Exogenous 6-BA could reduce the expression level of OsIPT6, whereas ABA could promote much more OsIPT6 transcripts. Therefore, the expression of OsIPT6 was not only controlled by the growth progression, but also affected by 6-BA and ABA.3. Based on the cloned promoter region of OsIPT6, the binary expression vectors that fused the OsIPT6 promoter and a series of segments of this promoter for controlling the reporter gene GUS expression have been constructed. The conserved motifs such as TATA box and CAAT box that generally locate at the upstream of the translation start codon ATG have also been identified in OsIPT6 promoter. In addition, the ABA responsive element, ABRE motif (consensus sequence with TACGTG), was figured out at two positions of 59 and -977, suggestive of their putative roles on responding to ABA signaling of OsIPT6.4. Using the DNA recombinant technique, the binary expression vector that fused the OsIPT6 open reading frame has been constructed. The transgenic tobacco plants integrated OsIPT6 open reading frame have been generated via Agrobacteria tumeficiens mediated transformation approach. Studies of OsIPT6-1 and OsIPT6-2, two transgenic lines with different expression levels of the target gene, demonstrated that OsIPT6 plays important roles on regulating the plant growth and leaf senescence. Compared to the control (CK1), OsIPT6-1, a transgenic line with none OsIPT6 transcripts detected, showed much lower leaf initiation speed and faster leaf senescence phenotype in the leaves that located at the positions of low or middle part in plants. In the meantime, the plants of OsIPT6-1 were much more dwarf than the control. It is suggested that a co-suppression probably occurs which interfere the homologous gene of OsIPT6 in the transgenic plants to be silenced. OsIPT6-2 showed high level expression of the target gene. The transgenic plants of OsIPT6-2 showed delayed leaf senescence and increased plant dry weight in contrast to the control plant. The OsIPT6-2 also had higher concentration of zeatin ribosides, an important substance of cytokinin type.5. The transgenic plants of OsIPT6-2 which had high expression level of the target gene also had higher photosynthetic pigment contents and soluble protein content, especially in the leaves at positions of mid- and lower part of the plant, and at the mid- and late growth stage. The over-oxidization degree of the cellular cytoplasm was lower in the OsIPT6-2.6. Based on sequence of the AtMKK9, a mitogen-activated protein kinase kinase that play vital roles on regulation of leaf senescence in Arabidopsis, a homologous gene of AtMKK9 in cotton referred to GhMKK9 has been cloned. The binary expression vectors fused the GhMKK9 open reading frame (ORF) with sense and anti-sense orientation have been constructed and the transgenic tobacco plants fused the binary constructs have been generated, which provide a basis for further analysis of the gene function via the transgenic approach in the future.