| Under water culture condition, we analyzed the effect of nitrogen forms on the growth of Malus hupenensis, on the MhIPT3 expression by quantitative real-time PCR and on the contents of Z+ZR,iP+iPA, IAA, as well as ABA by ELISA..1.In an attempt to isolate MhIPT3 gene, a 689 bp EST sequences were used to design the gene-specific primers (GSPs) for 5′RACE and 3′RACE. The entries from the apple EST database (DT001742 ), showed good alignment with the Arabidopsis thaliana IPT genes. As a result, the full-length of 1314bp of MhIPT3 cDNA sequence was obtained (DQ792508). The nucleotide of MhIPT3 contains 248-bp 5′non-coding, a 963-bp open reading frame encoding a molecular mass of 37.3 KDa protein with 321 amino acids, and a 151-bp of 3′non-coding region; The deduced amino acid sequence of Malus hupenensis MhIPT3 showed 34.22%, 53.27%, 35.34%, 55.29%, 35.11%, 47.43% and 38.14% identity to those of AtIPT isozymes from Arabidopsis thaliana, AtIPTl, AtIPT3, AtIPT4, AtIPT5, AtIPT6, AtIPT7 and AtIPT8, but exhibited only 25.10% and 25.52% identity to those of Arabidopsis thaliana tRNA IPT isozymes, AtIPT2 and AtIPT9, respectively. Malus hupehensis MhIPT3 contains GATGTGKS (amino acids number 40-47) sequence known as the ATP/GTP binding motif which is universally observed in ATP-consuming enzymes including ATP-binding cassette transporters; The MhIPT3 forms a cluster with LjIPT3, AtIPT3 and BrIPT3, but a separate cluster with other IPT enzymes including Arabidopsis thaliana tRNA (AtIPT2 and AtIPT9). The MhIPT3 appears to be evolutionary more related to AtIPT3, AtIPT5 and AtIPT7 than AtIPT4, AtIPT6 and AtIPT8. Further, MhIPT3 could be amplified from genomic DNA and cDNA of pingyitiancha, and fragment size and sequenced results of the amplification products were the same (data not shown), indicating that introns are absent in the ORF of MhIPT3. In order to determine the function of endogenous expansin MhIPT3 in growth and development of plants cell, a construct containing full-length cDNA of MhIPT3 gene in sense and atisense orientation driven by the constitutivecauliflower mosaic virus 35S promoter was assembled and introduced into tobacco and'Royal Gala'plants. An expression vector carrying full-length cDNA of MhIPT3 gene in sense orientation with vector pET-30a(+) was constructed and introduced into E.coli., then induced expression proteins from E.coli loading MhIPT3 gene.2.Quantitative real-time PCR analysis revealed that MhIPT3 gene expressed at different levels in roots, stems and leaves. When NO3- was re-supplied to nitrogen-limited seedlings, MhIPT3 transcript rapidly accumulated within 2 h. The manner of MhIPT3 induction corresponded well with that of the accumulation of iP+iPA and Z+ZR in roots. Supplementing NH4+ did not induce MhIPT3 expression. These results suggested that nitrate up-regulates expression of MhIPT3. The levels of IAA were similar between nitrate treatment and ammonium treatment. The amounts of ABA by supplied nitrate were upper than ammonium treatment in 24 hours, then decreasing lower than ammonium treatment. The leaf areas did not exist significant difference between the treatment and the control in 24 hours after nitrate supply, but the leaf areas of nitrate treatment were larger than ammonium treatment in 72 hours and 168 hours.
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