Cloning And Functional Analysis Of A Protein Phosphatase 2C Gene (IrisPP2C1) In The Flower Of Iris Tectorum And Genome-wide Analysis Of PP2C Family In Arabidopsis

A major mechanism of signal transduction is reversible protein phosphorylation mediated by protein kinases and protein phosphatases, involved in regulating many biological processes. Protein phosphatases (PPs) function in protein phosphoregulation and are involved in diverse environmental stress responses and developmental processes. To date, a genome-wide overview of the PP2C gene family in plants is not yet available.The availability of the complete Arabidopsis genome sequences allows a timely, systematic genome-wide comparative analysis of gene families of dicots. In this study, we performed multiple BLAST algorithms to search against the corresponding data set using the known PP2C proteins got from several database. Then, we eliminated the proteins that did not contain the PP2C domains after detecting by SMART and Pfam. And also, we studied PP2C gene family of Arabidopsis by using bioinformatic technology and methods.A PP2C gene IrisPP2C1 (Iris tectorum Protein Phosphatase 2C 1, Eu366253) was isolated from the flowers of Iris (Iris tectorum). To study the function of IrisPP2C1, The recombinant construction of the IrisPP2C1 and GFP fusion gene was introduced into onion (Allium cepa) epidermal cells. The results showed that he IrisPP2C1-GFP fusion protein accumulated mainly in the nucleus. Then, transgenic Arabidopsis plants with IrisPP2C1 gene were identified by using PCR method. Finally, the function of transgenic homozygotes was tested. The main results are as follows:1) According to the conserved motifs of plant protein phosphatase 2C gene, degenerate oligonucleotides were designed. A full cDNA sequence of PP2C gene from Iris tectorum flower was cloned by RT-PCR. It had 1198 bp and coded 393 amino acid residues. The accession number is EU366253 in GenBank. The results of BLAST showed that IrisPP2C1 had the common conserved domains of PP2Cs. 2) Southern blot showed that the IrisPP2C1 gene was a low copy in the Iris tectorum genome, and there was probably a small PP2C gene family. Northern blot showed that the expression of IrisPP2C1 gene was induced by ABA in Iris flowers.3) To examine its subcellular localization, IrisPP2C1 was fused in frame to the 5'terminus of the green fluorescent protein (GFP) reporter gene under the control of the cauliflower mosaic virus 35S promoter (CaMV 35S). The recombinant constructs of the IrisPP2C1 fusion gene and GFP alone were introduced into onion (Allium cepa) epidermal cells. The result indicated that the IrisPP2C1-GFP fusion protein accumulated mainly in the nucleus. Thus, IrisPP2C1 is a nuclear-localized protein, which is consistent with software predication.4) The coding region of IrisPP2C1 was introduced into the vector pBI121 under the control of the CaMV 35S promoter and then transformed into wild-type Arabidopsis (Col-0) by floral dip method. Transgenic plants with IrisPP2C1 gene were identified by PCR technology. The transgenic plants showed dwarf phenotype, smaller leaves and dark green color compared with wild-type Arabidopsis plants.5) To investigated the sensitivity of 35S::IrisPP2C1 Arabidopsis in response to ABA during germination and root growth, exogenous ABA (0, 2, 5, 10μM) were added in culture medium. The results revealed that exogenous ABA inhibited the germination of Arabidopsis seeds of both wild type and the transgenic plants compared with those of untreated. However, the inhibitory effects on the transgenic lines were more obvious than that on the control. Similarly, ABA at 5~10μM also inhibited the root growth of both the wild type and the transgenic plants, but the transgenic plants were much sensitivity to ABA than the wild type plants. These results indicated that over-expression of IrisPP2C1 gene in Arabidopsis endowed the plant with more sensitivity to exogenous ABA.6) 80 PP2C family genes were identified by using multiple BLAST algorithms from the Arabidopsis genome.7) The phylogenetic tree of PP2C gene family was generated with the neighbor-joining method. Based on the statistical support of each branch, we divided the Arabidopsis PP2C family into 13 subfamilies, which were supported by the analysis of gene structures and protein motifs. Further study indicated that an ancestral set of PP2C genes defining each subfamily already existed before the dicotyledon divergence.8) Twenty motifs were identified among related proteins within the PP2C family using the MEME motif search tool. Some motifs were widespread among PP2C proteins and the others were specific to only one or two subfamilies. The different motif distribution in the respective protein sequences may provide an anchor to study the divergence of gene function in different subfamilies.