Molecular Cloning And Functional Analysis Of OsBIPP2C1 And OsBIPP2C2, Two Novel Protein Phosphatase 2C Genes In Rice

In the previous work of our lab, over 200 differential expressed cDNA from rice induced by benzothiadiazole (BTH) were cloned and identified through suppression subtractive hybridization. Among them, HIBI-n9 and BIHI-w2 with inserts of 414 bp and 755 bp, respectively, showed significant similarities to genes encoding known plant phosphatase 2C proteins. In order to elucidate the biological function of these two putative phosphatase 2C genes and their role in rice disease resistance, this study cloned and identified full-length cDNA of the genes and designated as OsBIPP2Cl and OsBIPP2C2, respectively.The full-length cDNA of OsBIPP2Cl was isolated through a combination of virtual cloning and RACE. Rice ESTs matching to BIHI-w2 sequence were retrieved from dbEST of GenBank database and a single long sequence was assembled. The assembled sequence is of 1900 bp long and contains an open reading frame (ORF) of 1710 bp, which predicts to encode a protein of 569 amino acids with a calculated molecular weight of 58.7 kD and the isolectric point of 4.1. A signal peptide is predicted to locate from the first amino acid to the 39th amino acid at the N-terminus of the OsBIPP2Cl protein. The predicted protein sequence contains a protein phosphatase 2C domain, approximately 255 aa in length at its C terminus. In this domain, most of the amino acids that are required for binding Mg2+/Mn2+ ions to maintain catalytic activity were identified. The OsBEPP2Cl shares weak sequence identity to other PP2Cs identified so far, showing 13-16% of identity to Arabidopsis ABI2 and AtPP2CA, tobacco NtPP2Cl and Medicago MP2C. So it is a new protein, we called it OsBEPP2Cl (Oryza_sativa L. BTHjnduced p_rotein phosphatase 2C 1, OsBIPP2Cl). The OsBIPP2Cl gene exists as single copy on the rice chromosome 3. As there is no phosphatase 2C gene identified in rice so far, we therefore concluded that OsBIPP2Cl is a novel gene encoding phosphatase 2C in rice.OsBIPP2Cl was rapidly activated as soon as 6 hr after treatment by spraying with 0.3 mM BTH solution, maintained a relatively high level within 24 hr, and thereafterdeclined gradually to the normal level at 72 hr after treatment. Inducible expression of OsBIPP2Cl was detected as early as 6 hr in BTH-treated rice seedlings after inoculation with the blast fungus, Magnaporthe grisea. This inducible expression was maintained at a relatively low level within the first 24 hr after inoculation. The significant induction was observed during the second 24 hr and then declined to normal level. In contrast, no obvious fluctuation of OsBIPP2Cl gene expression was detected in water-treated rice seedlings after inoculation. Meanwhile, OsBIPP2Cl was only slightly up-regulated by treatment with SA, but was strongly induced by 5 mM H2O2 within 12 hr after treatment. Moreover, OsBIPP2Cl was also found to be induced by salts, heavy metals and ABA, implying its multiple roles in stress acclimation. This is a common characteristic of some other stress-responsive PP2Cs such as ABI1 and ABE.Coding sequence of OsBIPP2Cl was cloned to downstream of CaMV-35S promoter in the plant binary vector CHF3. Constructed vector was transformed into tobacco by Agrobacterium-mediated leaf disc transformation. A total of 13 transgenic lines were obtained through screening of antibiotic resistant re-generating seedlings and PCR detection. Northern blotting analysis showed that OsBIPP2Cl was expressed in most of the transgenic lines. Preliminary results showed the Tl plants of the OsBIPP2Cl-overexpressing lines enhanced the resistance to tobacco mosaic virus (TMV) and tobacco blank shank (Phytophthora parasitica var. nicotianae) diseases. Moreover, the transgenic Tl plants also showed an enhanced tolerance to salt and osmotic stress. The Tl generation of the transgenic lines showed a variable sensitivity to ABA as illustrated by germination rate of the seeds and seedling growth under ABA treatments.Another PP2C gene was cloned by a similar way to that used in isolation of OsBIPP2Cl and was named as OsBIPP2C2 (Oryza...