Isolation, Molecular Clone, Characterization And Functional Analysis Of An Aquaporin (JcPIP2) In Jatropha Curcas

Jatropha curcas L, a Euphorbiaceae plant species, thrives in many tropics and sub-tropics areas. It is heavily resistant to drought and can be planted even in desert areas. In order to explore the molecular basis of drought resistance in Jatropha curcas L, we isolated an aquaporin from Jatropha curcas (JcPIP2). It is considered that aquaporins are related to drought tolerance in plant though the relationship between aquaporins and drought resistance still remains elusive.1. Isolation and molecular clone of JcPIP2Plasma membrane proteins were purified by aqueous polymer two-phase partition method. From plasma membrane proteins of Jatropha curcas, a 29kD band was isolated belonging to plant plasma membrane intrinsic proteins (PIPs) by LC-MS/MS analysis. Degenerate primers were designed according to the peptide: NPYNHLLGGGANSVNTGYSK. The cDNA was obtained by RT-PCR and RACE. The exon material of JcPIP2 contains a coding sequence of ORF (open reading frame) composing of 843 nucleotides. 5' and 3' untranslated sequences of JcPIP2 gene were 39bp and 103bp separately. The length of DNA of JcPIP2 ORF was 1273bp including three introns. GenBank accession No. is EF030420. 2. Bioinformatics Analysis of JcPIP2Comparison of the coding proteins showed high identity between JcPIP2 and other plant PIPs. The 94ï¼…identity was found between JcPIP2 and Ricinus communis PIP. The 86ï¼…identity was found between JcPIP2 and spinach SoPM28A whose protein structure had been reported. Analysis of predicted hydropathy plot and TMHMM posterior probabilities for JcPIP2 indicated that JcPIP2 comprised six transmembrane helices and five hydropholic loops. It showed that JcPIP2 was a model membrane protein for the high hydropholic. Prediction of three-dimensional structure of JcPIP2 showed it's similarity with the structure of spinach SoPM28A. Phosphorylation sites were analyzed to be five, and it was the basis for the study of phosphorylation regulation of JcPIP2 activity.3. Southern blotGenomic DNA was prepared from young leaves using the method of CTAB and completely digested by restriction enzymes EcoR I, BamH I, Sac I and Kpn I separately. The blot results revealed that more than two hybridized bands were found in each digested product.4. Northern and Western blotNorthern blot analysis revealed that its transcripts expressed in all tested tissues. Immunodetection of JcPIP2 with anti-JcPIP2 antibody indicated that this protein ubiquitously located in all tested tissues of the plant too.5. Analysis of JcPIP2 activityTo determine water transporting activity of JcPIP2, the cRNA of JcPIP2 was heterologously expressed into Xenopus oocytes expression system. Oocytes injected with JcPIP2 cRNA had a swelling rate 10 times higher than that in oocytes injected with water. The result showed that JcPIP2 encoded a functional water channel protein. 6. Sub-cell location of JcPIP2 in Xenopus oocytes We synthesized the JcPIP2-GFP cRNA and injected it into Xenopus oocytes expression system. The result of heterologously expression indicated that it only located on the membrane of Xenopus oocytes. It also proved that JcPIP2 was a membrane protein.7. Changes of abundance and expression of JcPIP2 under water deficitProtein levels increased in Jatropha curcas with the aggravation of the degree of drought stress. In addition, the transcripts of JcPIP2 were highly expressed in roots too.8. Phosphorylation regulation of water transport activity of JcPIP2JcPIP2 encoded a functional water channel protein as demonstrated by heterologously expressed in Xenopus oocytes. The water transport activity was regulated by phosphorylation. The phosphorylation sites were identified by mutants of JcPIP2 expression in Xenopus oocytes.