Scientia Agricultura Sinica ›› 2015, Vol. 48 ›› Issue (5): 831-840.doi: 10.3864/j.issn.0578-1752.2015.05.01

• CROP GENETICS & BREEDING·GERMPLASM RESOURCES·MOLECULAR GENETICS •     Next Articles

Isolation and Functional Analysis of Phosphate-Responsive Gene OsRCI2-9 in Oryza sativa

REN Hong-yan, WEI Qiang   

  1. College of Life Sciences, Shaanxi Normal University, Xi’an 710119
  • Received:2014-09-09 Online:2015-03-01 Published:2015-03-01

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Abstract: 【Objective】 The objective of this study is to analyze the function of OsRCI2-9 gene and reveal its mechanism of tolerance to low phosphorus stress.【Method】By comparative analysis of transcription profiles in rice transgenic plants with over expression (OsPHR2 (O)) and repression (OsPHR2 (Ri)) of OsPHR2 (the Pi-signaling regulator), OsRCI2-9 gene which belongs to RCI2 family induced under the OsPHR2 (O) background, was isolated. Firstly, the AffyMatrix data were validated by real-time PCR. The primers were designed according to the sequence on the TIGR web and its full length cDNA was amplified and the DNAStar was used to find its open reading frame. The transmembrane domain were predicted using the TMHMM2.0 software. Then the protein sequence was used to search the homologs in Arabidopsis thaliana and Zea mays from Phytozome 9 database by BLASTP program. Multiple alignment and homology analysis were performed among OsRCI2-9 and RCI2s in A. thaliana and Z. mays by usingClustal X 1.8 software and these sequences were used to construct the maximum likelihood tree by using MEGA 5.2.1. The cis-regulatory elements in promoter were searched in PLACE database. Also the expression patterns of OsRCI2-9 under various abiotic stresses were analyzed using the RT-PCR and the real-time PCR. Finally, the transgenic plants with overexpression of OsRCI2-9 driven by CaMV35S promoter was developed for phosphate starvation resistant experiment and the available phosphate were detected.【Result】OsRCI2-9 is located on chromosome 6 of O. sativa. The full-length of OsRCI2-9 was 237 bp with 2 exons and 1 intron and consisted of 78 amino acids. It was observed that the OsRCI2-9 protein has two transmembrane domains. Multiple alignments and homology analysis showed that it has a high sequence similarity with ZmRCI2-2, ZmRCI2-7 and ZmRCI2-8. It was also found that the OsRCI2-9 promoter has many cis-acting elements including the dehydration, cold, ABA, cytokinins, gibberellin signaling and phosphate starvation responsive elements. It was further revealed that the expression of OsRCI2-9 is up-regulated by phosphate starvation and also slightly up-regulated by potassium and iron starvation but down-regulated by nitrogen starvation in roots. Further analysis showed that OsRCI2-9 overexpressed transgenic plants were growing slowly and its tiller number was reduced compared to wild type under the full phosphate conditions, but no significant difference between them were detected under the phosphate starvation conditions. Measurements of the available phosphate showed that the OsRCI2-9 overexpressed transgenic plants significantly increased the soluble phosphate concentration both under the full phosphate conditions and the phosphate starvation conditions.【Conclusion】 The OsRCI2-9 gene significantly participates in the regulation of phosphorus homeostasis in O. sativa.

Key words: Oryza sativa, RCI2 family, stress responses, phosphate

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