水稻磷胁迫响应基因OsRCI2-9的克隆及功能鉴定

doi:10.3864/j.issn.0578-1752.2015.05.01

摘要/Abstract

摘要： 【目的】分析水稻OsRCI2-9的功能，进而解析其耐低磷胁迫的作用机制，以利于磷高效作物的改良与选育。【方法】通过对比分析水稻磷信号中心调控因子OsPHR2超表达（OsPHR2(O)）与干涉（OsPHR2(Ri)）转基因植株和野生型植株基因芯片数据，发现一个在OsPHR2(O)背景下诱导表达的RCI2家族基因——OsRCI2-9，首先利用实时荧光定量PCR对芯片数据进行验证。然后根据TIGR上的基因序列设计引物，扩增其全长cDNA；进而利用DNAStar来寻找其开放阅读框，并利用TMHMM2.0对蛋白序列的跨膜结构进行预测；在Phytozome中利用BLAST搜索拟南芥和玉米中的同源基因，并将OsRCI2-9与拟南芥RCI2、玉米RCI2家族的蛋白序列利用Clustal X 1.8进行多重序列比对和同源性分析；利用MEGA 5.2.1软件的最大似然法对同源蛋白构建进化树；利用植物顺式作用元件数据库PLACE分析启动子序列中的顺式作用元件；利用RT-PCR和实时荧光定量PCR检测OsRCI2-9在不同胁迫处理条件下的表达模式；将OsRCI2-9的cDNA序列连入带有CaMV 35S启动子的pCAMBIA1300表达载体中构建其超表达载体并发展转基因材料，对其表型进行分析，并对转基因植株的有效磷进行测定。【结果】OsRCI2-9位于水稻第6染色体上，基因全长237 bp，由2个外显子和1个内含子组成，编码78个氨基酸。OsRCI2-9含有2个跨膜结构域，其在序列上与玉米ZmRCI2-2、ZmRCI2-7和ZmRCI2-8同源性较高；通过启动子序列分析发现OsRCI2-9启动子含有干旱、低温，ABA、细胞分裂素、赤霉素及磷饥饿响应等元件；RT-PCR和实时荧光定量PCR发现OsRCI2-9受到缺磷处理的强烈诱导，在根中OsRCI2-9还受到缺钾和缺铁的少量诱导及缺氮的微量抑制。进一步研究发现在正常磷浓度下，OsRCI2-9超表达转基因植株与野生型相比，表现为生长缓慢，分蘖数减少，而在低磷条件下，表型与野生型差别不大。通过测定有效磷含量发现OsRCI2-9超表达转基因植株在正常磷浓度与缺磷条件下较野生型植株有效磷含量均有明显的提高。【结论】OsRCI2-9参于了植物体内磷平衡调控。

关键词: 水稻, RCI2家族, 胁迫响应, 磷

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

任鸿雁，魏强. 水稻磷胁迫响应基因OsRCI2-9的克隆及功能鉴定[J]. 中国农业科学, 2015, 48(5): 831-840.

REN Hong-yan, WEI Qiang. Isolation and Functional Analysis of Phosphate-Responsive Gene OsRCI2-9 in Oryza sativa[J]. Scientia Agricultura Sinica, 2015, 48(5): 831-840.

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