Identification and characterization of Rubisco activase genes in Oryza punctata

Rubisco activase (Rca), a specific chaperone, catalyzes the in vivo activation of Rubisco, and thus plays a major role in plant photosynthesis. Although the genes encoding Rubisco activase have been studied in many model or economic plants, few studies have analyzed their homologs in the plants that closely related to crop species. In this study, an Rca gene was identified and characterized in a wild relative of rice, O. punctata Kotschy ex Steud. The gene was 2747 bp long and possessed six exons and five introns with 47% GC content. Further, cDNA sequencing produced two transcripts RcaL and RcaS that differed in the sequence by an inclusion of 99 bp at the carboxy terminus of RcaL. Sequence comparison between the two transcripts and the genomic DNA showed that there was a 20-bp alternative splicing event that occurred at the fifth intron of the gene leading to the synthesis of a short polypeptide. The leaf transcriptome analysis showed that RcaS had a higher expression level than that of RcaL in a normal growth environment. In addition, the yeast two-hybrid assays displayed the small isoform of Rubisco activase in close contact with the large subunit of the Rubisco in this species, supporting the side-on binding model of interaction between two proteins. This study broadens our understanding of the molecular characteristics of some essential genes in photosynthesis.

核酮糖-1,5-二磷酸羧化酶/加氧酶激活酶（Rubisco activase，Rca）是一类特异性分子伴侣，可催化核酮糖-1,5-二磷酸羧化酶/加氧酶（Rubisco）的体内激活过程，因此在植物光合作用中发挥关键作用。尽管目前已在多种模式植物与经济作物中对编码Rca的基因开展了研究，但针对作物近缘植物中其同源基因的分析却较为匮乏。本研究在水稻的野生近缘种Oryza punctata Kotschy ex Steud.中鉴定并表征了一个Rca基因，该基因全长2747 bp，包含6个外显子与5个内含子，GC含量为47%。进一步的cDNA测序结果显示，该基因存在两种转录本RcaL与RcaS，二者的序列差异在于RcaL的羧基末端多包含一段99 bp的序列。对两种转录本与基因组DNA的序列比对结果表明，该基因的第五内含子处发生了一段20 bp的可变剪接事件，由此产生了较短的多肽产物。叶片转录组分析结果显示，在正常生长环境下，RcaS的表达水平高于RcaL。此外，酵母双杂交实验结果显示，该物种中Rca的小亚型可与Rubisco的大亚基紧密结合，这一结果支持了两种蛋白质之间以侧对侧方式结合的相互作用模型。本研究加深了我们对光合作用中部分关键基因分子特征的认知。