| Ribulose-1,5-bisphosphate carboxylase/oxygenase(Rubisco) is a pivotal enzyme that initiates the first step of carbon fixation and photorespiration. Although Rubisco accounts for up to half of the soluble protein in the leaves of plants, its carboxylation efficiency is still low. Activity of Rubisco is an important factor for the efficiency of carbon assimilation, but it is regulated by Rubisco activase (RCA). RCA is a nuclear-encoded soluble chloroplast protein, it is essential for the activation of Rubisco and hydrolysis of ATP. The RCA plays an important role in regulating plant growth and development, and has been the focus of agricultural bio-engineering since first reported in Arabidopsis.In the present study, the RCA gene was cloned based on the maize inbred B73whole genome sequence. The relationship between peptides and genes was investigated using Western blot, MALDI-TOF/TOF and specificity antibody preparation. Transiently expressed GFP-protein fusions were conducted to definitude the sub-cellorientation of Zmrcal and Zmrca3. Expression of RCA genes was assayed by Real-time RT-PCR and RT-PCR. Correlation and regression analysis were conducted to further elucidate the relationship between RCA genes and maize yield. The results may provide theoretical basis for the use of the genes in maize breeding. The main findings were as follows:1. We identified two maize RCA genes on chromosome4. These two genes contained different ORFs. The one with the shorter ORF was the previously reported gene Zmrcal, whereas the other with the longer ORF had never been reported in maize. We tentatively designated this new maize RCA gene as Zmrca3. Zmrcal contains1299bp open reading frame encoding a mature protein of381amino acids, with a calculated molecular mass of42.70kD. In contrast, the Zmrca3contains1389bp open reading frame encoding a mature protein of411amino acids, with a calculated molecular mass of45.96kD.2. Protein expression assays suggested that both Zmrca1and Zmrca3could contribute to the in vivo RCA polypeptide in maize. The recombinant proteins of50.1kD and52.8kD were detected by MALDI-TOF/TOF. The leaf proteins of43.8kD and46.7kD were detected by MALDI-TOF/TOF. The Zmrcal and Zmrca3corresponded to short and long polypeptide respectively.3. The temporal and spatial expression characterizations were investigated by semi-quantitative RT-PCR and Real-time RT-PCR. The Zmrcal and Zmrca3transcripts accumulated mostly in leaves and, to a lesser extent, in stems, tassel spikelets, and immature ears, whereas no or a very low signal was detected in roots. During the dark period, transcripts of the Zmrcal and Zmrca3gradually increased, with the highest levels detected at8:30in the morning. Following this peak, the accumulation of these two transcripts declined gradually, reaching their lowest levels at20:30. Zmrcal and Zmrca3were expressed at a relatively low level in the early growth stages of maize, up to tasselling, but with more accumulation during the grain filling stages. Expression of the two genes could be induced by moderately high temperature.4. Subcellular localization prediction with TargetP1.1and WOLF PSORT program indicated that Zmrcal and Zmrca3were located in chloroplasts. To examine the subcellular localization of the Zmrcal and Zmrca3protein in the plant cells, we fused the coding regions of Zmrcal and Zmrca3in frame to the coding region for the N-terminal side of GFP, and the fusion gene was expressed under the control of the35S promoter of Cauliflower mosaic virus (CaMV). GFP fused to either the GFP-Zmrcal or GFP-Zmrca3proteins was detected in chloroplasts.5. In the123inbred lines, linear correlation showed that the expression levels of both genes were positively correlated with grain yield at significance levels of0.01and0.05respectively. In addition, the expression levels of Zmrcal and Zmrca3could jointly explain24%of the grain yield variation by regression analysis. |
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