| Rice is one of the most important crops in the world. The population in our country, rice yield and grain consumption per capita topped the list. Recently, the decreased of field area and the increasing extreme weather and other nature disaster spur increasing photosynthesis per leaf area. However, the key point of increasing photosynthesis is not the light reaction, but the carbon allocation and assimilation. Most of previous researchers concentrated on single gene which acted on only one site in CO2 transporting route or assimilation and gave some useful instructions. Based on the original work of previous researchers about ictB, FBP/SBPase and SBPase gene those could enhance photosynthesis and growth. In this study, ictB and FBP/SBPase related to CO2 transporting and assimilation derived from cyanobacteria were co-introduced into rice, and expected that they should act on inorganic carbon transporting and carboxylation in Calvin cycle respectively, then further increase carboxylation efficiency under natural field conditions. In this study, T5 -T7 plants obtained by self-fertilization were selected by molecular detection and investigated by physiological, biochemical and leaf anatomy analysis. The results indicated ICTB and FBP/SBPase showed synergistic interaction in carbon flow and assimilation route and then promoted photosynthetic, physiological and biochemical indexes. The main results were listed below:Cyanobacterial full-length ictB and FBP/SBPase cDNA were cloned into the vector pCAMBIA1301 containing a CaMV 35S promotor, chloroplastic transit signal sequence rbcs (ribulose bisphosphate carboxylase small subunit) and nos terminate sequences to give single (ictB and FBP/SBPase respectively) and binary (ictB-FBP/SBPase) genes overexpression recombinant plasmids, and empty construct plasmid were then transformed into Agrobacterium tumefaciens EHA105. The Oryza sativa spp. indica vs. 9311 (wild-type) was transformed with the resultant plasmids by the standard Agrobacterium mediated method as described previously and gave 56 homozygous transgenic rice lines.14 lines were selected by hygromycin, molecular and screened in paddy field and attributed into two-gene, one-gene and empty construct groups. The constructs had inserted in the genomes of transgenic lines with only one copy of each gene and correct proteins were detected by southern blot and western-blot analysis respectively.The results of physiological and biochemical analysis in paddy field indicated that mesophyll conductance, net photo synthetic rate and carboxylation efficiency in three groups were higher about 10.5-36.8%,13.5-34.6% and 21-33.9% compared to wild type respectively. The response curve of Pn to varying Ci was assessed by measuring the CO2 uptake indicating that the difference became significant between transgenic groups and WT as the Ci increased above 200 ?mol CO2 mol-1, and the changing range of Pn in ICTB, FS, ICTB+FS groups were 31.3-31.9 (mean value 31.6±0.16),33.4-34.5 (mean value 34.1±0.39) and 35.4-37.3 ?mol CO2 m-2s-1 (mean value 36.8±0.6) respectively, while that of WT and empty construct was 26.6-26.8 (mean value 26.7±0.07) and 27.3-28.3 (mean value 27.7±0.32) respectively, this indicated that the Pn of three transgenic groups were higher than that of WT about 18.4%,27.7%,37.8% at the CO2 saturated points respectively. As C, levels exceed 400 ?mol CO2 mol-1, the value of Pn tended towards to stabilization and attained saturated situation in all lines. These results suggested that the higher ability to perform maximal photosynthetic rate in transgenic groups was owing to the functions of these two genes since WT and empty construct groups had almost the same performance. The photosynthetic ability increased orderly by the expression of ictB, FBP/SBPase and ictB+FBP/SBPase which showed the function on improving photosynthetic capacity exerted by FBP/SBPase was greater than that of ictB and additive effect was observed in ICTB+FS groups (ictB+FBP/SBPase).The results of subcellular localization showed that ICTB was a membrane protein lied in plasmalemma with or without RBCS signal peptide, this indicated its natural final address was plasmalemma, and the positive roles of ICTB in transcellular carbon delivery to chloroplasts and improving gmin liquid phase, this also indicated subcellular localization of a protein is intrinsic to its function. While FBP/SBPase was localized in chloroplast and would promote CO2 assimilation in the transgenic lines based on our results, and they exerted synergetic interactions at different sites in the route of CO2 transporting and assimilation respectively thus improved the whole photosynthetic efficiency.The results of leaf anatomy properties showed that no significant difference of leaf thickness, leaf mesophyll thickness, mesophyll wall thickness, chloroplast size, chloroplasts per mesophyll area, Sc/Smes, cross-sectional area occupied by the sclerenchymatous, vascular bundle and intercellular airspace were observed between three transgenic groups, empty construct and WT. The same trend was observed for stomatal density and stomatal index which demonstrated that the target genes we introduced did not change leaf anatomical properties and the increased gm and Pn in transgenic lines did not result from the leaf anatomy properties. The expression of ictB and FBP/SBPase had also exerted positive effect on the LMA and WSC compared with that of WT and this indicated that increased LMA mainly resulted from the increased WSC, and the increased WSC produced by improved photosynthesis was not used to build the structural components such as support tissues and cell wall and probably only accumulated as chemical substance in mesophyll cells.Compared to WT, similar chlorophyll content were observed in leaves of three transgenic groups which resulted in almost the same value of Jmax, meant the capacity of photosynthetic machinery to convert light energy to biochemical energy and RuBP regenerative capacity were similar and enough for increasing gm and Pn. The possible reason for increased gm and Pn in transgenic groups was not related to the capacity of converting light energy directly but mainly due to the biochemical function of these two proteins alone or combined.Rice grain yield is mainly determined by three components, including number of panicles per plant, grain number per panicle, and grain weight. The results of yield traits investigation demonstrated that only number of panicles per plant in three transgenic groups showed significant increment compared with that of WT while the other two components didn't show. Plant height also exhibited obvious enhancement with respect to WT plants with the same increasing trend from ICTB to FS and ICTB+FS groups in that order, while the number of filled grains per panicle, seed-setting rate and kilo-grain weightiness did not exhibit significant difference between three transgenic groups and WT. The results inferred neither over-expressing of the two target genes nor only one of them was enough to improve all the yield traits, and the potential problem for improving rice yield in the transgenic lines might be unresolved due to the limitations of our practice, but provided a promising genetic improvement by transformation of photosynthetic related genes for promoting rice yield potential.In summary, the photosynthetic parameters increased significantly in only one gene over-expressing groups or combined, but the effect of FBP/SBPase was better than that of ictB and additive effect was observed in ICTB+FBP/SBPase group. The results indicated these two genes should play positive roles in synergistic interaction for improving photosynthetic capacity and yield traits and laid the groundwork for subsequent research work. |
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