| Wheat is one of most important crops in the world, which productivity ability and supply-demand situation always impact national economic development and the food safety. With the development of the society and the increase of population, demand for food keeps increasing in the past, today and future. Photosynthesis is the basis of all green plants, and enhancement of light use efficiency is essential for higher yield in major crops. Comparative to C3 crops, C4 crops, such as Zea mays,Sorghum vulgare and Saccharum officinarum have CO2 concentration mechanism with higher light compensation point, lower CO2 compensation point, and weaker photorespiration, especially under high light intensity, high temperature and drought. Introduction of C4 key enzyme genes into C3 plants to improve their photosynthetic characteristics and grain yield has been an active research area in plant biology at home and abroad.Phosphoenolpyruvate carboxylase (PEPC) is one of the key enzymes in C4 photosynthetic pathway, it main distribute to cytosol of mesophyll cell in C4 plants. The C4 pathway forms CO2 concentration mechanism, providing CO2 for C3 pathway in bundle sheath cells, severs as a"CO2 pump". Pyruvate, orthophosphate dikinase (PPDK) is potentially rate limiting in C4 pathway, also plays important roles in C4 pathway. PPDK is located in the chloroplast of mesophyll cell, catalyzing the reproduction of PEP that is CO2 first receptor. To date, many studies indicated that the photosynthesis efficiency and the grain yield of C3 crops can be improved by introducing C4-specific pepc gene into C3 plants.In order to obtain high photosynthetic efficiency transgenic wheat germplasms with C4 photosynthetic characteristics, a full-length cDNA for PEPC and a full-length cDNA for PPDK were isolated from maize inbred line Z561, which was high photosynthetic efficiency and PEPC activity. Furthermore, plant high efficient expression vector p3301-pepc containing maize pepc gene and select marker bar gene was introduced into wheat mediated by particle bombardment transformation. Hopefully, we may thereby be able to provide the theoretical and technical support for the improvement of photosynthetic efficiency of wheat varieties and accomplish high photosynthetic efficiency transgenic breeding.The results were as follows:1. A full-length cDNA for PEPC was isolated from Zea mays by homology-based cloning technology. The nucleotide sequence was characterized that the sequence length was 3053 bp, containing one ORF with 2910 bp which coded a peotide 970 amino acids. After alignment on NCBI by Blast, the identity of the cloned fragment with pepc genes from X15238 was 99%. There were 16 differences in the nucleotide sequence, resulting in 5 differences in the amino acid sequence, and which had no effect on the PEPCase activity. After alignment on DNAMAN program, the identities of the cloned fragment with PEPC genes from Saccharum officinarum C4-form, Sorghum vulgare C4-form and Setaria italica C4-form were about 91%,90.2% and 83.2%, respectively; and that from Oryza sativa C3-form, Sorghum vulgare C3-form, Setaria italica C3-form and Echinochloa crus-galli C3-form were about 74.1%,75.1%,76.8% and 75.7%. Alignment and phylogenetic analysis of the amino acid sequence deduced from the fragment and the PEPCase sequence of other plants retrieved from GenBank were carried out by DNAMAN program showing the sequence was closer to Saccharum officinarum C4-form and Sorghum vulgare C4-form than to Setaria italica C4-form. The function sites of the PEPC protein were analysised using Interproscan program on EMBL , and the resultes showed that the amino acid sequence of Zea mays C4-form PEPC protein had seven conserved domains.2. A full-length cDNA for PPDK was isolated from Zea mays by homology-based cloning technology technology. The nucleotide sequence was characterized that the sequence length was 3014bp, containing one ORF with 2757 bp. After alignment on NCBI by Blast, the identity of the cloned fragment with ppdk genes from J03901 was 92.94%.3. The full-length cDNA sequence coding for maize PEPCase gene (GenBank accession number: FJ415327) was cloned into the high-efficiency binary expression vector pCAMBIA3301, named as p3301-pepc.4. The binary vector p3301-pepc was introduced into six wheat varieties using particle bombardment transformation method. A total of 409 PPT resistant plants were obtained, among them the expected 668bp fragment was present in 357 of the 409 PPT resistant plants. The integration of maize pepc gene in transgenic wheat plants was confirmed by Southern blotting analysis. The copy number of different transgenic lines was analyzed by real-time fluorescence quantitative PCR. Among the fifteen positive transgenic lines, seven had one copy number, three had two copies, two had three copies, one had five copies, one had eight copies and one had seventeen copies. RT-PCR analysis results showed that the foreign gene could expressed normally in transgenic wheat plants, and expression strength in transgenic plants was distinct remarkably. Physiological studies showed that the transformants displayed higher photosynthesis than non-transformed controls.5. Genetic analysis of T1 transgenic lines showed that the foreign pepc gene could stability transmit to the progeny, with the segregation ratios of most transgenic lines following Mendelian manner. Expression level of the maize C4-spcific pepc gene in T1 transgenic lines was determined firstly by assaying the activity of PEPC, and the results displayed that partly transgenic lines had higher PEPC activities than untransformed control, with the highest enhancement of 2.17 fold. SDS-PAGE electrophoresis analysis of leaf protein in transgenic and untransformed plants revealed that expression level of pepc gene was different in transgenic lines, in accord with the results of PEPCase activity; Photosynthetic physiological analysis Under field condition showed that net photosynthesis rates of most T1 transgenic lines were significant higher than non-transformed control, the highest photosynthesis was (31.95μmolCO2m-2s-1) distinctly higher than the max value reported under normal condition in Huaihuaihai planting area, and increased by 26% than non-transformed control.The results described above provid important research materials, theoretical and methodology for high photosynthetic efficiency transgenic wheat breeding.
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