Improvement Of Wheat (Triticum Aestivum) By Genetic Engineering

Wheat is one of the most important crops in the world. The food security of human being depends heavily on the production of wheat. In China the mainly breeding objects of wheat are high yield, high quality and multiple disease resistance.A. The most basic goals of wheat breeding is to increase the yield. A method for increasing wheat yield is to improve the efficiency of photosynthesis. Wheat belongs to C₃ plants according to its photosynthesis pathway. Its photosynthesis rate is 1/3 to 1/2 of maize's, which belongs to C₄ plants. To transfer maize C₄ traits in to wheat could increase its biomass and yield. It will be a possible method for wheat high yield breeding. The rate-limited enzymes in C₄ photosynthesis pathway with maize and sorghum had been identified. Among them both PEPC and PPDK are the enzymes specifically and highly expressing. In this research the intact C₄ type of pepc and ppdk genes derived from maize were transfer to wheat genome.1. Long PCR was a very useful tool to amplify intact genes of PEPC and PPDK which are very long. Several chemicals had been tried to optimize long PCR system and found betaine was the best Betaine, as an amino acid analogue with small tetraalkylammonium ions, could remarkably improve the amplification of long targets from the plant genome. The suitable concentration of betaine was between 1.0 mol/L and 2.5mol/L. We could effectively amplify a 9 kb DNA segment from maize genome DNA and 16 kb DNA segment from plasmid. It was shown that different primers and different targets (different GC content) needed different concentrations of betaine. Betaine can reduce or eliminate non-special amplification. In the meantime we tried other additive chemicals, such as DMSO, glycerin, formamide. They were no notable results in long PCR.It is known that most intact genes are very long. And intact gene is very important for the gene to express specially and effectively.2. Intact C₄-pepc gene derived from Maize was amplified through LA-PCR and successfully sub-cloned into modified vector pGreen0029 to form a stable expression construct named as pBAC214 (12 kb), which contains CaMV 35S promoter driven bar gene as selection marker. Comparing the cloned DNA sequences (6.7 kb) with published maize C₄-pepc gene (GenBank accession E17154) sequences, the identity of DNA sequence alignment is 98.96%. There are only 49 differences between these two intact DNA sequences, of which 13 occur in the region of promoter, 18 in introns, and 18 in exons. The homology of mRNA sequence alignment is 99.38%, and the putative amino acids sequence identity is 99.38%. There are...