| Many compounds of protein and amino acids in organisms contain nitrogen, which is one of the most important mineral elements. Plant needs to absorb nitrogen from soil during their life, so lots of nitrogen fertilization are used to improve the crop products, it is said that about100million tons of nitrogen fertilization were fertilized in agriculture every year all over the world, which caused series of serious environmental and economical problems. Nowadays, people improve the usage of nitrogen of plants mainly through changing fertilizing methods or crop rotation, which have not solved the problems of low nitrogen usage of plant and some related issues. As we all know that plant take up nitrogen from soil is related to its own genes, ammonium transporters mediate the ammonium absorption, if we can find an efficient gene in ammonium absorption, then improve crops’ nitrogen usage through gene engineering technology is possible.The eutrophication of water bodies getting more serious day after day as the development of industry and agriculture, excessive accumulation of nitrogen and phosphor lead to fading and disappearing of submerged plant, simple aquatic plants’ community left which made the ecological system out of balance. Water hyacinth can excessively absorb the nitrogen and phosphor, study showed that using water hyacinth to restore water bodies can reduce nutrients level, clean the water, increase the dissolved oxygen content, and provide an available environment for submerged plant, which could improve the quality of the water bodies. It can reveal the restoration mechanism at molecular level through studied on water hyacinth’s nitrogen absorption mechanism. Water hyacinth can grow in any eutrophication of water bodies as the temperature is suitable, many economical and enviromental problems produced as its strong reproductive ability. Kinds of physical and biological methods are used to control water hyacinth but don’t work effectively, if we know how it absorbs nitrogen under eutrophication, maybe we can control water hyacinth from the nutrient metabolism aspect. Based on ammonium transporter amino acids sequences of other plants, such as rice, Triticum aestivum and so on, we designed a pair of degenerate primers to clone the conserved region of water hyacinth ammonium transporter gene, use RACE technology to amply the full-length cDNA. This novel AMT gene was1600nucleotide-long, encoding501amino acids, and9transmembranes were predicted by software. It shared high homology to Triticum aestivum about80%. EcAMT belongs to plant AMT1sub-family through blast alignment and phylogenetic analysis.Ligated the full-length cDNA to shuttle plasmid pYES2, then transformed it into yeast mutant31019b which can’t survive under YNB medium while ammonium concentration below5mM. The ammonium transporting function was identified through yeast complementation test, as the mutant31019b harbored EcAMT gene can survive well when the external ammonium concentrations below to5mM.Three EcAMT genes (labeled AMT1/AMT2/AMT3, respectively) expression effected by three different nitrogen status were analyzed by semi-quantitative and per. The results show that the EcAMT genes expression are different to each other under three kinds of nitrogen status, EcAMTl presents up-regulated clearly by nitrogen starvation after72h nitrogen starvation, but it shows time effect at the same time, that is to say that the expression of AMT1will change over time under different nitrogen status. Semi-quantitative experiment on AMT2and AMT3shows that AMT2present clearly ammonium induction and the expression of AMT3is almost not effected by nitrogen status. |
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