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Citrate synthase is one of the active enzyme regulating plants organic acids , is also involved in oxaloacetate and acetyl coenzyme A condensation to generate a key enzyme citrate . Citric acid to increase the plants can improve plant absorption of phosphorus in the soil as well as resistance to aluminum toxicity . Alfalfa is excellent forage legumes , the planting area of ??more than 30 million ha worldwide , China is the world's cultivated one of the countries that have more of alfalfa . A total of 14 provinces and autonomous regions nationwide planting alfalfa producing areas to the north, northwest , but rather limited alfalfa planted in the south , some alfalfa planted in the south suppressed in acidic soil Al 3 . Poisoning plants by means of genetic transformation citrate synthase gene into to intolerance aluminum plants , improvement of plant aluminum tolerance , so you can better fundamentally solve the Al 3 . Cloning First amplified from total RNA reverse alfalfa cDNA in cs conserved sequence of the gene using RACE technology , obtain MsCS cDNA of the whole sequence . The cDNA full sequence of 2031bp, including the 5 ' end of the non - coding region of 39bp 3 ' noncoding region of 434bp, contains a complete open reading frame contains 1551bp , encoding 517 amino acid residues , and the molecular weight of the protein encoded by 56.743 KDa. Related plant amino acid sequence of the citrate synthase gene DNAMAN deduced amino acid sequence of the gene of soybean , Arabidopsis , rice, sorghum citrate synthase gene deduced amino acid sequence for multiple sequence alignment analysis , results show that homology of more than 90% , having a high degree of homology , this gene , soybean , Arabidopsis , rice , and sorghum . Construction of the plant expression vector of the genetic transformation of tobacco to is First the pBI121 vector successful building MsCS gene plant expression vector pBI - MsCS ; then using Agrobacterium-mediated transformation of tobacco leaf disk transformation the desired MsCS gene superfamily amount of expression in transgenic plants . Antibiotic selection , 17 regenerated seedlings transformed . Regenerated plant leaf DNA was extracted for PCR detection , and the results were positive , and initial confirmation obtained six positive regenerated plants .
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