Beet Glutamine Synthase Gene Cloning

Nitrogen assimilation is very important for growth and development in plants.Inorganic nitrogen must be assimilated as organic nitrogen which can be absorbed andused by plants. Glutarnine synthetase is a key enzyme during this process.The experiment is to acquire the whole cytosolic glutamine synthetase gene (GS1)from Beta vulgaris. DNA was extracted from sugar beet leaves as template, primerswere designed according to GS1mRNA sequence (accession no AF343667) fromGenBank. Then GS gene was amplified via PCR method. The PCR product was ligatedinto T vector, tansformed competent cell of E. coli and sequenced. In order to check theresult of PCR, total RNA was extracted from leaves as RT-PCR template, primers weredesigned as described above, then amplified by RT-PCR method. Cloning and sequencingof RT-PCR product was the same as that of PCR product.During the expetiment, PCR system was optimized by cross experiment, and highfidelity enzyme was utilized which can minimize wrong base integration. GS1 gene isvery long, so it was cloned by subsection PCR method. Full-length GS1 gene sequenceand partial GS1cDNA sequence of sugar beet was successfully obtained. The wholeGS1gene is 9606bp in length, including 13 exons which are separated by 12 introns.Among these introns, the longest one is 3760bp, and the shortest one is 83bp. Thesequence similarity between exon part of GS1 gene and known GS1mRNA is 99.5%,there are only 5 base difference, cDNA sequence is obtained by RT-PCR method whichis 1068bp in length, and the sequences similarity between this cDNA and knownGS1mRNA is 99.6%, there are 4 base difference. Meanwhile, the sequence similaritybetween exon part of GS1 gene and GS1cDNA in the experiment is 99.9%, there is onlyone base difference. We can see the results in this paper are pretty reliable.In this experiment, full-length GS1 gene was acquired firstly from sugar beet. Thenthe regulatory region can be separated based on the result, which provides molecularbiological foundation for GS gene expression, regulation and implement of highammonia assimilation in sugar beet...