Molecular Cloning Of Vitamin Biosynthesis Related Enzyme Genes From Glycine Max And The Genetic Transformation To Potato

In silico cloning or virtual cloning, which is developing with the development of genome, EST projects and bioinformatics, is a rapid pathway to clone gene by assembling homologous genome or EST sequences of target genes with the help of computer, and then to confirm it by RT-PCR. In silico cloning was applied first in human due to the abundant human genome or EST information, but it is rarely used in plants because of less sequences.In this paper, we used the EST Blast method to clone Glycine max tyrosine aminotransferase gene TAT and dehydroascorbate reductase gene DHAR. Semiquantitative RT-PCR was adapted to detect the expression of these two genes in different time treated by illumination. RT-PCR was adapted to detect the expression of these two genes in different tissue treated by illumination. Moreover, the TAT gene was transformed to potato leave discs and stem segments by Agrobacterium tumefadens strain of LBA4404. The aim of this study is to establish available cloning gene approach based on the high similarity of homologous gene sequences in different species and improve the quality of potato through genetic engineering.1 Cloning and analysis of Glycine max TAT gentA novel Glycine max tyrosine aminotransferase gene TAT full-length cDNA of 1709bp (GenBank Accession, DQ003328) was cloned by Blasting the Glycine max EST database with the Arabidopsis thaliana homologous TAT gene cDNA sequence as a querying probe.Searching the cDNA sequence for potential coding regions by ORF finder (NCBI), an entire open reading frame(ORF) of 425 amino acids was found with a MW of 46.7KDa and a pI of 5.00. It was detected with a potential start codon at the 226^th site and a stop codon at the 1503^th site of the sequence. To examine the accuracy of in silico cloning cDNA sequence, this sequence was cloned by RT-PCR and sequenced. The result confirmed that the in silico cloning cDNA sequence was accurate.The deduced amino acid sequence of Glycine max TAT was aligned against related TAT sequences of other species such as Arabidopsis thaliana, Oryza sativa, Medicago truncatula and Solenostemon scutellarioides. The multiple sequence alignment showed the identity of 97%, 94%, 86% and 80%, respectively. The results revealed the putative peptide encoded by Glycinemax TAT is significant similarity in sequence of other plants. Phylogenetic tree of plant TATs is mainly consistent with the classification system of these plants.Use Semiquantitative RT-PCR, we can find out the illumination time impact the expression of TAT. With the illumination time increasing, the expression of TAT will be increased. TAT expression in Glycine max leaves and stems are higher than roots. Rarely TAT expression in Glycine max roots was found.2 Cloning and analysis of Glycine max DHAR geneA novel dehydroascorbate reductase gene DHAR of Glycine max was cloned and identified which was blasted by search of the Glycine max EST database with the homologous gene cDNA of Arabidopsis thaliana.This sequence was confirmed by RT-PCR, molecular cloning and sequencing. The full length of Glycine max DHAR gene cDNA is 955bp(GenBank accession, DQ006810).The cDNA sequence encodes dehydroascorbate reductase (DHAR) of 259 amino acid which with a MW of 28.9KDa and a pI of 5.06. It also contains a complete ORF of 777bp which with a potential start codon at the 36^th site and a stop codon at the 815^th site of the sequence.Compared with Arabidopsis thaliana, Medicago truncatula, Lotus corniculatus, Brassica oleracea, Lycopersicon esculentum and Oryza sativa, we found the gene is almost conserved and the multiple sequence alignment showed the identity of 66.28%, 79.92%, 81.37%, 67.05%.. 63.06% and 60.66%, respectively. Phylogenetic tree of plant DHARs is mainly consistent with the classification system of these plants. These results revealed that it is a convenient technique for cloning novel gene by searching EST database with homologous gene of model plants such as the Arabidopsis thaliana.Semiquantitative RT-PCR revealed that increasing illumination time will increase the expression of DHAR. DHAR expression in Glycine max leaves and stems were detected by RT-PCR, but the expression was very low in roots.3 Potato genetic transformation of Glycine max TAT geneThrough mediated by Agrobacterium tumefaciens LBA4404, Glycine max TAT gene was transformed into potato leave discs and stem segments. 33 explants were regenerated on selective media with Kanamysin. Two of them was confirmed by PCR as transgenic plants.Further works are in processing.