| In silico cloning is developing with the project of genomic and EST. It is a new method of gene cloning, which is applying with bioinformatics. Lower cost, rapid, simple and clear objective are all advantages of in silico cloning, compared with traditional strategies. With the human genomic project implement, scientists have cloned many human functional genes by in silico cloning, but it is rarely used in plants because of less sequences.In this paper, we used the EST Blast method to clone Glycine max pyridoxal kinase (PK) and pyridoxine biosynthesis protein (PDX) genes. Also, we took Glycine max PDX gene into potato by genetic transformation. The objective 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 metabolic engineering.1 cloning and analysis of Glycine max PK geneAccording to the relative conservation of homologous gene, using Arabidopsis thaliana pyridoxal kinase cDNA sequence as a query probe to blast Glycine max EST database, a soybean PK gene cDNA of 1102bp was obtained (GenBank Accession, DQ006813). It contained a complete ORF, from 119bp to 1045bp, encoded 308 amino acids. 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 PK was aligned against related PK sequences of other species such as Solanum tuberosum, Arabidopsis thaliana, Oryza sativa, Triticum aestivum and Brassica napus. It showed the identity of 81% 75% 78% 79% and 76%, respectively. The results revealed the peptide encoded by Glycine max PK is significant similarity in plants. Phylogenetic tree of plant PKs is mainly consistent with the classification system of these plants.Using Semiquantitative RT-PCR, we found out the effect of the illumination time impacting the expression of PK. It revealed that the expression of PK was showing no difference with the illumination time increasing. In another word, PK gene was not regulated by illumination. Another conclusion was PK expressed in Glycine max leaves stems and roots.2 cloning and analysis of Glycine max PDX geneAccording to the relative conservation of homologous gene, using Arabidopsis thaliana pyridoxinebiosynthesis protein cDNA sequence as a query probe to blast Glycine max EST database, a soybean PDX gene cDNA of 1280bp was obtained (GenBank Accession, DQ139265). It contained a complete ORF, from 20bp to 955bp, encoded 311 amino acids. To examine the accuracy of in silico cloning cDNA sequence, this sequence was confirmed by RT-PCR, genomic PCR, molecular cloning and sequenced. The result confirmed that the in silico cloning cDNA sequence was accurate.The deduced amino acid sequence of Glycine max PDX was aligned against related PDX sequences of other species such as Lotus corniculatus var. japonicus, Nicotiana tabacum, Arabidopsis thaliana, Medicago truncatula, Oryza sativa and Triticum aestivum. It showed the identity of 93% 92% 92% 92% 85% and 70%, respectively. The results revealed the peptide encoded by Glycine max PDX is significant similarity in plants. Phylogenetic tree of plant PDXs is mainly consistent with the classification system of these plants.Semiquantitative RT-PCR proved that the expression of PDX was showing no difference with the illumination time increasing. Another conclusion was PAX expressed in Glycine max leaves stems and roots.3 potato genetic transformation of Glycine max PDX geneGlycine max PDX gene was transformed into potato leave discs and stem segments by Agrobacterium tumefaciens LBA4404. 42 explants were regenerated on selective media with Kanamysin, and 6 of them were confirmed by PCR as transgenic plants. Put the transgenic plants in the soil and further works are in process.
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