Study On Gene Cloning In Alhagi Sparsifolia, Swainsona Salsula Based On The Chip Hybridizations Results

Alhagi sparsifolia Shap. classified to Alhagi Gagneb. is perennial plant as bee and medical plant and it is tolerant to drought, salt and waterlogging as forage. While Swainsona salsula Taub. classified to Swainsonia Salisb form Leguminosae. Has the anticancer function and immunoregulatory activity. As a neutral plant tolerated to salt, it also can improve soil conditions.Through analysis Alhagi sparsifolia Shap. and Swainsona salsula Taub. hybridizing the chip hybridization results of Medicago truncatula by Bioinformatic, we screened two genes non-symbiotic hemoglobin (nSHb) and formate dehydrogenase (FDH) from Alhagi sparsifolia Shap.and one gene isoflavone reductase (IFR) from SwainsonasalsulaTaub.We extracted total RNA from Alhagi sparsifolia and Swainson asalsula Taub, and reverse transcribed it to cDNA using oligo dT, then designed the primers according to the nSHb, FDH and IFR gene sequence in Medicago truncatula genebank, amplified the three full length cDNAs using RT-PCR technique, named them AsnSHb, AsFDH and StIFR.. As follows :The length of AsnSHb is 483bp encoding 160 amino acids, AsFDH is 1170 bp encoding 389 amino acids and StIFR is 957bp encoding 318 amino acids. Through blasting with Medicago sativa database the homology of nucleotide is 92.50%, 89.41% and 81.82% respectively, the homology of amino acids is 91.51%, 92.03% and 83.39% respectively. Furthermore, we constructed three expression vectors and transformed them to Nicotiana tabacum and Arabidopsis thaliana through Agrobacterium transformation. Our results would lay the foundation of molecular information to study the function of the protein coded by nSHb, FDH and IFR.We devised the primers with Bglâ…¡and Speâ… restriction enzyme sites, amplified ORF of three cDNAs using PCR, then inserted them into the vector pCAMBIA1₃0₂ by Bglâ…¡and Speâ… sites. After identifying the three recombinant plasmids using Bglâ…¡and Speâ… restriction enzyme digestion we transformed the plasmids into Agrobacterium rhizogenes EHA105 and screened by rifampicin. Furthermore, we transformed these three genes to Nicotiana tabacum and Arabidopsis thaliana by the way of Agrobacterium transformation.