Cloning Of StGAPC And AtGAPC2 Genes And Transformation Of Potato

Cytosolic glyceraldehyde 3-phosphate dehydrogenase is an enzyme prevalent in biology, mainly involved in glycolysis reaction. In recent years, show the GAPC mRNA expression levels response to salt, hot, low oxygen, low phosphorus abiotic stress environment and increased anti-stress capacity of plant by GAPC gene transfer.Potato as one of the most important food crop has played a very important role in food security, which is often vulnerable to high temperature, drought, and low phosphorus stress in growth process, so enhance stress resistance capacity by transgenic technology have great significance. This study consists of three parts:1. According to known sequences from Genebank, cloned potato and Arabidopsis GAPC gene and constructed plant expression vector.2. Research the shoot regeneration of four varieties of potato microtuber slices:ChuanYu10, Mira, Favorita, ChuanYu Zao and optimize the system.3. Transgenic StGAPC and AtGAPC2 gene to potato By Agrobacterium dip slice of potato microtuber into and screened positive plants. The main result:(1) The genes encoding cytosolic glyceraldehyde 3-phosphate dehydrogenase of Solanum tuberosum L. (StGAPC) and Arabidopsis thialiana Col. (AtGAPC2) cDNA were cloned from seedlings by RT-PCR. Both of the StGAPC and AtGAPC2 cDNA were 1017 bp in length, encoded a protein subunit of 338 amino acids, and they were identical to the sequences in Genebank. Bioinformatic analysis demonstrated that between StGAPC and AtGAPC2, the nucleotide sequence and amino acid similarity 84% and 92%, respectively. The 3D structure of StGAPC and AtGAPC2 were highly similitude with Oryza Sativa GAPDH C chain (2e5rC), contained a conserved GAPC domain; CDS sequences of GAPC showed that the same family plants have high homology attributed to the same branch in the evolution, with the existing classification system corresponds. pBI121 as based vector, we construct gene expression vector driven by CaMV35S promoter. The recombinant vector was introduced into Agrobacterium strain EHA105 and determined positive clones by PCR.(2) The regenerative capacity of potato microtuber growth in dark condition was weaker than that in light. The research of plant growth regulator combinations on shoot regeneration of four species showed that:IAA addition will cause severe hairy of tuber slice. The regeneration capacity of different varieties significantly different and respond differently to the same hormone. ChuanYulO regeneration strongest, the bud formation via two ways, one of the best combination of direct regeneration was:MS+2mg L-16-BA +0.25mg L-1TDZ+0.1mg L-12,4-D, shoot induced percentage 45.53%; the best combination which induced the formation of callus then multiple bud was:MS+1mg L-16-BA+3mg L-1TDZ+0.05mg L-12,4-D+0.05 mg L-1GA3, the shoot induced percentage 61.69%. The regeneration way of Mira and Favorita were direct shoot regeneration, and the best combination of induced buds was the same as ChuanYu10, but the rates of induced buds were low only 20.38% and 14.10%, respectively. ChuanYu Zao slices in MS+0.5 mg L-16-BA+1 mg L-1TDZ combinations can form a large number of green bud primordial, but those would quickly browning wither and die so we can't get useful buds. Treatments by removing the Sucrose out of medium, cutting slices surface after remove bud eye, can greatly improve the ChuanYu10 and Mira direct regeneration bud rate, respectively, up 81.23% and 65.33%, a basis for further gene transformation.(3) StGAPC and AtGAPC2 were transformed into potato by Agrobacterium-mediated method, respectively. We got 90 kanamycin resistant shoots by 75 mg·L-1 kanamycin selection system, but none of those lines could grow roots in MS medium contain 50 mg·L-1 kanamycin. PCR amplification of resistance marker gene nptⅡ, more two bands were obtained, while amplification of GAPC gene (1029bp) we obtained 2000bp band. There are currently 65 lines transferred into the foreign fragment, initially identified as positive plants. Several positive lines with low phosphorus stress(100μmol L-1), the bottom leaves of ChuanYulO began to yellow after 8d, showed a slight P-deficiency symptoms while transgenic StGAPC lines of ChuanYu10 with normal leaf did not appear deficiency symptoms, but root growth slightly slower. Both transgenic AtGAPC2 lines of Mira and untransgenic plants, in the low phosphorus treatment appeared deficiency symptoms such as dark green leaves, purple stems, but the growth potential of transgenic lines significantly better than that of control which showed the dead state. After 24d the biomass of transgenic material was significantly higher than that of untransgenic material, and the biomass between Mira and ChuanYu10 was not significant.In summary, in this study we cloned StGAPC and AtGAPC2 genes. The optimized Shoot regeneration system as genetic transformation regeneration system, we initially obtained lines having low phosphorus tolerance, but whether this improved resistance caused by over expression of the target gene and the mechanism requires further study.