Gene Cloning Of Phosphite Dehydrogenase From Soil Pseudomonas Sp. And The Unique Characterization Of Its Transgenic Tobacco Plants

Phosphorus(P) is one of the essential macro-elements for plant growth &development and involved in a variety of plant metabolic processes. In soil, phosphate has a rich reservoir, but is largely fixed into the insoluble form by metal chelation because of its high reactivity, resulting in low level of phosphorus available for plant uptake. Therefore, the lack of available phosphorus in soil has become an important limiting factor for the sustainable development of agriculture, and it is urgent for improving the ability of plants to absorb phosphorus from soil or seeking alternative phosphorus-fertilizers to phosphate. Phosphite dehydrogenase(PTDH), existing in some bacteria, can oxidize phosphite into phosphate. If introducing this enzyme into the target plant specie, the corresponding transgenic plants should be able to directly utilize phosphite as phosphorus fertilizer. Regarding this, we attempted to amplify a PTDH gene named Ps Ptx directly from the soil metagenome, and then introduce it into tobacco by Agrobacterium-mediated transformation, and finally perform a unique characterization of the resultant transgenic tobacco plants under the stress conditions of phosphite. The major results of this work are shown as follows:(1) Gene cloning of PsPtx and construction of its expression vectors Using the soil metagenome as DNA template, the full length of Ps Ptx gene was successfully amplified by its specific primers via two rounds of PCR program. It was then subcloned into a p BI121-derivative plasmid to achieve the plant expression vector p BI121(Ps Ptx) that was further verfied by sequencing. The coding region sequence(CDS) of Ps Ptx is 1011 bp that encodes a protein of 336 aa(with the theoretic molecular weight of 36.5 k Da). The conserved domain prediction indicated that the Ps Ptx-encoded protein belonged to a member of phosphite dehydrogenase,containing the conserved NAD+ binding motif and catalytic residues. Furthermore,phylogenetic tree analysis showed that the Ps Ptx gene was originated from an undefined Pseudomonas species. In addition, through IPTG induction, the Ps Ptx gene could be efficiently expressed with high solubility in its recombinant Escherichia coli strain.(2) Identification of the Ps Ptx transgenic tobacco plants and gene expression analysis of Ps Ptx The plant expression vector p BI121(Ps Ptx) was transformed into the Agrobacterium tumefaciens strain LBA4404. Through the leaf disc transformation by Agrobacterium infection and subsequent multiplex PCR identification, the positive transgenic tobacco plants of Ps Ptx gene were obtained. Using Trizol reagents the total RNA were extracted from the tissues(root, stem, leave) of wild-type and Ps Ptx transgenic tobacco lines(Ps Ptx-3,4,8), and reversely transcribed as the PCR templates for amplification of Ps Ptx and internal 18 S r RNA using the respective gene-specific primers. The results showed that Ps Ptx was expressed in all tested tissues of its transgenic tobacco plants.(3) Feature analysis of Ps Ptx transgenic tobacco plants a. Seeds of transgenic lines Ps Ptx-3, 5, 7 and WT tobacco were germinated and grown on 0.1x MS solid media supplemented with different concentrations of phosphate(Pi) or phosphite(Phi) as the sole phosphorus(P) source. The results showed that the growth of both root and aboveground parts were remarkably inhibited in WT under the Phi stress, with an increasing extent with the concentration of Phi. In contrast, the Ps Ptx transgenic tobacco was hardly affected by Phi, with much vigorous growth and more biomass.b. A further test was conducted by comparing the growth of seedlings from transgenic line Ps Ptx-3 vs. WT tobacco in solid media containing 100 mg·L-1Kanamycin(Kan) or 3 m M Phi. The results showed that Phi and Kan had nearly common inhibition on WT tobacco, but were similarly tolerated in the transgenic line Ps Ptx-3.c. A growth competition test was also performed in greenhouse by sowing the seed mixture of Ps Ptx-transgenic line and wild-type tobacco(with the seed number ratio of 1: 100) in a mixture of sand and vermiculite(1:1) containing 120 mg·kg-1of phosphate(Pi) or Phi as phosphorus(P) source. The result also showed that the growth of WT tobacco was severely inhibited by Phi, but all Ps Ptx-transgenic lines could normally grow in the P nutrition condition using only the Phi-fertilizer.In conclusion, these data imply that the Ps Ptx transgenic plants can utilize Phi for plant growth & development by converting it into phosphate(Pi). In other words,Phi can be possibly used as an alternative P nutrition to phosphate(Pi) for Ps Ptx transgenic plants. Furthermore, the Ps Ptx gene can be used as a new selection marker for plant transgenic studies. Obviously, this work has established a solid basis for the future studies particularly applying the Ps Ptx gene to engineer the major crops.