Construction And Functional Analysis Of Recombinant Bacteria For Expression PeaT1 In Bacillus Subtilis

PeaT1, a protein elicitor that is from Alternaria tenuissima, can promote plant growth and trigger systemic acquired resistance in plants. Bacillus subtilis is a kind of natural plant endophyte with non-toxic, antagonism to plant pathogen. In this research, recombinant B. subtilis was constructed for introducing the induction resistance of PeaT1 and the colonization characteristics of B. subtilis to plants. The integration, transcription and expression of recombinant B. subtilis were identified by molecular biological methods. The colonization and the biological functions of recombinant B. subtilis in plants were also analyzed.1. The recombinant expression vector pHY43N-peaT1 with P43 promoter sequence, nprB signal peptide-encoding sequence and peaT1 gene was constructed in shuttle vector pHY300-PLK. In order to accomplish the extracellular expression of PeaT1 in B. subtilis, P43 promoter sequence and nprB signal peptide-encoding sequence were cloned from B. subtilis chromosomal DNA. The two sequences and peaT1 gene were spliced by overlapping extension. This product was cloned into the E. coil B. subtilis shuttle vector pHY300-PLK. The recombinant expression vector pHY43N-peaT1 plasmid was constructed that was confirmed by restriction enzyme digestion and DNA sequencing analysis.2. The PeaT1 protein was expressed extracellularly in B. subtilis WB800. The expression vector pHY43N- peaT1 was transformed into B. subtilis WB800 by electroporation. Recombinant B. subtilis were confirmed by polymerase chain reaction (PCR). The transcription was verified by reverse transcription polymerase chain reaction (RT-PCR). SDS-PAGE and Western blotting analysis showed that protein elicitor PeaT1 was expressed extracellularly in B. subtilis.3. The colonization of B. subtilis WB800-PeaT1 was verified in rice, wheat and tomato plants. Gnotobiotic plant assays revealed that B. subtilis WB800-PeaT1 can colonization in wheat and rice as same as B. subtilis WB800 and last for 30 d at least. The colonization characteristics of B. subtilis WB800 -PeaT were not influenced by transformation of PeaT1.4. Biological function assays indicated that recombinant B. subtilis WB800-PeaT1 is able to promote wheat growth, enhance drought tolerance and increase tomato disease resistance to gray mould. Soaking wheat seed with WB800-PeaT1, the function of promoting growth on wheat is better than B. subtilisWB800. Wheat root activity increased by 1.4 times comparing with WB800 when wheat seed was soaked with WB800-PeaT1 and drought tolerance coefficient of wheat increased from 43.42% to 63.94%, which indicated that WB800-PeaT1 improved wheat drought tolerance might correlate with the increasement of root activity. The results revealed that the resistance to tomato gray mould was increased and the induced efficiency reached 70.07% when tomato treated with WB800-PeaT1.