Abstract: 【Objective】L-proline in root exudates of cotton is one of the key factors affecting the colonization of biocontrol microorganisms. Previous studies showed that L-proline could improve the biofilm formation ability of Bacillus subtilis NCD-2. Through high-throughput sequencing technology, we explored the regulatory genes related to the biofilm formation and biocontrol potential of strain NCD-2, which laid a foundation for further understanding the molecular interaction between cotton root exudates and the strain.【Method】Transcriptome (RNA-seq) and isotope labeled relative quantitative proteomics (iTRAQ) were analyzed after 24 hours of co-culture with 10 mg·mL^-1 L-proline and strain NCD-2, respectively. RT-qPCR was used to verify the expression of some differential genes in different metabolic pathways.【Result】Transcriptome analysis showed that 1 071 DEGs were obtained after L-proline and NCD-2 co-culture, of which 602 genes were up-regulated and 469 genes were down regulated. Go analysis showed that 49, 14 and 30 functional items were significantly enriched in biological processes, cell components and molecular functions, respectively. KEGG pathway is mainly enriched in compound metabolism, flagellum assembly, bacterial motility or chemotaxis. Proteomic analysis showed that total of 211 differentially expressed proteins were detected compared to the control. Hierarchical cluster analysis was carried out to group proteins, among which 118 proteins were up-regulated, which 93 proteins were down-regulated. Go analysis showed that 13 and 8 functional items were significantly enriched in biological process and molecular function, respectively. KEGG pathway is mainly enriched in amino acid metabolism, carbohydrate metabolism, flagellum assembly and ABC transporter. Further transcriptional-proteomics analysis revealed that 112 differentially expressed genes (or proteins), including 38 down-regulated genes (or proteins) and 74 up-regulated genes (or proteins), were detected. Go was enriched in 9 aspects, including nutrient reservoir activity, catalytic activity, cell membrane, localization, cellular lipid metabolic process,  oxidation-reduction process, sigma factor activity, transport activity and spore formation. KEGG pathway is mainly enriched in energy metabolism, ABC transporters, antibiotic biosynthesis, flagellum assembly, motility or chemotaxis and two-component system. 26 differentially expressed genes was verified by RT-qPCR, the results showed that there were some differences in the expression level, but the expression trend was basically consistent with that of RNA-seq and iTRAQ.【Conclusion】The interaction between L-proline in cotton root exudates and Bacillus subtilis NCD-2 was a complex biological process, which depended on multiple genes in different metabolic pathway networks. It was clear that differential expressed genes (or proteins) of the two-component system, antibiotic biosynthesis, energy metabolism, motility or chemotaxis, flagellum assembly and in ABC transporter pathway may play an important role in the interaction between cotton root exudates and Bacillus subtilis. The results lay a foundation for further study on the molecular interaction between L-proline and B. subtilis NCD-2.

Key words: L-proline, Bacillus subtilis, transcriptome, proteome, interaction relationship