Biological Characteristics Of Bacillus Altitudinis 6ww6 And Mechanisms Of Salt Tolerance Based On Proteomics And Modification Proteomics

Bacillus altitudinis belongs to the Bacillus genus of Firmicutes and is a common plant probiotic.Bacillus altitudinis 6ww6 was isolated from saline-alkali land in Binhai and has a good growth-promoting effect on wheat and corn under saline-alkali and non-saline-alkali conditions.In this paper,the salt tolerance characteristics,motility,and biofilm formation ability of the 6ww6 strain were studied.Through genome alignment analysis,TBLASTn search,NR library search verification,and elimination of other homologous result sequences,the orphan genes of strain 6ww6 were finally obtained and primers were designed for PCR identification,and its specific molecular identifier was screened out.The salt tolerance mechanism of the strain 6ww6 was analyzed by proteomics and protein modification omics.The research results serve as a sufficient theoretical basis for the development and application of this strain.The main findings of the study are shown below.(1)Salt tolerance ability of strain 6ww6 was tested and found that it could not only grow in liquid LB with a Na Cl concentration of 1-11%,but also had good motility and biofilm formation ability in a salty environment.It is ensured that it can migrate to the rhizosphere of crops in saline-alkali land and colonize to form biofilm to improve the adaptability of crops to saline-alkali environment.(2)Strain 6ww6 can be used as the preferred strain of microbial fertilizer.To strengthen the intellectual property protection of strains,a rapid detection method for strains was established.Five specific genes were screened out: J939?13195,J9319?05960,J9319?13355,J9319?05965 and J9319?13350.Primers 5960 F and 5960 R can singlely amplify the target gene J9319?05960 of strain 6ww6,which is determined to be the specific molecular marker of strain 6ww6.(3)Proteomics data showed that 457 proteins were up-regulated,and 470 proteins were down-regulated in strain 6ww6 under 11% Na Cl salt stress.Through analysis,it was found that these differentially expressed proteins were significantly enriched in ribosome,purine metabolism,amino acid biosynthesis,two-component system and other pathways;In addition,the expression of proteins related to choline,betaine,and trehalose biosynthesis and transport was significantly up-regulated.The gene encoding betaine dehydrogenase J9319?RS14850was introduced into Escherichia coli BL21 to expression and found that the growth rate of BL21 in LB medium with Na Cl concentration of 3% and 5% could be significantly increased.(4)4D-Lable acetylation modification proteomics analysis was used to find out the acetylation modification differential proteins of strain 6ww6 in the salt environment,of which776 proteins were up-regulated and 509 proteins were down-regulated.The analysis found that after the strain 6ww6 was subjected to salt stress,acetylation modification levels of proteins in some functional pathways changed greatly,such as ribosomal proteins,glycolysis/gluconeogenesis,carbon metabolism,and DNA repair.In addition,the acetylation motif analysis found that in addition to the similar acetylation motifs currently found in fungi,bacteria,mice,etc.,acetylation motifs unique to this strain were also found,such as KacV,KacG,KacW,Kac*Y,HKac,E*KacK,KacP,SKac,Kac**H,HKac.In particular,some basic amino acids appeared abundantly in the-10-+10 region of the acetylation modification site,and these motifs were closely related to the enzymatic and non-enzymatic acetylation modification of strain 6ww6 protein.Studies have shown that under salt stress,strain 6ww6 can regulate its own life activities by regulating protein expression and acetylation modification level,such as weakening protein translation,cell motility,energy metabolism,etc.to reduce intracellular energy loss under stress environment.Enhance the metabolism of nucleotides such as purines and the degradation of fatty acids to provide energy for cells to resist stress.In addition,strain 6ww6 also regulates the intracellular osmotic pressure by regulating the synthesis of osmotic substances such as proline,glutamic acid,glutamine,betaine,trehalose,etc.so that the strain can adapt to the salt environment and restore cells normal life activities.