Functional Analysis Of Small RNAs In Xanthomonas Campestris Pv. Campestris

Bacterial small RNA（s RNA）,also known as non-coding RNA（nc RNA）,is a group of RNA molecules that are 50-500 nucleotides in length,normally do not encode proteins,but has independent functions.s RNA acts as a post-transcriptional regulator and plays an important role in various bacterial cellular physiological processes.Although a large number of s RNAs had been identified in different bacterial species,few of them has been functionally characterized.Previously,612 s RNAs were identified from（Xanthomonas campestris pv.campestris）strain 8004（Xcc 8004）in our lab,but only 65 of them has been functionally characterized.The lab’s goal is to finish the functional identification of all 612 s RNAs gradually.The task of this study is to finish the functional identification of 10 s RNAs by using over-expression.To obtain the over-expression strain of a s RNA,the s RNA gene was firstly cloned into the over-expression vector p BBad18K and then introduced into the Xcc wild-type strain.Next,the expression level of the target s RNA in the over-expressing strain was determined by semi-quantitative RT-PCR.The results showed that the expression level of the target s RNA in the over-expression strain was 1.7 to 17.9 times higher than that of the empty vector-carrying wild-type strain（WT/p B）,indicating the successful of over-expression strain construction.To determine the biological function of the target s RNA,the growth,virulence,extracellular polysaccharide and extracellular enzyme production,smarming motility,metal tolerance,osmotic pressure and protein denaturant tolerance of the s RNA over-expression strains were we examined and compared to the wild-type strain WT/p B.The results showed that:（1）in minimal medium,the over-expression strain of s RNA-Xcc039,s RNA-Xcc132,s RNA-Xcc279,s RNA-Xcc308 and s RNA-Xcc333 grow significantly faster,while the over-expression strain of s RNA-Xcc096 grow significantly slower than the wild-type strain;indicating that these s RNAs are involved in the growth of Xcc.（2）The over-expression strain of s RNA-Xcc071 and s RNA-Xcc132 showed a significantly enhanced tolerance to Co²⁺compared to the wild-type strains,indicating that the two s RNAs are related to the metal ion tolerance of Xcc.In addition,to identify Hfq-dependant s RNA,in this study,EMSA was used to detect the in vitro binding of the 10 s RNAs with Hfq proteins.The results showed that s RNA-Xcc010,s RNA-Xcc039,s RNA-Xcc096,s RNA-Xcc132,s RNA-Xcc143,s RNA-Xcc279,and s RNA-Xcc308 can directly bind to Hfq in vitro,suggesting that these s RNAs may be Hfq-dependent s RNAs.In summary,in this study,the biological function of 10 Xcc s RNAs was characterized and obtain the following innovative results:（1）six growth-related s RNAs were identified in Xcc and（2）two Co²⁺tolerance-related s RNAs were identified in Xcc.These results provide the first step to understanding the role of s RNA in the growth and metal stress of Xcc.