Study On Hfq Protein And SRNA In Pseudomonas Syringae MB03

Pseudomonas syringae is a common plant pathogen that is widely distributed and numerous.There are currently more than forty pathogenic species,and there are signs of its survival even under the ice in Antarctica,so scientists have paid great attention in recent years.Among them,the tomato pathogenic subspecies Pseudomonas syringae pv.tomato.DC3000 is the most concentrated,and DC3000 has become a model strain for studying the interaction between bacteria and plants.In addition,some P.syringae containing ice nucleus proteins can cause plants to form frost at-5?to-2?and cause damage to the plants.This study used the laboratory to freeze frozen tissues from plants.A strain of P.syringae MB03 with high ice nucleus activity was isolated.Small non-coding RNA?sRNA?in bacteria is a type of small ribonucleic acid fragment that has no encoding function.It is between 40 nt and 500 nt in length.Most of them are located between genes,and they affect the life activities of bacteria playing a regulatory role.Hfq is a ubiquitous regulatory protein in bacteria.Its main role is to act as a molecular chaperone for sRNAs,assisting sRNAs to perform its role,making its effect more stable and effective.The main topic of this project is the role of some sRNAs in Pseudomonas syringae in regulating related phenotypes?respectively:oxidative stress tolerance,nematode feeding preference,T6SS activity,biofilm,motility,etc.?and The regulatory mechanism of sRNAs involved in bacterial oxidative stress tolerance has been further explored.First,16 sRNAs in P.syringae were predicted by bioinformatics,which were P1,P9,P11,P14,P15,P16,P24,P26,P27,P31,P36,Crc Z₁₀,Crc Z₄₂,Prr F₁₀,Prr F₃₃,and found the specific position and fragment sequence of them and the hfq gene encoding the regulatory protein in MB03.Because P.syringae MB03 can produce ice nucleus proteins to promote the survival of bacteria in low temperature environments,in order to demonstrate the role of sRNAs in the survival of bacteria in low temperature environments,wild-type cultures were grown at different temperatures?6?,16??.Pseudomonas syringae MB03 was subjected to q RT-PCR after shaking for 24 hours.The results showed that most of the sRNAs in the experiment showed a down-regulated expression,but P9,P26,P27,and Prr F₁₀ were significantly up-regulated.For the above phenotypes,select seven sRNAs that can be implemented,namely P1,P9,P11,P14,P15,Prr F₁₀,and Prr F₃₃.Design upstream and downstream primers to construct the knockout strains:?P1,?P9,?P11,?P14,?P15,?Prr F₁₀,and?Prr F₃₃.The oxidative stress tolerance experiment found that the?P11 strain significantly increased tolerance to oxidative stress,which was manifested in the?P11 survival ability was significantly increased by 50 times compared to wild-type MB03 under 0.3 m M/L H₂O₂ treatment.At the same time,the inhibition zone experiment proved that the oxidative tolerance of P11 interrupted strains was enhanced.Therefore,it is speculated that P11 may negatively regulate the oxidative stress tolerance of P.syringae MB03,and then verified by q RT-PCR experiments to obtain?P11.The genes related to oxidative stress?peroxidase,catalase,SOD,etc.?did appear to be significantly up-regulated.At the same time,in the?-galactosidase activity test,the mixed plaque of?P11 and DH5??pUC18?showed the fastest discoloration on X-Gal plates,which proved that the T6SS activity of?P11 was lower than that of wild type.T6SS activity was lower,indicating that P11 positively regulates the activity of the type VI secretion system ofP.syringae MB03.In addition,in the nematode feeding preference experiment,biofilm formation experiment,and swimming?swamming?experiment,the interrupted strains showed different differences from the wild-type P.syringae MB03,respectively.In summary,the sRNAs in P.syringae MB03 bear different regulatory functions of life activities.