Expression Of Pathogenic Genes From Pseudomonas Syringae MB03 And Nematicidal Activity Against Caenorhabditis Elegans

the disease caused by plant-parasitic nematodes(PPNS)have become the second-largest agricultural damage which is only next to fugal disease in the world,Far more than bactrial and viral disease,and The annual economic losses of the agricultural crop up to billions of dollars.When plant parasitic nematodes destroying plants roots and tissues by feeding,it also induces other soil microorganisms' infections,mainly fungi and Bacteria to increase damage and plants death.The most effective way to control nematodes disease in the past few decades is chemical nematicides,but it is likely to cause the irrecoverable damage to the environment.Due to much concern about health and environment safety have lead to remove several nematicides.Therefore an urgent need to replace chemical pesticides with alternative means of control of biological control which is non toxic and more environment friendly.In this study,we regard first time reported the Nematicidal activity of P.syringae MB03 isolated by our own lab which has toxic to C.elegans and M.incognita as background,C.elegans as a model organism.We adopted two strategies to screen the potential virulence genes of P.syringae MB03.In this study,totally 57 virulent genes successfully cloned and Expressed in E.coli to carry out Nematicidal activity.First we checked the virulence genes by using the induced recombinant bacteria for nematicidal activity to C.elegans,Then go for further analysis.Among 57 cloned genes 10 high toxic activity of recombinant bacteria were identified and 8 recombinat proteins were purified such as:GL003493?GL005008?GL000116?GL001656?GL001678?GL004219?Spr03(GL001094)and YbgIO3(GL001171).Furthermore,in order to confirm which conserved domain play a major toxic role of the MCP proteins,we have studied the domain truncation of this kind of proteins and totally get 11 proteins.After the toxicity test of the 11 purified proteins to the nematodes,the conclusion are as follows:the only MCP protein showed no nematicidal activity while those purified conserved domain proteins similar to both MCP and HAMP are lethal to C.elegans.The LC50 value of purified recombinant proteins of GL003493,GL005008 and GL004759MCP+HAMP are 390.86 ?g/ml,492.54 ?g/ml and 511.89?g/ml respectively;and the other proteins GL004219 and GL000116 show a certain insecticidal activity,and the LC50 value are measured as 274.19 ?g/ml and 320.89?g/ml.The protein GL001656 and GL001678 has no nematicidal activity.Interestingly The protein Spr03((GL001094)and Ybg103(GL001171)showed the highest activity among these proteins,and measured LC50 value of the protein Spr03(GL001094)and Ybg103(GL001171)are 147.90 ?g/ml and 192.60 ?g/ml.Moreover,these two proteins also have a certain effect on brood size,locomotion,L1 growth inhibition,and the behavioral response of C.elegans.