Construction Of Genetic Knockout System By Endophytic Bacteria 336X With Biological Control Ability

Endophytic bacteria refers to a kind of microbial that can colonise in the healthy plant tissue and establish the harmonious relations with host plant. Endophytic bacteria can promote the growth of plants and improve plants resistance, etc. At present, Endophytic bacteria research has become a hot spot of research at home and abroad. Using endophytic bacteria on wheat to control plant soil-borne diseases, which is safe, effective etc, has become the various countries' phytopathologists research hot spot.Studying on biocontrol mechanism of biocontrol strain found the theoretical basis of producting a stable, efficient and easy to save biocontrol agent and applying to fields. Separating biocontrol genes which is related to biocontrol and researching the functions of genes at the molecular level is an important way to find out biocontrol mechanism of biocontrol strain.Serratia proteamaculans 336X used in this research is Gram-negative bacteria separated from wheat root in our laboratory.It is a biological control agent against take-all disease of wheat. Biocontrol efficacy of 336X against wheat take-all disease can amount to 85%. Wheat endophytic bacteria 336X can produce chitinase and protease. Both of them are cell wall degrading enzymes.It also can form biofilm, iron carrier, Induction system resistance, etc. Serratia proteamaculans 336X shows well colonization in the wheat rhizosphere. It has the the ability of movement and chemotaxis which are associated with biocontrol ability.In order to study the functions of Genes which are related with biocontrol ability of endophytic bacteria 336X, further understand the biocontrol mechanism of biocontrol strain 336X.This study starts from two directions. On the one hand use the sacB gene that encodes levansucrase mark the carrier.When sacB gene express in 336X, it will has the sensitivity of sucrose. Construct ring carrier genetic knockout system of wheat endophytic bacteria 336X.With the methods knock out flagellar movement of related gene motA, get four motA knockout mutations. Similarly knock out chemotaxis genes CheW and get three CheW knockout mutations.All above provide a powerful evidence for the feasibility of the system.On the other hand, use Red Recombination System to construct linear carrier genetic knockout system of wheat endophytic bacteria 336X. At first get engineering bacterium 336X-1 through genetic transformation for 336X, which makes linear carrier in 336X-1 can occur. Further determine the homologous recombination 336X-1 on the temperature sensitivity. Construct the linear targeting C1-Km-C1 that is related with chiA gene.Through the electroporation method introduce the linear targeting fragment above into engineering bacterium 336X-l,and get two Chitinase A knockout mutations. Also, construct the linear targeting phy-Km-phy that is related with assume phytase gene of 336X. Through the knockout method above get four assume phytase gene knockout mutations.Above provide a powerful evidence for the feasibility of the system.Finally, compare the two knockout systems and analyze the advantages and disadvantages of each.The study above lay the theoretical foundation of researching the function of genes at the molecular level and stating biocontrol mechanism of biocontrol strain.