Studies On The Siderophores Related Genes Of Rice Endophytic Streptomyces Hygroscopicus OsiSh-2

Iron is essential for the normal growth and development of most living things on Earth.Although iron is abundant in nature,most of it exists in the form of insoluble hydroxides and oxides,which are difficult to be absorbed and utilized by organisms.Siderophores are small molecular compounds that can chelate ferrivalent iron efficiently.In recent years,more and more studies have shown that siderophores play an important role in the interaction between microorganisms and the environment.Microorganisms use siderophores to effectively absorb and transform iron for their own use.The ferricarriers compete with other microorganisms for iron to achieve antagonism.Siderophores of beneficial microorganisms can also help host plants absorb iron and improve the tolerance of plants to iron-deficient environments.Therefore,it is of great significance to explore the types,functions and modes of action of siderophores produced by functional microorganisms for the exploitation of microbial siderophores.In this study,we focused on the high-yield siderophores of Streptomyces hygroscopicus OsiSh-2 in rice by bioinformatics analysis combined with a series of physiological,biochemical and molecular biological techniques.A series of studies were carried out on the types of siderophores produced by siderophores and their action modes in response to iron deficiency stress and antagonism to magnaporthe grisea,and the following results were obtained:1.There are four potential siderophores in the OsiSh-2 genome.Anti SMASH analysis showed that there were four potential siderophores in OsiSh-2 genome,which were located in Scaffold 5,Scaffold 7,Scaffold 14 and Scaffold 25.Scaffold 5and Scaffold 25 are 100%similar to the known hydroxamic acid type siderophores Desferrioxamine B and Coelichelin respectively,hereinafter referred to as Desand Coe.The Scaffold 7 and Scaffold 14 gene Scaffolds are not annotated as known compounds,but contain partial elements similar to the carboxylate type siderophores Staphyloferrin B,which have been tentatively named Sh-2-S1 and Sh-2-S2.Further comparison of the core synthetic genes of the above four gene Scaffolds showed that Des,Sh-2-S1 and Sh-2-S2 all had non-ribosomal peptide synthase（NRPS）independent biosynthesis mechanism,and only Coe was NRPS dependent biosynthesis mechanism.The presence of at least two different types of siderophores in the OSISH-2 genome was observed.2.Osish-2 can respond to changes in iron concentration in the surrounding environment by regulating biosynthesis of different siderophores.OsiSh-2 started siderophores biosynthesis from the third day without adding iron,and its maximum yield reached 87.01%,which was relatively high compared with other reported endophytic actinomycetes.However,only 30μm of Fe Cl₃ was added to the medium to completely inhibit the production of siderophores,indicating that OsiSh-2 is highly sensitive to changes in iron concentration in the environment.The transcription levels of four key synthesis genes ShDesD（Des）,Sh Cch H（Coe）,Sh Csg1（Sh-2-S1）and Sh Csg2（Sh-2-S2）in Osish-2 were detected by fluorescence real-time quantitative PCR.It was found that in the iron deficiency environment,The four key siderophore synthesis genes of OsiSh-2 were induced to express,and the transcription level of Sh Cch H with siderophore Coe was the largest,which increased 19.27 times in iron-poor medium compared with iron-added environment on day 7.3.By constructing siderophores mutant strains,it was preliminarily confirmed that different siderophores play different roles in affecting OsiSh-2 growth and resistance to stress.The genetic operation system for siderophores related gene knockout and overexpression of OsiSh-2 was determined and optimized.Siderophores gene Scaffolds or key core synthetic genes were knocked out by homologous recombination and conjugative transfer techniques,and three siderophores corresponding to the synthetic defective mutant were successfully obtained:Δdes D,Δcch H,Δcsg2 and a siderophore overexpressed strain DesD-OX.The ferritic yield ofΔdes D mutant decreased to a certain extent,and the ability of melanin production was inhibited in the spore ofΔdes D mutant,but the antagonistic ability ofΔdes D and DesD-OX mutant against magnaporthe griseae did not change compared with the wild type.Δcsg2 siderophores yield was almost unchanged compared with wild type.However,the antagonistic ability to magnaporthe grisea decreased significantly.Compared with wild type,Δcch H siderophores yield decreased,growth rate slowed down,antagonistic ability to magnaporthe grisea significantly decreased,and sensitivity to oxidative stress significantly decreased,increasing the tolerance of hydrogen peroxide.In summary,four siderophores of OsiSh-2 were identified in this paper,and all of them were involved in OsiSh-2’s response to stress,and the genetic operating system of OsiSh-2 siderophores related gene knockout and overexpression was optimized.And revealed the role of Des,Coe and OsiSh-2 siderophores secreted by OsiSh-2 in OsiSh-2 growth,siderophores yield and rice blast resistance mechanism.It provides technical support and theoretical basis for further enriching the functional spectrum of microbial siderophores and OsiSh-2 for disease prevention and promoting vitality.