| Iron(Fe)is an essential element for all eukaryotic cells and is required for important cellular functions such as DNA synthesis and repair,respiration,and detoxification of free radicals.Iron is abundant in the Earth's crust but is poorly bioavailable due to its low solubility in aerobic environments at neutral pH.During long-term evolution,certain plants and microorganisms have evolved specific mechanisms to chelate insoluble iron through release of siderophores,which are low molecular weight organic compounds with high affinity for iron,which convert insoluble iron in the soil into bio-available iron.Although we have done some researches on extracellular siderophores in aspects of biological control and growth promotion,the molecular mechanism of its synthesis and promotion are still unclear and need further study.Trichoderma spp.is one of the most promising biocontrol microorganisms in the current green agricultural production and can produce siderophores that have the functions of absorption,storage and intracellular transport of iron,and have an important effect on the growth and development of the thalli itself.In this study,we use Trichoderma asperellum T6 with high-yield siderophores,which was separated and identified by my laboratory,to carry out the following researches:1.To identify the key genes involved in siderophores biosynthesis in T.asperellum,firstly,through continuous cultivation of the wild-type strain(WT)in iron-deficient media and determination of the daily siderophores production,it was found that on the 5th day,the siderophores production reached a maximum value of 37.53%.Secondly,through the determination of the unit siderophores production in media with the presence or absence of iron element,it was found that the unit siderophores production in the presence of iron was 96.43% lower than that in the case of iron deficiency.Conversely,the dry weight of mycelium increased by 125.86 % compared with that of iron deficiency.Simultaneously,the expression level of sidA gene of T.asperellum in the presence or absence of iron was analyzed by RT-PCR.The gene expression level of iron deficiency was higher than that of iron.The above two experimental results showed that a clear correlation between sidA gene and siderophores production,and it is preliminarily considered that sidA gene may participate in the synthesis of siderophores produced by T.asperellum.2.The sequence of sidA was obtained from the T.asperellum genome database(http://genome.jgi.doe.gov/Trias1/Trias1.home.html),and SMART was used for domain analysis to further confirm the correctness of the sidA.The Double-jiont PCR technique was used to knock out the sidA gene,which is the major siderophores gene of T.asperellum,based on the principle of homologous recombination.At the same time,the gene deletion strain ?sidA was constructed to study the biological function of the siderophores.By obtaining the knockout mutant ?sidA,it will be clear whether sidA gene is a key gene for siderophores synthesis of T.asperellum.(1)To determine the concentration of hygromycin B against T.asperellum,a series of concentration gradients were set on PDA and TB3 solid media respectively.The growth diameter was measured at 40 h and the inhibition rate was calculated.The results showed that 300 ?g/m L hygromycin B can completely suppress the growth of hyphae in 40 h.(2)According to the sidA Knockout fragment of T.asperellum constructed by Double-joint PCR technique,the principle of homologous recombination and protoplast transformation,the sidA knockout mutant ?sidA was obtained eventually.The gene knockdown and the single copy integration were verified by anchor PCR and Southern blot.3.After obtaining the correct ?sidA of T.asperellum,its phenotypes were analyzed and studied.The results are as follows:(1)In order to further determine that the sidA gene is a key gene for the siderophores synthesis of T.asperellum,the unit siderophores production of the wild-type(WT)strain and two mutant strains(?sidA)cultured in iron-deficient LNM medium for 5 days was determined.It was found that the unit siderophores production of the two mutant strains decreased by 38.67% and 36.65%,respectively.The results showed that the sidA gene is a key gene for the synthesis of siderophores in T.asperellum.(2)In order to study the effect of siderophores on the growth of T.asperellum,the colony growth assays of WT and ?sidA in PDA,LNM,MM,MM-Fe(iron-deficient MM medium)was performed in this study,and found no significant difference on iron-rich PDA and MM media;when they grown on iron-deficient LNM and MM-Fe media,WT grew slightly faster than ?sidA.The above results indicated that the siderophores can promote the growth of Trichoderma spp.in an iron-deficient environment.(3)In order to clarify the effect of knockout of the sidA gene of the siderophores on the sporulation and spore germination of T.asperellum,spore germination of WT and ?sidA were observed under microscope at 9 h and 12 h,and taking pictures.It was observed that the spore germination rates of ?sidA decreased by an average of 96.26% and 46.40%,respectively.The sporulation of two ?sidA growing on PDA for 10 days also decreased by 33.01 % and 41.02 %,respectively.The results showed that the sidA gene of the siderophores can affect the sporulation and spore germination of T.asperellum.(4)In order to study the effect of sidA gene on resistance of T.asperellum,the plate growth assays of WT and ?sidA under NaCl,KCl,DS(D-Sorbitol)and SDS stress were performed.The inhibition rates of ?sidA were higher than those of WT under NaCl,KCl,DS and SDS stress.The results showed that the knockout of sidA gene reduced the resistance of T.asperellum T6 to various stress factors. |
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