Cloning And Functional Analysis Of StNPP1 Gene Of Setosphaeria Turcica

The pathogen of northern corn leaf blight（NCLB）is Setosphaeria turcica（Luttrell）Leonard et Suggs.It causes very serious harm to maize production and belongs to the worldwide maize disease.NCLB first broke out in Italy,and at the beginning of the 20th century,it had spread to all maize production areas around the world,and it once became an important fungal disease that seriously harmed maize production.NPP1（necrosis inducing Phytophthora protein 1）is an effector protein related to Phytophthora in parsley and Arabidopsis.Through whole genome sequencing and gene function prediction,StNPP1 is screened in Setosphaeria turcica.In this paper,based on the research object of StNPP1 gene,the expression quantity of StNPP1,the cloning of StNPP1,the bioinformatics analysis of coding proteins,and the effect of StNPP1 on the pathogenicity of Setosphaeria turcica.The specific research results are as follows:1.Screening of effector protein and detection of StNPP1 expression in Setosphaeria turcica.The effector protein StNPP1 was screened out by sorting out the functional effector proteins in B.maize.The results of q RT-PCR detection of StNPP1 gene expression showed that StNPP1 gene was highly expressed in the early stage of infection.After 6 hours of inoculation,comparing with the control group inoculated with maize,the expression level of StNPP1 was up-regulated by 4.30 times in the treatment group.With the extension of the course of disease,the expression begins to decrease gradually,but it reached the peak at 48hours.Comparing with the control group,the expression level of the treatment group was up-regulated by 6.85 times.Overall,The expression level of StNPP1 was always higher than the control group,indicating that StNPP1 may play a positive regulatory role in the process of strain-host interaction.2.Cloning and bioinformatics analysis of StNPP1 geneObtained the StNPP1 gene sequence of B.maize by T-A cloning technology.The whole length of StNPP1 was 770 bp.The prediction and analysis of a series of bioinformatics software showed that the total number of amino acids encoded by StNPP1 effector protein was 229,the molecular weight of the encoding protein was about 24.26 k Da,and the theoretical isoelectric point PI was 6.95.The effector protein contains a total of 3386 atoms.It was predicted that its molecular formula was C₁₀₈₇H₁₆₇₅N₂₈₉O₃₂₈S₇and its fat index is 79.65.It was predicted that the protein was a hydrophobic protein with signal peptide and no transmembrane structure.It was speculated that it was a secretory protein.In addition,there were no complex structures such asβ-bridge and Bend region in the secondary structure of StNPP1 effector protein,and the model of the secondary structure was relatively simple.The phylogenetic tree analysis of the effector protein StNPP1 showed that it had the highest homology with Et28A family proteins of maize,and the consistency was as high as 100%,but the homology with other pathogenic fungi was low.3.The proof for the StNPP1 gene of Setosphaeria turcica inhabited the immune response of plantsThe recombinant plasmid p GR107-StNPP1 was successfully constructed through T-A cloning,PCR product purification,gel recovery,single enzyme digestion,homologous linker primer design,seamless connection,Escherichia coli transformation and other tests,then the expression vector p GR107-StNPP1 was transferred into Agrobacterium GV3101.The recombinant plasmid induced by Agrobacterium can inhibit programmed cell necrosis caused by INF1 elicitor on tobacco leaves,It was preliminarily proved that StNPP1 gene can inhibit plant immune response.4.Functional analysis of StNPP1 gene of S.turcicaTwo VIGS infection cloning systems were successfully constructed:StNPP1-pCN81-240and StNPP1-pCN81-250.The Agrobacterium solution and Agrobacterium containing p C101and p C301 were mixed at a ratio of 1:1:1,Agrobacterium infiltrated the leaves and inoculats tobacco,resulting in the systemic infection of tobacco.After inoculating the crude extract of tobacco leaves virus into maize inbred line B73 by acupuncture,mosaic leaves and slight photobleaching symptoms appeared 18 days after inoculation.On maize plants inoculated with VIGS vectors StNPP1-pCN81-240 and StNPP1-pCN81-250,the relative expression levels of StNPP1-pCN81-240 and StNPP1-pCN81-250 were both down-regulated.These results showed that the vector could induce the silencing of StNPP1 gene in maize,and the silencing efficiency of StNPP1 gene was as high as 65.8%when the fragment of StNPP1-pCN81-250 was silenced.The silencing of StNPP1 gene weaked the infection of pathogenic bacteria,the gene can help the pathogen to complete the infection and colonization of the host plant.