The Overexpression Mutant Preparation And Functional Analysis Of StCHS5 Gene In Setosphaeria Turcica

Chitin,synthetized catalytically by chitin synthetase,is the main component of the fungal cell wall and an ideal target for fungicide.there are 8 chitin synthase genes in the genome of Setosphaeria turcica.Among 8 chitin synthase genes the expression level of StCHS5 is the highest in the stage of appresorium development.No conidia are produced in StCHS5 knockout mutants.The StCHS5 overexpression mutants were created in this paper aiming to clarify the function of the gene by comparing growth and development as well as pathogenesis of StCHS5 knockout mutants,wild type,and StCHS5 overexpression mutants.The main findings are as follows:1.StCHS5 belongs to the class Ⅳ and subfamily Ⅱ in the chitin synthase gene family of Setosphaeria turcica.StCHS5,a full length of 5780 bp and a CDS sequence of 5301 bp,located at the scaffold₁8:484659-490439（-）position in the genome contains 2 introns and 3 exons.StCHS5 protein is located on the cell membrane and contains actin domain,cytochrome b5 domain,and chitin synthetase catalytic domain Ⅱ;its molecular formula is C8760H13669 N2377O2632S78,which encodes 1766 amino acid residues,with a relative molecular mass of 196898.92Da,the isoelectric point（PI）is 5.76,the fat index is 84.98,the average hydrophilicity is-0.252,and the instability index is 40.08,which are unstable proteins.The StCHS5 protein consists of 37.94%α-helix.4.64%β-turn,12.46%extended chain,and 44.96%random coil.2.StCHS5 gene and green fluorescent GFP gene were amplified by fusio n PCR method.Using the PBARKS I plasmid as a vector,the StCHS5 gene overexpression vector PBARKSI-GFP-StCHS5 was successfully constructed by seamless ligation.StCHS5 gene overexpression transformants were gained by s creening protoplast with glufosinate-resistant Bar gene and green fluorescent-la beled GFP gene.Transformants were verified by glufosinate resistance screenin g,Bar sequence amplification,GFP sequence amplification,and Real-Time-PC R.Four strains of StCHS5 gene overexpression were obtained and named OEStCHS5-1,OE-StCHS5-2,OE-StCHS5-3,OE-StCHS5-4,respectively.3.Compared with the wild type,the colony growth rate is faster,colony colour is lighter,melanin content of the ΔStCHS5 mutant mycelium is less,the mycelium cells are longer,and no conidia are produced;The colony growth rate of the OE-StCHS5 mutant is slower,colony colour is darker,melanin content of the OE-StCHS5 mutant mycelium increases,mycelium cells become shorter,and no conidia are produced.This results showed that StCHS5 gene is involved in the growth and development of pathogens.4.In the PDA media containing Congo Red,CFW and SDS,the colony growth of the ΔStCHS5 mutant was significantly better than that of the wild type and OE-StCHS5 mutant,indicating that the deletion of StCHS5 reduced sensitivity of theΔStCHS5 to cell wall interfering substance such as Congo Red,CFW,SDS.The chitin content in the mycelium cell wall of ΔStCHS5 mutant was significantly lower than that of wild type,and the number of protoplasts released by hydrolyzing mycelium cells using cell wall decomposing enzyme increased significantly.In contrast,the chitin content in the mycelium cell wall of OE-StCHS5 mutant was significantly increased and the number of the protoplasts released by hydrolyzing mycelium cells using cell wall decomposing enzyme are lower than the wild type.In addition,in the ΔStCHS5 mutant,chitin accumulation in the mycelium cell tip was less than that of the wild type,while the OE-StCHS5 mutant in the mycelium cell tip had higher chitin accumulation than the wild type.The above results indicate that the StCHS5 mutation may affect the chitin synthesis and assembly between chitin and other components in the cell wall,which in turn affect structure of the cell wall as well as the sensitivity of the cell to cell wall interfering substance and the ability of cell wall anti-enzymatical hydrolysis,by affecting the synthesis or accumulation of chitin in the cell wall,and then affect the sensitivity of the cell to interfering substances and the degree of enzymatic hydrolysis.5.Compared with the wild type,the ΔStCHS5 mutant mycelium could not develop normal appressorium and invasive nail structures,and could not penetrate cellophane.However,in the OE-StCHS5 mutant,the formation of appressorium and invading nail structure was delayed,and the ability to penetrate cellophane was lower than that of the wild type.StCHS5 gene affects the formation of appressorium and the ability of penetrating cellophane.