Functional Analysis Of Endochitinase Gene VDECH From Verticillium Dahliae

Chitin constitutes 3%-60% of fungal cell wall.Most fungal cell wall could be degraded by chitinase,which could,prevent or interrupt the infection,colonization and expansion of fungi in plants.Fungal chitinase is an important member of chitinase family with excellent application potential in biological control of plant diseases.In this paper,the endochitinase gene VDECH was cloned from the genomic DNA of Verticillium dahliae strain Vd080,and the recombinant protein was expressed in vitro.The prokaryotic expression product VDECH showed the function of elicitor,which induced plant primary immune response,caused hypersensitivity reactions(HR)in both cotton and Arabidopsis,and induced the production and accumulation of hydrogen peroxide as well.Besides,VDECH could up-regulated expression of defense related genesof cotton and Arabidopsis and innduce systemic acquired resistance of cotton and improve resistance to the main pathogen,V.dahliae.In addition,VDECH can also directly inhibit the conidia germination of V.dahliae and Fusarium oxysporum.The main results are as following:1.The endochitinase gene VDECH was cloned from V.dahliae strain Vd080,and the VDECH gene was located on the first chromosome of V.dahliae and consisted of 1902 bases,including a 54 bp intron and two exons.The size of the protein was 615 amino acids,the molecular weight was 63.9 k Da and the isoelectric point was 6.35.2.The prokaryotic expression vector p Cold-TF-VDECH was constructed and transformed into E.coli BL21 for protein expression.With different IPTG induction and temperature conditions,determined concentration of IPTG of 0.6 m M and induction temperature of 16 °C for the best induction conditions.Purified recombinant protein with His-tag was obtained by column purification.3.VDECH can induce plants to produce primary immune responses.5μg/m L and above of VDECH can induce Arabidopsis and cotton leaves produce typical HR phenotype,and induce the production and accumulation of hydrogen peroxide.4.VDECH has a broad spectrum of temperature tolerance,after 4 °C-100 °C treatment,still have chitinase activity and could induce plant hypersensitivity reactions.Chitinase showed activity the highest at 50 °C,0.563 U/mg.5.VDECH could induce cotton system acquired resistance(SAR),and significantly improve the cotton resistance against V.dahliae.The disease control efficiency reached to 51.43% after 3 days of the VDECH treatment to leaves.6.VDECH could induce the expression of plant resistance-related genes and activate the defense response.The expression of PDF1.2,LOX,PR1,PR2,PR5,SOD and POD genes in Arabidopsis were significantly up-regulated,and PDF1.2 was up-regulated 196 times after 48 hours induction.At the same time,the expression levels of PAL,4CL,cadherin synthase gene,acidic chitinase,basic chitinase,PPO and POD were also significantly up-regulated in cotton.7.VDECH can effectively inhibit the conidia germination of Verticillium dahliae and Fusarium oxysporum.After 36 h of incubation with VDECH,the inhibition rate of germination was recorded at 99.57% for V.dahliae,and 96.89% for F.oxysporum.These results indicate that VDECH could be recognized by the plant and elicit defense responses with plant resistance increasing.VDECH is an effective inhibitor of conidia germination,which might play critical role in the interaction between plants and pathogens.In summary,VDECH has the potential and application prospect to improve plant resistance and control diseases.